type 2 collagen research

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• Published: 29 January 2016

Efficacy and tolerability of an undenatured type II collagen supplement in modulating knee osteoarthritis symptoms: a multicenter randomized, double-blind, placebo-controlled study

• James P. Lugo 1 ,
• Zainulabedin M. Saiyed 1 &
• Nancy E. Lane 2  

Nutrition Journal volume  15 , Article number:  14 ( 2015 ) Cite this article

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Undenatured type II collagen (UC-II) is a nutritional supplement derived from chicken sternum cartilage. The purpose of this study was to evaluate the efficacy and tolerability of UC-II for knee osteoarthritis (OA) pain and associated symptoms compared to placebo and to glucosamine hydrochloride plus chondroitin sulfate (GC).

One hundred ninety one volunteers were randomized into three groups receiving a daily dose of UC-II (40 mg), GC (1500 mg G & 1200 mg C), or placebo for a 180-day period. The primary endpoint was the change in total Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) from baseline through day 180 for the UC-II group versus placebo and GC. Secondary endpoints included the Lequesne Functional Index (LFI), the Visual Analog Scale (VAS) for pain and the WOMAC subscales. Modified intent-to-treat analysis were performed for all endpoints using analysis of covariance and mixed model repeated measures, while incremental area under the curve was calculated by the intent-to-treat method.

At day 180, the UC-II group demonstrated a significant reduction in overall WOMAC score compared to placebo ( p  = 0.002) and GC ( p  = 0.04). Supplementation with UC-II also resulted in significant changes for all three WOMAC subscales: pain ( p  = 0.0003 vs. placebo; p  = 0.016 vs. GC); stiffness ( p  = 0.004 vs. placebo; p  = 0.044 vs. GC); physical function ( p  = 0.007 vs. placebo). Safety outcomes did not differ among the groups.

UC-II improved knee joint symptoms in knee OA subjects and was well-tolerated. Additional studies that elucidate the mechanism for this supplement’s actions are warranted.

Trial registration

CTRI/2013/05/003663 ; CTRI/2013/02/003348 .

Peer Review reports

Introduction

Osteoarthritis, which entails the destruction of joint cartilage and remodeling of the adjacent bone, is the most common form of arthritis affecting more than 25 million Americans [ 1 ]. Current therapies for OA include various over the counter analgesics, a number of nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of corticosteroids or hyaluronic acid, plus tramadol and other opioid analgesics to relieve severe pain [ 2 , 3 ]. While these therapies can alleviate symptoms in the near term, their ultimate impact on the pathophysiologic progression of OA is limited [ 4 ].

Previous studies reported UC-II to be efficacious for the treatment of arthritis [ 5 , 6 ]. More recently, a statistically significant improvement in knee joint function over placebo was also reported in a clinical study comprising a group of healthy individuals, supplemented with UC-II, and who developed transient knee joint pain upon strenuous exercise [ 7 ]. These same individuals also took longer to experience pain after 120 days of supplementation. Based on these observations, the current study was designed to evaluate the efficacy of UC-II in knee OA subjects compared to placebo and to GC, which is a widely available supplement that is used for reducing joint pain.

Materials and methods

Investigational products.

The study product UC-II® (Lot 1204004) was derived from chicken sternum. It was manufactured under current good manufacturing practice (cGMP) conditions using a patented process that preserved its native structure (Chick Cart Inc., Fort Smith, AR). Both glucosamine hydrochloride (GH) and chondroitin sulfate (CS) were purchased through Wilke Resources (Lenexa, KS). The Wellable group (Shishi City, Fujian) manufactured GH under cGMP and according to United States Pharmacopeia 26 specifications. Sioux Pharm (Sioux Center, IA) manufactured bovine-derived CS under cGMP. UC-II and GC were encapsulated in opaque, size “00” capsules with sufficient amounts of excipients (microcrystalline cellulose and silicon dioxide) such that they were sensory identical to placebo. InterHealth Nutraceuticals provided all study materials. All American Pharmaceutical (Billings, MT) verified the amount of active ingredients in the study capsules. Study materials were kept in a secure cabinet with access restricted to the site coordinator, the dispensing pharmacist, and the principal investigator.

Study design

The objective of this randomized, double-blind, placebo-controlled clinical study was to evaluate the ability of UC-II to improve knee symptoms in OA subjects, as measured by overall WOMAC score, compared to placebo and to GC. The trial was conducted at 13 centers in southern India. Because of a limitation in synovial fluid sampling procedures at multiple clinical sites, the study was conducted under two separate study protocols. Study protocols were approved by each center’s Institutional Ethics Committee (IEC), and listed on the clinical trial registry of India as study protocols 003663 and 003348. Enrollment, randomization, and follow-up visits were identical for both protocols, and were carried out at days 1 (baseline), 7, 30, 60, 90, 120, 150 and 180 (Table  1 ). All investigators attended the same investigator meetings, used identical intake and data reporting forms, and were trained and monitored by the same group of clinical research associates.

Efficacy measurements were assessed at all visits and included WOMAC, VAS, and LFI indices. The knee flexion range of motion (ROM) test was performed at each visit. Subject diaries and study product were provided at all visits, except day 180 and were collected at all follow-up visits. Subjects were instructed to record daily the consumption of study product, use of rescue medication, as well as concomitant medications in the subject dairy for the entire duration of the study. Blood and urine were collected at screening and day 180. Pregnancy testing was done at screening and follow-up visits. Adverse events (AEs) were recorded using each subject’s diary inputs plus site visit questionnaires administered by intake personnel at all study visits.

Clinical endpoints

The primary endpoint was defined as the change in total WOMAC score from baseline through day 180 for the UC-II group versus placebo and GC. Secondary clinical endpoints for both protocols were similar and included the change from baseline through day 180 versus placebo and GC for all endpoints including the following scores: (1) mean VAS; (2) mean WOMAC subscales; (3) LFI; and (4) knee flexion. Another endpoint included the change from baseline to day 180 for the serum biomarker cartilage oligomeric matrix protein (COMP). In protocol 003348, additional secondary endpoints included the change in serum biomarker, C-reactive protein (CRP) plus synovial fluid biomarkers interleukin (IL)-6, and matrix metalloproteinase (MMP)-3 from baseline to day 180.

Study subjects

A total of 234 subjects were screened and 191 randomized (Fig.  1 ). Study inclusion criteria were 40–75 years-old male and female subjects, a body-mass index (BMI) of 18–30 kg/m 2 , moderate-to-severe OA by physical examination (crepitus, bony enlargements, joint swelling, etc.) in one or both knees, knee pain for at least 3 months prior to the start of the study, an LFI score between 6 and 10 and a VAS score of 40–70 mm 7 days after withdrawal from excluded medications, plus a knee radiograph that was graded as Kellgren and Lawrence (K-L) radiograph score of either 2 or 3 [ 8 ]. All OA diagnoses were confirmed by each study site investigator and noted in the subject’s case report form (CRF). In the case of bilateral knee involvement, the index knee used for the study was the one that presented with the most severe OA symptoms at baseline. Detailed inclusion–exclusion criteria are summarized in Table  2 .

Enrollment and randomization flow chart

Ethics, consent and permissions

Subjects were recruited after they reviewed, understood the study details, and then signed the IEC-approved consent form. The study conformed to the Declaration of Helsinki (version 1996).

Randomization & blinding

Block randomization, consisting of nine individuals per block, was executed in a 1:1:1 ratio using random numbers generated by an independent statistician (SPSS version 16.0). Knowledge of the randomization code was limited to the statistician plus one QA monitor unrelated with the study. Each investigator was given opaque, sealed envelopes denoting single patient identity numbers, randomization codes, and supplementation regimen to be opened in case of an emergency. The code was broken after the clinical database was locked.

Dosing regimen

Subjects ingested two blue pills in the morning with breakfast and two white capsules before bedtime. For the UC-II cohort, the two morning capsules were placebo, while the evening capsules contained 20 mg each of UC-II totaling 40 mg, which is identical to previously used clinical dose levels [ 5 , 7 ]. This dose delivered 1.2 mg of undenatured type II collagen as determined by a newly developed and validated extraction-ELISA protocol (AIBiotech, Richmond, VA & Chondrex, Redmond, WA). For the GC group, the morning and evening doses delivered 750 mg of GH plus 600 mg of CS each totaling a daily dose of 1,500 mg of GH plus 1,200 mg of CS. The placebo group ingested identical numbers of blue and white capsules containing excipients only. Study bottles were labeled according to ICH-GCP and applicable local regulatory guidelines.

Prior and concomitant therapies

Prior medications were documented at the screening visit by the study investigator. At each visit, study personnel reviewed subject diaries and questioned each participant on the use of any concomitant medications including those on the prohibited list. Prohibited medications included ibuprofen, aspirin, other NSAIDS, or any other pain relievers (OTC or prescription), plus any dietary supplements (excluding vitamins) that could support joint health. All concomitant medications used during the study was documented in the subject’s medical record by the study investigator then transcribed into their CRF by study personnel.

Rescue medications

Acetaminophen was allowed at a dose of 500 mg twice daily. Participants were instructed to not take this medication within 48 h of an evaluation visit. Usage levels and timing was entered at each visit into the subject’s medical record by the study investigator. Study personnel transcribed this information into the subject’s CRF.

Compliance and safety

Subjects were instructed to bring their bottles to each visit. Remaining capsules were counted and recorded in the subject’s CRF and accountability log. As a secondary measure of compliance, subjects completed a diary indicating daily dosing of the study products. Safety assessments were performed at all visits by the site investigator and staff (see Table  9 ).

Study evaluations

WOMAC scores were determined using the WOMAC VA3.1 questionnaire containing 24 items grouped into three categories: pain, stiffness, and physical function (score range 0–2400). Each respective WOMAC subscale mean scores was determined by dividing the subscale score by the number of questions (5, pain; 2, stiffness; 17, physical function) it contained. The mean VAS score was determined using a VAS questionnaire containing 7 pain-related questions (score range 0–700), and then dividing the overall score by seven. LFI score was determined using an LFI questionnaire that assessed pain, walking distance, and activities of daily living, (score range 0–24). Knee flexion was measured using goniometry with the subject lying in the prone position and the leg to be tested positioned along the edge of the table [ 9 ].

Synovial fluid biomarkers

Synovial fluid (~0.5 mL) was aspirated from the knee joint using an appropriate sized needle (18–24 gauge, depending on joint size). Harvested fluid was stored frozen until tested. IL-6 and MMP-3 levels were determined using the corresponding Duoset ELISA kits (R&D Systems, Minneapolis, MN).

Serum biomarkers

COMP levels (Quantikine ELISA, R&D Systems) were determined in both study protocols. CRP levels (Latex COBAS INTEGRA, Roche Diagnostics GmbH, Mannheim) were assessed in protocol 003348. Serum was stored frozen until analyzed. Interassay and intrassay coefficients of variation for COMP and CRP were <5 %.

We verified, using 2-way analysis of variance (ANOVA), that the results of the two protocols could be combined into a single analysis by demonstrating there was no group by study interaction and that the magnitude of the efficacy observed under the two protocols was similar.

A modified intent-to-treat (mITT) analysis was used to assess the efficacy and safety outcomes that was defined a priori . This included all subjects who were randomized, consumed study product, and had at least one completed post-baseline visit. An analysis of covariance (ANCOVA), that included supplementation as a fixed factor and the corresponding baseline value of the variable being tested as a covariate, was used for assessing the overall statistical significance of the primary and secondary endpoints. Following ANCOVA, the Tukey-Kramer multiple comparison test was used for determining pairwise statistical significance and calculating 95 % confidence intervals. Also, a mixed model repeated measures (MMRM) analysis of the primary endpoint was performed to account for the multiple assessments obtained during this study. In addition, the method of trapezoids was used to calculate incremental area under the curve (iAUC) for all study groups. For iAUC estimation, missing values were imputed using the expectation-maximization algorithm in SAS. Rescue medication usage between groups was compared using logistic regression followed by pairwise comparisons using the Tukey-Kramer test. In addition, a stratified analysis of the primary endpoint was performed according to baseline serum COMP levels above and below the median value for this biomarker. Differences were considered significant if the resultant p-value was ≤0.05. An independent statistician performed the analyses and other calculations using SAS version 9.3 (Cary, NC).

Demographics and baseline characteristics

Two hundred and thirty-four subjects were screened and 191 subjects who met the eligibility criteria were randomized to placebo ( n  = 62), GC ( n  = 66), or UC-II ( n  = 63) (Fig.  1 ). Per mITT criteria, 5 subjects were excluded from all analyses because they failed to present at any post-randomization visits resulting in an absence of clinical data. Table  3 summarizes the demographics of the remaining 186 subjects that were eligible for efficacy and safety analyses. Baseline subject characteristics, OA severity, serum CRP, COMP, IL-6 and other characteristics were similar among the three groups.

Subject dropouts

One hundred and sixty four subjects completed the study: 53, placebo; 57, GC; and 54, UC-II. The 27 dropouts, which included the five subjects mentioned previously, were allocated across the three cohorts as follows: 9, placebo; 9, GC; and 9, UC-II. The final dropout rate was 14 %. Subjects’ dropout reasons are summarized in Fig.  1 . No subject withdrew from the trial due to an adverse event attributable to any study product.

Study product compliance

Compliance with daily dosing of study capsules exceeded 90 % for all cohorts (data not shown).

Total WOMAC score

The UC-II supplemented group had statistically significant changes in the total WOMAC score compared to placebo (−551 vs. −414; 95 % CI −232 to −42; p  = 0.002) and GC (−551 vs. −454; 95 % CI −190 to −3; p  = 0.04) at day 180 (Fig.  2a , Table  4 ). When the total WOMAC results were analyzed, using MMRM, to account for treatment by time interactions, there remained a statistically significant difference between the UC-II and the placebo groups (−514 vs. −397; 95 % CI −210 to −24; p  = 0.0097; Table  4 ). An iAUC analysis also yielded statistically significant differences between the UC-II group versus placebo (−2042 vs. -1479; 95 % CI −1012 to −113; p  = 0.0098; Table  4 ). No significant changes were observed between the GC and placebo cohorts regardless of the type of analytical model used.

Total WOMAC score ( a ), Mean VAS ( b ), Total LFI ( c ) in the UC-II, GC and placebo groups over the 180-day study period. Values are presented as mean ± SE. *Significant difference between the UC-II ( n  = 54) and the placebo ( n  = 53) group, p  < 0.05. †Significant difference between the UC-II ( n  = 54) and the GC group ( n  = 57), p  < 0.05

Total WOMAC score based on baseline COMP levels

We found that subjects supplemented with UC-II, and presented with baseline COMP levels ≥285 ng/mL (median), had a greater reduction in the total WOMAC score than both placebo and GC groups with similar COMP levels under all modeling conditions (Table  5 ). For study participants with baseline COMP levels <285 ng/mL, no significant differences between the study groups were noted. Interestingly, we did observe a smaller placebo effect among individuals with baseline COMP levels ≥285 ng/mL as compared to those with <285 ng/mL (28 % vs 32 %). Despite this, UC-II efficacy, as defined by a reduction in overall WOMAC score, was higher in subjects with COMP levels ≥285 ng/mL versus subjects with COMP levels < 285 ng/mL (43 % vs 36 %).

WOMAC mean subscores—pain, stiffness and physical function

At day 180, we observed significant reductions in all three WOMAC subscales for UC-II group compared to placebo (Table  6 ): pain (24.0 vs. 17.0; 95 % CI −11.1 to −2.8; p  = 0.0003), stiffness (23.8 vs. 17.8; 95 % CI −10.4 to −1.6; p  = 0.004), and physical function (22.5 vs. 17.3; 95 % CI −9.3 to −1.3; p  = 0.007). The UC-II cohort also had significant reductions in WOMAC pain (24.0 vs. 19.2; 95 % CI −8.9 to −0.7; p  = 0.016) and stiffness (23.8 vs. 19.4; 95 % CI −8.7 to −0.1; p  = 0.044) at day 180 compared to GC.

Mean VAS score

The UC-II supplemented group had a significant decrease in mean VAS score at day 180 (Fig.  2b ) versus both placebo (22.6 vs. 17.0; 95 % CI −9.5 to −1.8; p  = 0.002) and GC (22.6 vs. 18.4; 95 % CI −8.0 to −0.4; p  = 0.025). In contrast, the GC group was not significant compared to placebo at any time.

A significant reduction was observed in the LFI score for the UC-II group at day 180 versus placebo (2.9 vs. 2.1; 95 % CI −1.4 to −0.2; p  = 0.009; Fig.  2c ). UC-II supplementation also has a greater improvement in LFI score versus GC (2.9 vs. 2.2; 95 % CI −1.4 to −0.2; p  = 0.008). No significant change was observed between the GC and placebo cohorts. Improvement in the total LFI score for the UC-II group was attributed to a significant reduction in the LFI subscale for daily activities at day 180 ( p  = 0.004 vs. placebo; p  = 0.013 vs. GC, data not shown).

Knee flexion

No significant differences were observed between the study groups (data not shown).

A significant increase in the final CRP levels versus baseline occurred in all three cohorts ( p  = 0.001). However, no statistical difference between the three cohorts (Table  7 ; p  > 0.05) was noted. The scientific reason behind this increase is not well understood. A significant decrease in serum COMP levels was seen in all groups versus baseline ( p  = 0.04) with no significant changes between groups.

Similar non-significant decreases in IL-6 and MMP-3 levels were noted for all cohorts (Table  7 ).

Rescue medication usage

The number of subjects that used rescue medication was significantly lower in the UC-II group compared to placebo (Table  8 ; p  = 0.001). Sixty individuals used rescue medications, at least once, during the study. Twenty-eight of these users were from the placebo group, 21 and 11 were from the GC and UC-II cohorts, respectively.

Safety assessments

No clinical or statistically significant changes were reported for any of the hematologic, blood biochemistry or vital signs results (Table  9 ). No significant changes were noted for the urinalyses results (data not shown).

A total of 45 AEs were reported during the 180-day study period: 9, placebo; 28, GC; and 8, UC-II (Table  10 ). The majority (62 %) of these occurred in the GC group. Fifteen of 45 events were classified as possibly related to supplementation, 14 of which belonged to the GC group and 1 to placebo. The 14 possible events linked to GC supplementation were primarily gastrointestinal in nature. The eight AEs noted for the UC-II cohort were deemed not related to supplementation. One individual in the GC group was removed from the study due to a respiratory tract infection (cough & fever). This infection was classified as an SAE. The event was investigated by the attending physician and center staff and judged as not related to GC consumption.

We assessed the ability of UC-II to improve joint symptoms in moderate-to-severe knee OA subjects. The results presented herein demonstrate that individuals consuming UC-II presented with better clinical outcomes versus those supplemented with placebo or GC. Analysis of the WOMAC subscales showed that reductions in all three WOMAC subscales contributed to the improvement in the overall WOMAC score observed in subjects supplemented with UC-II. In contrast, GC supplementation failed to induce a statistically significant improvement in the WOMAC, VAS or LFI scores versus placebo. These results confirm previous findings by Crowley et al. [ 5 ], which reported greater reduction in knee OA symptoms after 90 days of UC-II supplementation than what was observed with GC.

An interesting finding that emerged from this study is that stratification, according to baseline COMP levels, appears to have selected for individuals that responded better to UC-II supplementation. A greater reduction in knee OA symptom scores was observed among individuals with baseline serum COMP levels ≥285 ng/mL and supplemented with UC-II. This improvement was of sufficient magnitude that statistically significant outcomes for UC-II were observed versus both placebo and GC supplementation under all the statistical analyses we employed (ANCOVA, MMRM and iAUC). COMP, a cartilage turnover marker, is released into serum by chondrocytes and synovial cells [ 10 – 12 ]. Levels of this biomarker have been shown in several studies to have modest correlation with OA severity. However, serum COMP levels in groups of OA subjects overlap with levels observed in healthy populations, and this has limited the use of COMP as a prognostic marker for OA progression [ 12 – 14 ]. While the biologic or clinical significance to these findings remains to be understood, we find this preliminary observation an interesting one suitable for further investigation and confirmation.

The etiology behind UC-II’s impact on OA symptoms is not known. However, undenatured type II collagen has been shown to protect animals against the onset of joint damage in both OA and RA experimentally induced arthritis models [ 15 – 18 ]. This protection is hypothesized to occur via the induction and migration of T regulatory cell (Tregs) to the area of inflammation and damage [ 19 , 20 ]. The proposed role of Tregs may also have relevance to the moderation of OA symptoms, as in vitro studies have found that Tregs produce anti-inflammatory cytokines that stimulate chondrocytes to synthesize cartilage matrix components [ 21 – 23 ]. Additional studies that elucidate the precise mechanism through which UC-II mediates a reduction in knee OA symptoms are required.

The in vivo effects mentioned above may only be initiated by ingesting undenatured type II collagen as denatured (e.g., hydrolyzed) type II collagen fails to protect animals against the onset of arthritis [ 15 ]. This latter observation could explain why van Vijven and coworkers [ 24 ] concluded that there was insufficient evidence to support collagen for the treatment of OA as they combined data from all published clinical studies regardless whether native or denatured collagen was used in the trial.

We failed to observe any changes in knee ROM and distance walked regardless of supplementation. Improvements in these clinical outcomes are likely to be based not just on a symptomatic reduction in pain but also on physical improvements in the knee joint’s overall functionality. Until we undertake trials of longer duration, it remains an open question as to whether a slow acting supplement like UC-II can impact the biomechanical status of the OA knee sufficiently to improve knee ROM.

In the current study, GC supplementation did not significantly improve the signs and symptoms associated with knee OA. The scientific literature supporting the efficacy of GC is mixed, but there are various published studies which suggest that GC may moderate OA symptoms [ 25 – 27 ]. The GAIT study found that GC, and each component of GC individually, failed to impact OA symptoms as measured by the WOMAC pain scale; however, the placebo effect in that study was nearly 60 % which resulted in an underpowered study to determine differences between the treatments [ 28 ]. In contrast, a significant difference in knee pain was observed in the GC subgroup with moderate-to-severe knee pain compared to the placebo treated group [ 28 ]. To confirm the observation that GC may be more efficacious in subjects with moderate-to-severe knee OA pain, Hochberg and coworkers [ 29 ] performed a study in which OA subjects with moderate-to-severe knee pain, were randomized to GC or celecoxib for a period of 6 months. The results showed that GC treatment reduced WOMAC measured knee pain by 50 %, comparable to the results obtained with celecoxib [ 28 ]. It is worth noting that results such as these are not consistent across a number of studies for reasons yet to be determined [ 25 – 27 ].

In recent years, interest has focused on developing various biomarkers for monitoring OA progression and drug development [ 12 , 30 ]. We therefore assessed several biomarkers of inflammation (CRP, IL-6 and MMP-3) plus cartilage breakdown (COMP) and found no significant change for any of these biomarkers in this clinical trial. Since OA appears to impact the biology of several key components of the knee (e.g., synoviocytes, chondrocytes, etc.), the ability to achieve a significant change in any one biomarker could prove challenging for a slow acting supplement like UC-II. Also, multiple factors including ethnicity, physical activity, gender differences, and diurnal variation influence these biomarkers resulting in large variability in their levels [ 31 – 35 ]. Therefore, any change in these markers would have to occur as a result of a highly significant impact on the underlying pathophysiology of OA, given that the correlation between these biomarkers and OA pathophysiology are weak [ 12 ]. Such effects might be expected to occur more readily with a targeted agent [ 4 , 36 ].

This study found that UC-II, a nutritional ingredient containing undenatured type II collagen, significantly improved knee function in OA subjects by day 180, compared to placebo and to GC, and was well-tolerated. Based on the data presented herein, we believe that additional research is warranted both to confirm and to define these findings more extensively.

Abbreviations

adverse events

analysis of covariance

analysis of variance

current good manufacturing practice

confidence interval

cartilage oligomeric matrix protein

case report form

C-reactive protein

glucosamine hydrochloride plus chondroitin sulfate

incremental area under the curve

Institutional Ethics Committee

interleukin-6

intent-to-treat

Kellgren and Lawrence

Lequesne functional index

modified intent-to-treat

matrix metalloproteinase-3

mixed model repeated measures

nonsteroidal anti-inflammatory drugs

osteoarthritis

range of motion

• T regulatory cell

undenatured type II collagen

visual analog scale

Western Ontario McMaster Universities Osteoarthritis Index

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Acknowledgements

We gratefully acknowledge the support of Janet M. Peerson, M.S., University of California, Davis, for carrying out the statistical analyses shown herein and for helpful discussions. We acknowledge the support of the Laila Pharma Clinical team and the study investigators including Dr. Sundar Subramanian from V. S. Hospital, Tamilnadu; Dr. Meenakshi Sundaram from Vinayaka Mission Hospital, Tamilnadu; Dr. Balaji Thiruvadi and Dr. GM Bharat Kumar from Gram Clinical Research Karpagam Hospital, Tamilnadu; Dr. Saji Thomas from Little Flower Hospital and Research Center, Kerala; Dr. K. Balakondiah from Bollineni Superspeciality Hospital, Andhra Pradesh; Dr. Siva Prasad from Apollo Hospitals, Andhra Pradesh; Dr. K Rajapandian from Apollo Speciality Hospitals, Tamilnadu; Dr. K. Vasu from Pujitha Hospital, Andhra Pradesh; Dr. MAVV Prasad from Vijaya Super Speciality Hospitals, Andhra Pradesh; Dr. P. Ashok Kumar from King George Hospital, Andhra Pradesh; Dr. P. Pavan Kumar from R. K. Hospital, Andhra Pradesh; Dr. G Satish Reddy from Prime Hospital, Andhra Pradesh. We thank Jonathan Hull, Ph.D. and Weiman Xu, Ph.D. for assisting with data assembly and manuscript preparation.

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JPL and ZMS are employees of InterHealth Nutraceuticals. NEL provided consulting services to InterHealth. This study was sponsored by InterHealth Nutraceuticals, Inc. Benicia, CA. The study was run and managed independently by Laila Pharmaceuticals Pvt. Ltd., India. Data collection was done by the clinical study staff at each respective site. Data analyses was performed by an independent statistician.

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Lugo, J.P., Saiyed, Z.M. & Lane, N.E. Efficacy and tolerability of an undenatured type II collagen supplement in modulating knee osteoarthritis symptoms: a multicenter randomized, double-blind, placebo-controlled study. Nutr J 15 , 14 (2015). https://doi.org/10.1186/s12937-016-0130-8

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• Published: 23 June 2022

Type II collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation

• Xinhua Li   ORCID: orcid.org/0000-0002-4387-3229 1 , 2 , 3 ,
• Shuting Yang 1 ,
• Gongsheng Yuan 1 ,
• Dian Jing 4 ,
• Ling Qin   ORCID: orcid.org/0000-0002-2582-0078 5 ,
• Hu Zhao 4 &
• Shuying Yang   ORCID: orcid.org/0000-0002-7126-6901 1 , 6 , 7  

Bone Research volume  10 , Article number:  46 ( 2022 ) Cite this article

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• Pathogenesis

Type II collagen-positive (Col2 + ) cells have been reported as skeletal stem cells (SSCs), but the contribution of Col2 + progenitors to skeletal development both prenatally and postnatally during aging remains unclear. To address this question, we generated new mouse models with ablation of Col2 + cells at either the embryonic or postnatal stages. The embryonic ablation of Col2 + progenitors resulted in the death of newborn mice due to a decrease in skeletal blood vessels, loss of all vertebral bones and absence of most other bones except part of the craniofacial bone, the clavicle bone and a small piece of the long bone and ribs, which suggested that intramembranous ossification is involved in long bone development but does not participate in spine development. The postnatal ablation of Col2 + cells resulted in mouse growth retardation and a collagenopathy phenotype. Lineage tracing experiments with embryonic or postnatal mice revealed that Col2 + progenitors occurred predominantly in the growth plate (GP) and articular cartilage, but a limited number of Col2 + cells were detected in the bone marrow. Moreover, the number and differentiation ability of Col2 + progenitors in the long bone and knee joints decreased with increasing age. The fate-mapping study further revealed Col2 + lineage cells contributed to, in addition to osteoblasts and chondrocytes, CD31 + blood vessels in both the calvarial bone and long bone. Specifically, almost all blood vessels in calvarial bone and 25.4% of blood vessels in long bone were Col2 + lineage cells. However, during fracture healing, 95.5% of CD31 + blood vessels in long bone were Col2 + lineage cells. In vitro studies further confirmed that Col2 + progenitors from calvarial bone and GP could form CD31 + vascular lumens. Thus, this study provides the first demonstration that intramembranous ossification is involved in long bone and rib development but not spine development. Col2 + progenitors contribute to CD31 + skeletal blood vessel formation, but the percentage differs between long bone and skull bone. The number and differentiation ability of Col2 + progenitors decreases with increasing age.

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Introduction.

Endochondral and intramembranous ossification are two processes involved in bone development in both humans and mice. The main difference between endochondral and intramembranous ossification is that the former forms bone through a cartilage intermediate, whereas the latter forms bone directly from neural crest-derived mesenchymal cells. Studies have shown that most bones in the body are formed through endochondral ossification, and only the bones of the cranial base and caudal cranial vault in the skull and clavicles ossify intramembranously. 1 , 2

Cartilaginous matrix proteins such as type II collagen alpha 1 (Col2α1), aggrecan (Acan) and Sox9 are markers of chondrocytes. Mutation of Col2α1, 3 , 4 , 5 , 6 Acan 7 or Sox9 8 , 9 causes severe skeletal malformation, including lower body weight, a deformed skeletal structure, severe growth retardation, malformation of endochondral bones and intervertebral discs, cleft palate and abnormal cartilage histomorphology. However, the contribution of type II collagen alpha 1-positive (Col2 + ) cells to skeletal development both prenatally and postnatally remains unclear.

Skeletal stem cells (SSCs) are defined by their multidifferentiation potential, such as their ability to differentiate into osteogenic, chondrogenic, and adipogenic progenies. 10 , 11 Using lineage tracing techniques 12 to trace the fate of leptin receptor-positive (Lepr + ) cells, 13 , 14 chemokine (C-X-C motif) ligand 12-positive (Cxcl12 + ) cells, 15 Gli1 + cells, 16 Prx + cells, 17 Nestin + cells, 18 Sox9 + cells, 19 Acan + cells 19 and Ctsk + cells, 20 recent studies have revealed that these cells are all SSCs and play important roles during bone development and homeostasis. Importantly, Col2 + cells have been found to contribute to chondrocytes, osteoblasts, early perichondrial precursors before Runx2 expression, Cxcl12-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells during postnatal life. Furthermore, Col2α1 is expressed by progenitors of the skeletal lineage during canonical endochondral bone formation in the cranial base. 19 , 21 However, how Col2 + progenitors contribute to skeletal development during aging remains unclear.

Here, by generating mice with ablation of Col2 + cells using a diphtheria toxin (DTA) transgenic line and genetic lineage tracing mouse models with tdTomato transgenic lines, we determined the function of Col2 + cells in the embryonic and postnatal stages. Our findings provide the first demonstration that Col2 + cells contribute to calvarial bone development, that intramembranous ossification derived from Col2-negative progenitors is involved in long bone and rib development but does not participate in spine development, and that Col2 + cells also contribute to blood vessel formation during skeletal development and fracture healing.

The ablation of Col2 + cells causes the death of newborn mice with loss of all vertebral bone and most other bones and cartilage

To understand the contribution of Col2 + progenitors to skeletal development, a new mouse model of Col2-cre;DTA +/− was created by breeding mice bearing a DTA transgene downstream of a floxed stop codon (DTA +/+ ) with Col2-cre mice. Cre- littermates served as controls (wild-type mice, WT mice) (Fig. S1A). On embryonic day 17.5 (E17.5), the Col2-cre;DTA +/− embryos survived and followed Mendelian law (19/80). Interestingly, we found that 70%–75% of mice died between E17.5 and the newborn stage. Approximately 25% of the newborn (16/72) Col2-cre;DTA +/− mice survived for minutes or a few hours after birth and then died due to oxygen insufficiency resulting from a lack of rib cages and other developmental defects.

To test the effect of Cre-mediated DTA ablation, we generated Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato mice. Lineage tracing detected abundant levels of tdTomato + cells in the spine, limbs, ribs, meninges and skull bone of Col2-cre;tdTomato mice (Fig. 1a ), but few tdTomato + cells were detected in the mutant mice compared with the WT mice. Notably, we found that many cells in the WT skull bone, particularly in the cartilaginous nasal capsule, were tdTomato + (Fig. 1b ), which confirmed that Col2 + cells contribute to skull bone and nasal capsule formation. Consistently, the cartilaginous nasal capsule was completely lost in the Col2-cre;DTA +/− ;tdTomato newborns, and the skulls of these newborns were smaller +/− than those of the control newborns. Most cells in the lower extremities and spine were labeled, although articular chondrocytes exhibited a weak signal (Fig. 1c, d ). In contrast, no tdTomato + cells could be detected in the spine region and lower extremities of Col2-cre;DTA +/− ;tdTomato newborns, which confirmed the effectiveness of cell ablation.

Spatial expression of Col2 + lineage progenitors in newborns and removal of the majority of Col2 + cells by DTA. a Fluorescence images showing the pattern of Col2 + lineage progenitors in middle sections of Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato newborn mice. b Fluorescence images showing the pattern of Col2 + lineage progenitors in transected skulls of Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato mice. White arrow, defective nasal capsule in a Col2-cre;DTA +/− ;tdTomato mouse. c Fluorescence images showing the pattern of Col2 + lineage progenitors in the lower extremities of Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato newborn mice. Note the complete loss of tdTomato + cells in the long bones of Col2-cre;DTA +/− ;tdTomato mice compared with wild-type newborn mice. d Fluorescence images showing the pattern of Col2 + lineage progenitors in middle sagittal sections of spines of Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato newborn mice. n  = 6 mice per condition; three independent experiments

The gross appearance of the Col2-cre;DTA +/− newborn mice showed severe dwarfism with extremely shortened limbs, tail and nose, intact but pale skin, cleft palate, and abnormal skull morphology 22 (Fig. 2a , Fig. S 1B, C ).

Ablation of Col2 + cells causes lethality in newborn mice due to the absence of endochondral bone and cartilage. a Gross appearance of Col2-cre and Col2-cre;DTA +/− mice at P0. The mutant mouse is small and has an extremely short four-limb pattern. b X-ray of P0 wild-type and mutant embryos. c Skeletons of Col2-cre and Col2-cre;DTA +/− mice at E17.5 and the newborn stage. Embryos and newborns were double stained with Alizarin red/Alcian blue. d Alizarin red/Alcian blue staining of skulls of Col2-cre and Col2-cre;DTA +/− newborns. Red arrow, complete loss of cartilage in Col2-cre;DTA +/− mice. e Alizarin red/Alcian blue staining of the mandibles of Col2-cre and Col2-cre;DTA +/− newborn mice. f Interior view showing the lack of mandibles in the skulls of Col2-cre and Col2-cre;DTA +/− newborn mice. Red arrow, bone loss in Col2-cre;DTA +/− mice. g Hindlimbs of Col2-cre and Col2-cre;DTA +/− newborn mice. Red arrow, bone loss in Col2-cre;DTA +/− mice. h Ribs of Col2-cre and Col2-cre;DTA +/− newborn mice. Red arrow, bone loss in Col2-cre;DTA +/− mice. i Clavicles of Col2-cre and Col2-cre;DTA +/− newborn mice. Red arrow, partial clavicle loss in Col2-cre;DTA +/− mice. n  = 6 mice per condition; three independent experiments

A skeletal radiograph examination of Col2-cre;DTA +/− newborns revealed that their vertebral bone was completely lost, and only parts of the skull, mandible, clavicle bone, and tiny bones of the hindlimbs and ribs were observed (Fig. 2b ). Consistent with the X-ray results, Alizarin red/Alcian blue staining of mutant embryos at E17.5 revealed the absence of cartilage and bone throughout the body skeleton and only limited Alizarin red staining in the craniofacial bone (Fig. 2c ), including the frontal, parietal, temporal, maxillary, interparietal and supraoccipital bones in the skull (Fig. 2d, f ) and mandible (Fig. 2e ). Interestingly, very small pieces of bone in the hindlimb (Fig. 2G ), small parts of the ribs (Fig. 2h ), and the clavicle bone were calcified with positive Alizarin red staining (Fig. 2i ).

Ablation of Col2 + cells disrupted bone and cartilage development

To shed more light on the function of Col2 + progenitors in mice, we performed sagittal sectioning of Col2-cre control and Col2-cre;DTA +/− newborns. In the Col2-cre newborns, all the bone, cartilage, brain, spinal cord, and organs were well organized and clearly identified (Fig. 3a ). However, the entire vertebrae and intervertebral disc, most of the long bone and other bones and all the cartilage were lost in the Col2-cre;DTA +/− mice (Fig. 3a ). Most interestingly, despite the severe bone loss in the skull and complete absence of the vertebrae and intervertebral discs observed in the mutant mice, their brain and spinal cord were located in a relatively narrow skull cavity and a “spinal canal”, respectively. However, the morphology of the spinal cord presented a spindle-like structure, which suggested that the spine or vertebral bone may be needed to guide the formation of normal spinal cord morphology.

Ablation of Col2 + cells results in absence of the spine and disruption of endochondral skeletal development. a Total view of middle sagittal sections of Col2-cre and Col2-cre;DTA +/− mice at P0. b H&E staining of the middle sagittal sections of Col2-cre and Col2-cre;DTA +/− mice at P0. c Von Kossa staining of the middle sagittal sections of Col2-cre and Col2-cre;DTA +/− mice at P0. d Von Kossa staining of the skull with transection. Yellow arrow, severe bone loss in the back of the skull of Col2-cre;DTA +/− mice. Yellow star, nasal cavity area in Col2-cre and Col2-cre;DTA +/− mice. e Von Kossa staining of craniofacial bone showing loss of the nasal cavity in mutant mice. f Higher magnification of Von Kossa staining showing similar sponge-like bone structures in both wild-type and mutant mice. g Von Kossa staining of bone in the hindlimbs of wild-type and mutant mice. h Higher magnification of Von Kossa staining of the hindlimbs of wild-type and mutant mice. Note that a small piece of well-calcified sponge-like bone structure can be detected in mice with ablation of Col2 + cells. i Von Kossa staining of the middle sagittal section of the spine of wild-type and mutant mice. Red arrow, severe vertebral bone loss in Col2-cre;DTA +/− mice. n  = 6 mice per condition; three independent experiments

To gain further insights into the bone changes, histological sections of the skeletons from newborns were examined by H&E (Fig. 3b , Fig. S 1D–F ) and Von Kossa staining (Fig. 3c ). Consistently, we found that the ablation of Col2 + cells resulted in the absence of most parts of the skeleton, including complete loss of the vertebrae and intervertebral disc and all body bones with the exception of parts of the skull and a tiny hindlimb bone, in the Col2-cre;DTA +/− mice. To determine the defects in finer detail, we further analyzed sections of the skull, hindlimbs and spine. The results showed that only the front skull bone could be detected in the Col2-cre;DTA +/− mice, and the nasal cavity and back skull bone were almost absent in these mice compared with the Cre control mice (Fig. 3d–f ), which indicated that Col2 + cells also play an important role in skull development. Most interestingly, a small piece of calcified sponge-like bone structure could be detected in the hindlimbs of the Col2 + cell-ablated mice (Fig. 3g, h ), which indicated that Col2 + cells dominantly contribute to long bone development but that Col2-negative (Col2 − ) cells also participate in long bone development. The Von Kossa staining results showed that vertebral bone mineralization was completely absent in Col2-cre;DTA +/− mice, which suggested that Col2 + cells are the major progenitors contributing to vertebral bone development (Fig. 3i ). Unexpectedly, even though almost all bones and cartilage were lost in Col2-cre;DTA +/− newborns, the toe and finger digits and intact patterns were normal in these mice, which suggested that limb pattern development is independent of Col2 + cells and bone development in mice (Fig. S 1B ).

The postnatal deletion of Col2 + cells resulted in mouse growth retardation and a type II collagenopathy phenotype

To assess the contribution of Col2 + cells to postnatal skeletal development in mice, we genetically ablated these cells by inducing the expression of DTA postnatally in Col2 + cells. Specifically, we applied tamoxifen (TM) to either TM-inducible type II collagen Cre (Col2-creERT) or Col2-creERT;DTA +/+ mice on postnatal day 3 (P3) and harvested the mice at four weeks of age. All Col2-creERT;DTA +/+ mice developed postnatal dwarfism with shorter limbs and body lengths (Fig. 4a–e ). Skeletal radiographs and whole-mount Alizarin red staining of the skeleton confirmed this observation and revealed skeletal defects, including epiphyseal dysplasia in the long bone, abnormalities of the capital femoral epiphyses and underdevelopment of the femoral head (Fig. 4d, h ). Representative micro-CT images showed epiphyseal dysplasia and substantially decreased bone mass in the long bones of Col2-creERT;DTA +/+ mice (Fig. 4f, g ). Quantitative analysis revealed that the percentage of bone volume to total bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) of the Col2-creERT;DTA +/+ mice were reduced to approximately 0.62-, 0.53-, and 0.5-fold and that the trabecular spacing (Tb.Sp) was increased 1.5-fold compared with those of the Col2-creERT controls (Fig. 4f ). Furthermore, Alizarin red/Alcian blue staining showed loss of bone and cartilage in the femur secondary ossification center in 4-week-old Col2-creERT;DTA +/+ mice (Fig. 4h ). Quantitative analysis demonstrated that the lengths of the femur and tibia bones of the mutant group were significantly shortened to 0.55 cm and 0.78 cm, respectively, compared with the lengths of 1.22 cm and 1.4 cm found in the WT mice (Fig. 4i ). Moreover, the postnatal ablation of Col2 + cells also impaired skull and long bone and cartilage development (Fig. 4j, k ).

Postnatal deletion of Col2 + cells causes mouse growth retardation and a type II collagenopathy phenotype. a , b Quantitative analysis of the body length and tail length of Col2-creERT and Col2-creERT;DTA +/+ mice from P3 to four weeks of age ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. c Macroscopic image of 4-week-old littermates. The mutant (right) mouse is small and exhibits dwarfism. d Total view of Col2-creERT and Col2-creERT;DTA +/+ mice double stained with Alizarin red and Alcian blue at four weeks of age. e X-ray of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. f Representative picture of the microCT 3D structure of the femur head of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. g Quantitative analysis of the BV/TV, Both, Tb.N, and Tb.Sp of the femurs of 4-week-old Col2-creERT (CreERT) and Col2-creERT;DTA +/+ (DTA) mice ( n  = 5 mice per group). The data are presented as the means ± s.ds. h Total view of the lower extremities of Col2-creERT and Col2-creERT;DTA +/+ mice double stained with Alizarin red and Alcian blue at four weeks of age. Yellow arrow, delayed secondary ossification development in Col2-cre;DTA +/− mice. i Quantitative analysis of the femur length and tibia length of 4-week-old Col2-creERT (CreERT) and Col2-creERT;DTA +/+ (DTA) mice ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. j Total view of the skulls of Col2-creERT and Col2-creERT;DTA +/+ mice stained with Alizarin red and Alcian blue at four weeks of age. k Total view of the forelimb of Col2-creERT and Col2-creERT;DTA +/+ mice stained with Alizarin red and Alcian blue at four weeks of age. l Safranin O/Fast Green staining of coronal sections of the femurs of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. m High-magnification image showing secondary ossification and cartilage in the femurs of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. n High-magnification picture showing the chondrocyte morphology of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. o Immunofluorescence staining of the GP and AC of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice for type 2 collagen. p High-magnification image showing type 2 collagen staining in 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. q Phalloidin staining showing the cartilage cytoskeleton of 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice. r Quantitative measurements of the percentage of cells with intact actin fragments relative to the total cells in Col2-creERT (CreERT) and Col2-creERT;DTA +/+ (DTA) mice ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. * P  < 0.05, ** P  < 0.01, *** P  < 0.000 1, NS not statistically significant

Postnatal deletion of Col2 + cells disrupted endochondral ossification and cell alignment

To gain more insight into the skeletal changes, we performed safranin O/fast green staining of tibial sections from 4-week-old Col2-creERT and Col2-creERT;DTA +/+ mice (TM injected at P3). The results further confirmed the significantly decreased bone mass in the tibia and the absence of secondary ossification centers in Col2-creERT;DTA +/+ mice (Fig. 4i ). Higher magnification examination of the secondary ossification center and growth plate (GP) in the tibia of Col2-creERT;DTA +/+ mice showed that the GP pattern was substantially disrupted, and the epiphyses were occupied by disorganized hypertrophic chondrocytes 22 (Fig. 4m, n ).

Immunofluorescence staining showed that the type II collagen matrix was enriched in articular cartilage (AC) and GP chondrocytes in 4-week-old WT mice (Fig. 4o, p ) and was substantially reduced in Col2-creERT;DTA +/+ mice, which indicated that Col2 + cells are essential for type II collagen production. Moreover, the F-actin immunostaining results showed that the cell alignment and pattern in GP chondrocytes were markedly disrupted. The quantitative analysis of these results demonstrated that the percentage of cells with intact actin filaments was decreased from 100% in the WT mice to 9.2% in the mutant mice. These findings indicate that the type II collagen matrix and Col2 + cells play important roles in cell patterning and GP organization (Fig. 4q, r ).

Spatial distribution of embryonic and postnatal Col2 + cells in the long bones and knee joints of mice

To investigate the contribution of Col2 + progenitors to skeletal development during the embryonic and postnatal stages, we traced the fate of Col2- expressing cells for different time periods in Col2-cre;tdTomato mice, in which Col2 + cells exhibit tomato fluorescence starting from the embryonic stage, and Col2-creERT;tdTomato mice, in which Col2 + cells express tomato fluorescence protein after treatment with TM at the indicated postnatal time. These findings demonstrated that Col2 + cells from both mouse strains and their descendants were permanently marked by the expression of the red fluorescent protein tdTomato.

To examine how Col2 + progenitors contribute to skeletal development in Col2-cre;tdTomato mice during the embryonic stage, we first examined the long bones and knee joints and found that tdTomato + cells were present in the AC, GP, bone surface, osteocytes, tendon and meniscus (Fig. 5a ). Interestingly, the tdTomato + fluorescence signal was weaker in the GP but stronger in the trabecular and cortical bone and meniscus at P0 and P8. Notably, the tdTomato + fluorescence was stronger in every compartment of the knee at P14 and P30 than at P0. With aging, tdTomato + fluorescence in the trabecular and cortical bone gradually decreased; however, tdTomato + fluorescence in the AC and GP strengthened from P90 to P180 and then decreased at P365.

Spatial distribution of embryonic and postnatal Col2 + cells in mouse long bones and knee. a Representative images from the lineage tracing of embryonic Col2 + cells in long bones and knee joints at different time points (P0, P8, P14, P30, P90, P180 and P365). b Representative images from the lineage tracing of postnatal Col2 + cells in long bones and knee joints at different time points (P6, P8, P14, P30, P90, P180 and P365). This analysis was performed by injecting 75 mg·kg −1 tamoxifen into P3 mice. To better present the data, the information for the Col2-creERT;tdTomato mice in each figure is presented as the harvest time (tamoxifen injection date + tracing time period)., e.g., P6 (3 + 3) means that the harvest date of Col2-creERT;tdTomato mice is P6, that tamoxifen was injected at P3 and that tracing was performed for three days. Because all Col2 + cells were activated starting from the embryonic stage in Col2-cre;tdTomato mice, we included only the date for the harvesting of mouse samples. For example, P8 means that the harvest date for the Col2-cre;tdTomato mice was P8. c Representative images from the lineage tracing of postnatal Col2 + cells in long bones and knee joints activated at different time points (P3, P21, P27, P87, and P362). Tamoxifen (75 mg·kg −1 ) was injected into mice at the indicated time points. d Representative images from the lineage tracing of Col2 + cells in long bones and knee joints at P90 after activation at different time points (embryonic stage, P3, P30, P60 and P87). e Representative images from the lineage tracing of Col2 + cells in the long bones and knee joints of mice at P30 after activation at the indicated time points (embryonic stage, P3, and P60). This analysis was performed by the injection of 75 mg·kg −1 tamoxifen into Col2-creERT;tdTomato mice at different time points. f Representative images of the long bones and knee joint of Col2-creERT;tdTomato mice at P30. This analysis was performed by the injection of 75 mg·kg −1 vehicle into Col2-creERT;tdTomato mice at P3. g Quantitative measurements of the percentage of tdTomato + cells with respect to the total cells in ( c ) ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. h Quantitative measurements of the percentage of tdTomato + cells with respect to the total cells in ( d ) ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. i Quantitative measurements of the percentage of tdTomato + cells with respect to total cells in ( e ) ( n  = 6 mice per condition; three independent experiments). The data are presented as the means ± s.ds. At least 1 000 cells of each sample were measured. The statistical significance was determined by one-way ANOVA and Student’s t test. * P  < 0.05, ** P  < 0.01, *** P  < 0.000 1, NS not statistically significant

To further examine the contribution of postnatal Col2 + cells to long bone and knee joint development, Col2-creERT;tdTomato mice were intraperitoneally (i.p.) administered TM at P3 and harvested at P6, P8, P14, P30, P90, P180, and P365. At P6 and P8, tdTomato + (Col + ) cells were predominantly detected in the AC, secondary ossification center, GP, and meniscus (Fig. 5b ). Interestingly, at P14 and P30, the pattern of Col2 + cells in the AC, GP and long bones of these mice was similar to that found in Col2-cre;tdTomato mice, and this pattern involved decreased tdTomato + fluorescence in the AC and GP but increased fluorescence in the cortical and cancellous bones. At P90, tdTomato + fluorescence was decreased in the cortical and cancellous bones but increased in the GP and AC. However, starting from P180, the numbers of tdTomato + cells in cortical and cancellous bones as well as the GP and AC significantly decreased with aging in both Col2-cre;tdTomato and Col2-creERT;tdTomato mice (Fig. 5a, b ).

The numbers of Col2 + progenitors decreased during aging

To further explore the effect of age on the fate of Col2 + progenitors, Col2-creERT;tdTomato mice were i.p. injected with TM at P3, P21, P27, P87, and P362, and sections were analyzed 3 days after each injection. As shown in Fig. 5c , tdTomato + cells were detected in articular chondrocytes, the GP and the meniscus after the administration of TM at P3. However, the number of tdTomato + cells in mice significantly decreased during aging. Approximately 98.1% of GP chondrocytes were tdTomato + in the mice administered the TM injection at P3, and the injection of TM at P21, P27, P87, and P362 decreased this percentage to 89.9%, 53%, 32.7%, and 23.2%, respectively. Similarly, approximately 97.8% of articular chondrocytes were tdTomato + in the mice that were injected with TM at P3, and the injection of TM at P21, P27, P87, and P362 decreased these percentages to 86.2%, 26.4%, 17.2%, and 9.1%, respectively (Fig. 5c, g ). In the meniscus, 38.1% of cells were tdTomato + in the mice that were injected with TM at P3, and the injection of TM at P21, P27, and P87 decreased this percentage to 31.1%, 7.8%, and 6.9%, respectively, whereas only a few positive cells were detected after the injection of TM at P362. The results demonstrated that the numbers of Col2 + progenitors decreased during aging.

To determine how age affects the number and differentiation ability of Col2 + cells, we compared the number of tdTomato + cells in the knee joints at P90 between Col2-cre;tdTomato mice whose Col2 + cells were activated at the embryonic stage and Col2-creERT;tdTomato mice injected with TM to turn on tdTomato protein expression at P3, P30, P60, and P87 (Fig. 5d ). The number of tdTomato + cells gradually decreased from the embryonic stage to P3, P30, P60 and P87. Specifically, at P90, 98.5% of GP chondrocytes were tdTomato + when Col2 + was activated at the embryonic stage; however, the percentage of these cells decreased to 98.2%, 55.4%, 47.1%, and 37.2% in the Col2-creERT;tdTomato mice injected with TM at P3, P30, P60 and P87, respectively (Fig. 5D, H ). Similarly, approximately 98.4% of articular chondrocytes were tdTomato + when Col2 + was activated at the embryonic stage, and the injection of TM at P3, P30, P60 and P87 decreased this percentage to 97.9%, 38.7%, 20.1%, and 17.2%, respectively. In the meniscus, 98.8% of cells were tdTomato + when Col2 + was activated at the embryonic stage, and the injection of TM at P3, P30, P60 and P87 decreased this percentage to 8.1%, 10.4%, 8.4%, and 6.9%, respectively. These results suggested that the numbers and differentiation ability of Col2 + cells decreased during the aging process.

To further confirm that the differentiation ability of Col2 + progenitors decreased with increasing age, we compared the tdTomato + cells in the knee joints over 1 month of tracing among mice whose Col2 + cells were activated at the embryonic stage, P3 and P60 (Fig. 5e ). Approximately 98.4% of articular chondrocytes were tdTomato + when Col2 + was activated at the embryonic stage, and this percentage decreased to 97.9% and 38.7% when TM was injected at P3 and P60, respectively (Fig. 5E, I ). Similarly, 98.5% of GP chondrocytes were tdTomato + when Col2 + was activated at the embryonic stage, and the injection of TM at P3 and P60 decreased this percentage to 98.2% and 55.4%, respectively. In the meniscus, 98.8% of cells were tdTomato + when Col2 + was activated at the embryonic stage, and the injection of TM at P3 and P60 decreased this percentage to 8.1% and 1.3%, respectively (Fig. 5e, i ). These results further confirmed that the differentiation ability of Col2 + progenitors decreased with age. No detectable td-Tomato fluorescence was detected in the control Col2-creERT;R26-tdTomato mice injected with vehicle, which confirmed the specificity of Cre recombinase activity (Fig. 5f ).

In addition, comparing type II collagen expression between embryonic and postnatal Col2 + cells revealed that the expression pattern of type II collagen in Col2-cre;tdTomato mice at P6 was similar to the pattern of Col2 + cells at P6 in Col2-creERT;tdTomato mice injected with TM at P3 (Fig. S 2 ). However, the type II collagen expression pattern in Col2-cre;tdTomato mice was completely different from the patterns of Col2 + cells at P30, P180 and P365 in Col2-creERT;tdTomato mice injected i.p. with TM at P27, P177 or P362 (Fig. S 2A–D ).

To further demonstrate the differentiation ability of Col2 + cells during aging, we performed in vitro primary cell culture. Col2 + chondrocytes were harvested from 5-day-, 1-month- and 5-month-old mice, and the differentiation ability was then tested through chondrogenesis and osteogenesis differentiation assays. The results showed that the differentiation potential of Col2 + cells decreased with increasing age. A quantitative analysis of the Alizarin red and Alcian blue staining results showed that Col2 + chondrocytes from P5 and 1-month-old mice had markedly stronger osteogenic and chondrogenic differentiation potential than Col2 + chondrocytes from 5-month-old mice (Fig. S 3 ). These results further indicate that the differentiation ability of Col2 + progenitors gradually decreased during aging.

CD31 + cells originating from Col2 progenitors expressed Col2 throughout their lifespan until the time points examined

Our results showed that the gross appearance of the Col2-cre;DTA +/− newborn mice exhibited a lack of blood vessel phenotype with pale skin (Fig. 2a , Fig. S 1B, C ). To further examine whether Col2 + cells contribute to blood vessel formation, we first traced the Col2 + cell locations in the skeleton of Col2-cre;tdTomato mice and found that Col2 + cells (tdTomato + ) were present in the GP, AC (chondrocytes) and bone (osteoblasts and osteocytes) of 4-week-old Col2-cre;tdTomato mice (Fig. 6a , Fig. S 4 ). Surprisingly, some CD31 + cells in blood vessels of the long and skull bones were also labeled with tdTomato + fluorescence (Fig. 6b–f ). Flow cytometry results showed that approximately 25.4% of CD31 + cells in the long bones of 4-week-old Col2-cre;tdTomato mice were Col2 + (Fig. 6b, c ), whereas almost all CD31 + blood vessel cells in skull bone were Col2 + (Fig. 6d ). Additionally, many CD31 + blood vessel cells in the brain, eyeball, heart, and skin were Col2 + (Fig. 6e , Fig. S 5A–C ), but none of the CD31 + cells found in the kidney vasculature during development were Col2 + (Fig. S 5D ). Consistently, we found that the ablation of Col2 + cells led to a marked decrease in the numbers of CD31 + blood vessels in the skull bone and eyes of Col2-cre;DTA +/+ ;tdTomato mice compared with those detected in the control Col2-cre;tdTomato mice (Fig. 6f , Fig. S 5E, F ). These results indicate that Col2 + cells can contribute to skeletal blood vessel formation.

Col2 + progenitors have multilineage differentiation ability, including CD31 + blood vessels. a Representative fluorescent images of the distal femur showing that cartilage cells, osteoblasts and osteocytes are Col2 + . Yellow arrow, osteoblasts and osteocytes of 4-week-old Col2-cre;tdTomato mice. b Flow cytometry analysis was performed using dissociated long bone marrow cells collected from 4-week-old Col2-cre;tdTomato mice. Representative dot plots show that some Col2 + bone marrow stromal cells were CD31 + ( n  = 3 mice per condition; three independent experiments). c Representative fluorescent images of the distal femurs of 4-week-old Col2-cre;tdTomato mice showing that some CD31 + cells in long bones were Col2 + cells. d Representative fluorescent images from the calvarial bone of 4-week-old Col2-cre;tdTomato mice showing that almost all CD31 + cells were Col2 + cells. e Representative fluorescent images from the craniofacial bone of 4-week-old Col2-cre;tdTomato mice showing that almost all CD31 + cells were Col2 + cells. Yellow arrow, overlapping staining of blood vessels. f The gross appearance of the head of Col2-cre and Col2-cre;DTA +/− newborn mice. Note that the mutant mice showed a marked loss of blood vessels in the skull. Yellow arrow, blood vessels in each group. g Representative picture showing that Col2 + cells from the calvaria bone of Col2-cre;tdTomato mice exhibit tube formation capacity. h Representative picture showing that Col2 + cells from the cartilage of Col2-cre;tdTomato mice exhibit tube formation capacity. i Representative picture showing that Col2 − cells from the cartilage of Col2-cre;tdTomato mice do not have tube formation capacity. All the data are reported as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. * P  < 0.05, ** P  < 0.01, *** P  < 0.000 1. NS not statistically significant

To further investigate whether Col2 + cells could form vascular lumens in vitro, we harvested Col2 + cells from the calvarial bone and cartilage of 1-month-old Col2-cre;tdTomato mice and tested the angiogenic tube formation capacity through angiogenesis assays. Indeed, Col2 + cells from calvarial bone and cartilage formed vascular lumens in vitro (Fig. 6g–i ).

Both hematopoietic and blood vessel endothelial cells are differentiated from hemangioblasts of mesodermal cells. 1 To determine whether Col2 + cells in the bone marrow contribute to hematopoietic cell-derived osteoclast formation, Bone marrow cells(BMCs) from Col2-cre;tdTomato mice were induced with RANKL/M-CSF. As shown in Fig. S 6 , osteoclasts did not exhibit tdTomato fluorescence. Moreover, TRAP staining for osteoclastogenesis assays confirmed that Col2 + cells cannot differentiate into osteoclasts.

Col2 + cells in the GP and AC displayed stem cell properties

Our results showed that Col2 + cells were located in the AC, bone marrow and GP and that the ablation of Col2 + cells completely disrupted skeletal development with the exception of craniofacial bone development. To determine whether Col2 + cells exhibit stem cell properties, we first performed a CFU-F activity assay. BMCs, GP chondrocytes and articular chondrocytes were isolated from 4-week-old Col2-cre;tdTomato mice, and the Col2 + cells were then sorted, as shown in Fig. 7a . The quantification of tdTomato + CFU-F colonies revealed that Col2 + cells from BMCs, GP chondrocytes and articular chondrocytes could all form CFU-F colonies (Fig. 7a ).

Col2-positive progenitors from different tissues have varied differentiation potential. a CFU-F assay of Col2 + bone marrow stem cells (BMSCs), GP progenitors and AC progenitors showing that every cell type can form CFU colonies. b Adipogenic differentiation of Col2 + BMSCs, GP progenitors and AC progenitors. c Osteogenic differentiation of Col2 + BMSCs, GP progenitors and AC progenitors. d Chondrogenic differentiation of Col2 + BMSCs, GP progenitors and AC progenitors. e Merged pictures showing Col2 + cells cultured from the calvaria of Col2-cre;tdTomato and Col2-cre;DTA +/− ;tdTomato newborns. Note that few Col2 + cells were detected in the Col2 + cell ablation group, confirming the effectiveness of the cell ablation technique. f Assay of the trilineage differentiation of cells cultured from the calvarial bone of Col2-cre and Col2-cre;DTA +/− newborns ( n  = 3 mice per condition; three independent experiments). g Different migration abilities of cells cultured from Col2-cre and Col2-cre;DTA +/− newborns. h Quantitative measurements of migration ability based on the results shown in ( g ) ( n  = 3 mice per condition; three independent experiments). All the data are reported as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. * P  < 0.05, ** P  < 0.01, *** P  < 0.000 1, NS not statistically significant

To further assess whether Col2 + cells have multiple lineage differentiation capabilities, Col2 + cells from the bone marrow, GP and AC were induced with osteogenic, chondrogenic and adipogenic media for the indicated times (Fig. 7b ). Interestingly, Col2 + cells sorted from BMCs could differentiate into osteoblasts, chondrocytes and adipocytes, whereas Col2 + cells from the GP and AC could differentiate only into osteoblasts and chondrocytes but not into adipocytes (Fig. 7b–d ). These findings suggested that Col2 + cells from BMCs are likely bone mesenchymal stem cells (BMSCs) at earlier stages, whereas a large population of Col2 + cells from the GP and AC were BMSCs at later stages. Most interestingly, the Alcian blue and Alizarin red staining of the cells showed that Col2 + cells from the GP exhibited markedly higher osteogenic and chondrogenic differentiation potential than Col2 + cells from articular chondrocytes and BMCs (Fig. 7c, d ).

Oct4 and Sox2 are stem cell markers. 23 , 24 To determine whether Col2 + cells express stem cell markers, we coimmunostained sections of the GP, bone marrow and AC tissues from newborn and 1-month-old Col2-cre;tdTomato mice to detect the expression of Oct4 and Sox2. Our data showed that Oct4 and Sox2 expression overlapped with Col2 + cells in the bone marrow, GP and AC (Fig. S 7A ), which confirmed that Col2 + cells from these three sources have stem cell properties. Moreover, our data showed that Oct4- and Sox2-positive cells overlapped with some Col2 + cells in the bone marrow but with markedly fewer Col2 + cells in the GP and AC than in the bone marrow of Col2-cre;tdTomato mice (Fig. S 7B, C ).

Col2-negative cells from the calvarial bone but not cartilage show high unipotent osteogenic potential

According to the lineage tracing results shown in Fig. 3c , both Col2 + and Col2 − cells are present in calvarial bone. To further test whether Col2 − cells also have differentiation potential, calvarial bone cells were isolated from the calvaria of newborn Col2-cre;tdTomato (control) and Col2-cre;DTA +/− ;tdTomato mice. We found that approximately 50% of cells in the control group were Col2 + , but almost no Col2 + cells were detected in the Col2-cre;DTA +/− ;tdTomato group, which confirmed the effectiveness of Co2 + cell ablation (Fig. 7e ). Moreover, we found that the cells of the control calvarial bone could differentiate into three lineages, namely, osteoblasts, chondrocytes and adipocytes. Interestingly, Col2 − cells could not differentiate into chondrocytes and adipocytes but exhibited markedly higher potential to differentiate into osteoblasts than control cells (Fig. 7f ). Additionally, the wound healing assay showed that Col2 − cells had stronger migration ability than the control cells (Fig. 7g, h ).

To investigate whether the differentiation potential differs between Col2 + and Col2 − cells, we isolated Col2 + and Col2 − cells from the bone marrow of the long bones of 1-month-old Col2-cre;tdTomato mice. We found that both Col2 + and Col2 − cells were capable of differentiating into osteoblasts and chondrocytes (Fig. S 8 ). A quantitative analysis of Alcian blue- and Alizarin red-stained cells showed that Col2 + cells from the bone marrow have markedly higher osteogenic and chondrogenic differentiation potential than Col2 − cells (Fig. S 8 ).

Col2 + progenitors contributed to chondrocyte differentiation and blood vessel formation during fracture healing

To test whether Col2 + cells contribute to postnatal chondrocyte differentiation and blood vessel formation, we created a closed femoral fracture with an intramedullary nail fixation model in 10-week-old mice as previously described 25 to trace Col2 + cells during fracture healing. We first induced tdTomato expression in Col2 + cells of Col2-creERT; tdTomato mice through the injection of TM at P3, induced a fracture in the mice at 10 weeks of age and harvested tissues two weeks after the fracture surgery. In the noninjured mice, the contralateral nonfractured femur exhibited prominent tdTomato + cells in the AC and metaphysis but few tdTomato + cells in the bone marrow (Fig. 8a ). However, strong tdTomato expression was detected throughout the fractured callus, including both bony and cartilaginous regions (Fig. 8b, c ). Immunofluorescent staining for CD31 and Col2α1 showed that 95.5% of the CD31 + cells and 43% of the Col2α1 + cells overlapped with tdTomato + (Col2 + ) cells in the callus area of Col2-creERT;tdTomato mice. In contrast, only 25.3% of CD31 + cells and 12.1% of Col2α1 + cells overlapped with tdTomato + (Col2 + ) cells in the intact long bones of Col2-creERT;tdTomato mice (Fig. 8b, c ). Immunofluorescent staining for blood vessels in the fractured callus showed that the percentage of CD31 + cells decreased markedly to 3.5% in Col2-creERT;DTA +/+ ;tdTomato mice compared with 12.5% in Col2-creERT;tdTomato mice (Fig. 8d ). These results indicate that Col2 + cells play an important role in blood vessel and bone regeneration during fracture healing.

Col2 + progenitors contribute to chondrocyte differentiation and blood vessel formation during fracture healing. a Illustration of the experimental design and representative images of femoral sections of control and fractured Col2-creERT;tdTomato mice. This analysis was performed by injecting 75 mg·kg −1 tamoxifen into mice at P3, subjecting the mice to fracture at 10 weeks of age, and harvesting the tissues after two weeks of healing. b Representative confocal images of femoral sections from the intact and fracture groups (post-injury) through the fracture site with immunofluorescence staining for CD31 (white) to demarcate the blood vessels ( n  = 3 mice per condition; three independent experiments). White arrow, fracture site. c Representative confocal images of femoral sections of the intact and fracture groups (postinjury) through the fracture site with immunofluorescence staining for CD31 (white) to demarcate chondrocytes ( n  = 3 mice per condition; three independent experiments). d Representative confocal images of femoral sections through the fracture site of Col2-creERT;tdTomato and Col2-creERT;DTA +/+ ;tdTomato mice with immunofluorescence staining for CD31 (white) to demarcate the blood vessels in +/+ ( n  = 3 mice per condition; three independent experiments). All the data are reported as the means ± s.ds. The statistical significance was determined by one-way ANOVA and Student’s t test. * P  < 0.05, ** P  < 0.01, *** P  < 0.000 1, NS not statistically significant

Embryonic type II collagen-positive (Col2 + ) cells reportedly represent an early mesenchymal progenitor that contributes to endochondral ossification. 6 , 19 The findings from this study provide the first demonstration that ablation of Col2 + progenitors starting at the embryonic stage causes lethality in newborn mice due to dyspnea and the absence of a skeleton. In contrast to the previous understanding that only flat bone is ossified intramembranously, we found that the ablation of Col2 + cells impaired calvarial bone development and led to complete loss of vertebral bone development and most of the long bone and cartilage. In addition, we found that Col2 + cells contribute to blood vessel formation in bone tissue and some other organs, including the brain, heart and skin. As mesenchymal cells differentiate into immature chondrocytes, which are marked by type II collagen, 26 at embryonic day 13.5 (E13.5), all the tdTomato + cells (including those in blood vessels and other tdTomato + cells) found at later stages should originate from cartilage (chondrocytes) at E13.5. Furthermore, our long-term fate mapping study revealed that postnatal Col2 + progenitors existed in the AC as well as in the bone marrow and GP and that the numbers and differentiation ability of these Col2 + progenitors decreased with increasing age.

The bones of the cranial base and caudal cranial vault ossify endochondrally, whereas the facial bone and rostral cranial vault ossify intramembranously. 1 , 27 , 28 Consistently, our results showed that Col2-negative cells exist in most craniofacial bones. However, in contrast to previous reports, 1 , 29 , 30 we also found Col2 + cells in craniofacial flat bones, including the mandible, premaxilla, parietal, frontal, jugal, palatine and temporal bones and clavicle, and participated in their development (Fig. S 9 ), and the ablation of Col2 + cells affected the formation of these bones. Endochondral and intramembranous ossification are two processes involved in bone development in humans and mice. Because Col2 + cells contribute to endochondral ossification, 4 , 6 ablation of Col2 + cells should remove all endochondral ossifications. Moreover, the bones that consist of Col2 − cells should be intramembranously ossified. Thus, our findings suggest that the endochondral ossification process may also contribute to skull bone development. This conclusion is supported by the findings reported by Akagami N et al., 21 who found that a large number of osteoblasts and suture mesenchymal cells in the calvaria express Col2. Similarly, the ribs, long bones and vertebral bones were previously reported to ossify endochondrally during development 1 , 2 , 31 ; however, we found that the ablation of Col2 + cells did not completely impair rib and long bone formation but caused complete loss of vertebral bone. These findings indicate that ribs and long bones may be ossified both intramembranously and endochondrally, that vertebral bone ossifies purely endochondrally, and that Col2 + cells are the major stem cells that drive vertebral development. During the spine and vertebral development process, Col2 + cells were first detected at E9.5 in the mouse sclerotome and notochord, 32 , 33 demonstrating that resident Col2 + populations develop from E9 to E9.5. Between E11 and 12, the unsegmented perichondral mesenchyme differentiates into the annulus fibrosus of the disc anlagen. Meanwhile, uncondensed mesenchyme develops into cartilaginous vertebral bodies. Starting from E14.5, the annulus is divided into fibrosus outer and cartilaginous inner parts, and then the inner annulus links the disc rudiments to the developing vertebral bodies. 34 Moreover, the ossification marker Cbfa1 was first detected at approximately E12.5 in condensed cartilage. 35 Rigueur D et al. confirmed that the vertebral column displays positive Alcian blue staining (cartilage) and is negative for Alizarin red staining at E14.5 (bone) but positive in a small area of E16.5 mouse embryosstaining. 36 Our data showed that the vertebral column was ossified at E17.5, and stage-specific ablation of Col2 cells at E17.5 resulted in loss of the vertebral column (Fig. 2c ). Thus, these results indicate that ossification of the vertebral column most likely commences at E15 or E16. 6 Based on the above findings, we speculate that Col2 + populations can contribute to multiple cell types in the spine, including vertebral cells and intervertebral disc cells. 37 Thus, ablation of Col2 + cells starting from E9.5 completely disrupts axial vertebral and intervertebral disc development.

The critical role of angiogenesis during endochondral and intramembranous ossification has been well established. 1 During endochondral ossification of long bone, the vasculature enters the avascular buffer layer of loose mesenchymal cells surrounding the mid-diaphysis of the cartilage model immediately before the initiation of angiogenesis and the mineralization of a bone collar within the perichondrium. The process of angiogenesis during endochondral ossification of long bones has also been well established, but the corresponding process during intramembranous ossification is generally assumed. Despite some similarities between calvarial and long bone osteogenesis in vascular invasion into the surrounding avascular loose mesenchyme and the association of invading vessels with mineralization, the fundamental difference in the osteogenesis of endochondral and intramembranous bones provides hints regarding the hypothetical differences in angiogenesis between the two types of bone formation processes. 38 In agreement with this theory, we found that almost all Col2 + cells in the blood vessels of skull bone were CD31 + , but only 25.4% of CD31 + cells in long bone were Col2 + cells, which suggested different cell origins for blood vessel formation in skull and long bones (Fig. S 10 ).

Blood vessels are composed of several different cell types. The inner layer of blood vessels is composed of endothelial cells (ECs), which are covered on the outer abluminal surface by perivascular (or mural) cells. 38 Angiogenesis requires extensive coordination among the different vascular cell types to ensure that new vessels are fully functional and stable. An increasing body of evidence indicates that blood vessels form and become specialized in an organ-specific manner and that this process is controlled by local microenvironmental signals, leading to specific molecular signatures in Ecs. 39 Through lineage tracing of Sox10 + cells, Wang et al. 39 first found that Sox10 + cells contribute to only a small number of blood vessels in the lung, spleen and kidney and do not contribute to the liver vasculature during normal development. Consistently, our results showed that Col2 + cells contribute differently to CD31 + blood vessels in different organs or tissues. Notably, all CD31 + cells in blood vessels in the brain, eyeball, heart, skin and skull bone were Col2 + cells (Fig. 6D, E , Fig. S 3 ). However, approximately 25.4% of CD31 + cells in long bones were Col2 + cells, whereas no CD31 + cells in the kidney vasculature were Col2 + cells during development. Interestingly, although our results suggest that the number of Col2 + blood vessels in long bones during normal development is lower than that in skull bones, the number of Col2 + blood vessels is markedly increased upon cell expansion following fracture. 39 Our findings thus provide the first demonstration that blood vessels in the skeleton partially originate from Col2 + progenitors.

SSCs are somatic stem cells that play important roles in the development, homeostasis, and regeneration of bone tissues. 40 These cells are generally defined as self-renewing cells with “trilineage” potential to differentiate into chondrocytes, osteoblasts and adipocytes. SSCs can be classified into three types depending on their location: bone marrow-derived SSCs, GP (a cartilaginous structure separating the primary and secondary ossification centers in growing bones) SSCs and perichondrial/periosteal (the connective tissue surrounding bone) SSCs. 12 , 41 Previous studies have demonstrated that Col2 + cells behave as SSCs and are present in the bone marrow, GP and perichondrium/periosteum. 12 , 42 Interestingly, we found that similar to Pthrp + cells, 43 Col2 + SSCs can differentiate into osteoblasts and chondrocytes but rarely differentiate into adipocytes in vitro. Most interestingly, Col2 + cells in the GP have stronger osteogenic and chondrogenic differentiation ability than those in the AC and bone marrow. In addition to these Col2 + cells, we also found Col2 + cells among calvaria-derived cells. 44 Interestingly, calvaria-derived Col2 + progenitors from Col2-cre;tdTomato control mice can differentiate into osteoblasts, chondrocytes, and adipocytes. However, Col2 − cells from the Col2-cre;DTA +/− mouse calvaria can differentiate only into osteoblasts. These results suggest that Col2 + cells in the calvaria might be progenitor cells with multipotent differentiation ability. Surprisingly, the comparison of Col2 − cells from Col2-cre;DTA +/− and Col2 + cells from Col2-cre mice revealed that Col2− cells have a stronger osteogenic ability than Col2 + cells, and this finding suggests that Col2 − cells may originate from neural crest cell lineages, which have been proven to have stronger potential for osteogenesis. 1 , 45 , 46 To further determine the characteristics of Col2 + cells and Col2 − cells, we isolated Col2 + and Col2 − cells from long bone marrow cells. Interestingly, we found that both Col2 + and Col2 − cells from bone marrow cells have osteogenic and chondrogenic differentiation potential. However, quantitative analysis showed that Col2 + cells have much stronger osteogenic and chondrogenic differentiation potential than Col2 − cells from 1-month-old mice. Further study would establish the relationship between Col2 − and Col2 + progenitors in the calvaria and long bone marrow, which is of interest. In addition, our data further showed that Col2 + cells have diverse roles in blood vessel formation between long bone and calvaria bone. Almost all Col2 + cells in calvaria bone contribute to blood vessel formation, but only 25.4% of Col2 + cells in bone marrow contribute to blood vessel formation. Further investigation will deeply analyze the precise contribution and mechanism of Col2 + cells to skeletal blood vessel formation in different bones.

The comparison of the differentiation potential of Col2 + cells from the bone marrow, GP and AC showed that bone marrow Col2 + cells have osteogenic, chondrogenic and adipogenic differentiation potential. However, Col2 + cells in the GP and AC have a markedly stronger capability for osteogenic differentiation than Col2 + cells from the bone marrow under the same differentiation conditions. One possible reason is that Col2 + cells in the bone marrow are SSCs at early stages, whereas Col2 + cells in the GP and AC are stem cells at later stages; thus, under comparable differentiation conditions, Col2 + cells in the bone marrow result in less osteoblast and chondrocyte formation than committed osteoblastic and chondrogenic precursor cells. Additionally, a very limited number of Col2 + cells are found in the bone marrow after sorting through flow cytometry. To increase the cell numbers, we collected 10x-fold more cells from the bone marrow than from the GP and AC. Thus, the sorting of these cells took a long time. As a result, to obtain sufficient numbers of cells, Col2 + cells from the bone marrow were cultured for a longer time than those from the GP and AC, which may affect their stem cell differentiation potential. Further study will interestingly identify the features of these different SSCs by single-cell RNA sequencing.

Our results showed that the numbers of Col2 + cells among articular chondrocytes decreased in an age-dependent manner, and this finding is supported by the fact that the numbers of pthrpr1 + columns in the GP gradually decreased with aging. 43 Interestingly, we further found that the Col2 + progenitors did not exhibit a decrease at a constant rate during aging; instead, Col2 + progenitors decreased rapidly during 2–3 weeks of age and then slowly decreased at later stages. The marked decrease in Col2 + cells from 2 to 3 weeks of age may result from genetic changes or the activation/deactivation of hormones/growth factors occurring during this stage. Comparing the gene expression changes in cells between 2 and 3 weeks with those between earlier and later stages could provide information on the mechanisms contributing to bone and cartilage regeneration and repair and age-related osteoarthritis.

Taken together, the results showed that Col2 + cells are involved in the development of calvarial flat bone and long bone. Col2 + cells are major progenitors that contribute to CD31 + blood vessel development in calvarial bone, long bone development and fracture repair. The number and differentiation ability of Col2 + progenitors in mice decrease with increasing age. The identification of Col2 + and/or Col2 − progenitors from the GP, AC and calvarial bone provides new promising therapeutic strategies for bone regeneration and repair and for the treatment of bone and cartilage diseases.

Methods and materials

All procedures regarding mouse housing, breeding, and collection of animal tissues were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania in accordance with the relevant guidelines and regulations established by the IACUC. All animals were of the C57BL strain, and all mice were housed under specific pathogen-free conditions. Col2-cre, 47 Col2-creERT, 48 R26-tdTomato, 49 and DTA +/+ 50 mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Col2-cre; DTA +/− and Col2-creERT; DTA +/+ mice were generated by breeding DTA +/+ mice with Col2-cre or Col2-creERT mice. Col2-creERT;DTA +/+ mice were injected with TM at the indicated time points to induce the postnatal deletion of Col2 + cells. To monitor the topography of Col2-cre and Col2-creERT function, we crossed Col2-creERT and Col2-cre mice with R26-tdTomato reporter mice and analyzed the patterns of tdTomato labeling in long bones, cartilage, and knee joints at different time points. The mice were euthanized by an overdose of carbon dioxide. TM (T5648, Sigma) solution was prepared and administered as previously described. 51 Briefly, TM was first dissolved in 100% ethanol (100 mg·mL −1 ) and then diluted with sterile corn oil to a final concentration of 10 mg·mL −1 . The TM-oil mixture was stored at 4 °C until use. The mice in both the control and experimental groups were administered the same dose of TM (75 mg·kg −1 body weight) according to the time points defined in the experimental protocol.

Samples were dissected under a stereomicroscope to remove soft tissues, fixed in 4% paraformaldehyde overnight at 4 °C, and then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 14 days at 4 °C. The decalcified samples were cryoprotected in 30% sucrose/PBS solution and then in 30% sucrose/PBS:OCT (1:1) solution overnight at 4 °C. The samples were then embedded in an OCT compound (4583, Sakura) under a stereomicroscope and transferred to a sheet of dry ice to solidify the compound. The embedded samples were cryosectioned at 6 μm using a cryostat (CM1850, Leica). 52

Safranin O/fast green staining

The mouse tibia was sectioned and stained with safranin O/fast green to visualize cartilage and assess the proteoglycan content, as described previously. 53 The samples on the slides were stained with Weigert’s iron hematoxylin and fast green and then with 0.1% safranin O solution.

Alizarin red/Alcian blue staining

The whole skeleton was subjected to Alizarin red/Alcian blue staining as reported previously. 51 Briefly, the skeletons of newborn mice ( n  = 3) were fixed with 90% ethanol and then stained with 0.01% Alcian blue solution (26385–01, Electron Microscopy Sciences) and 1% Alizarin red S solution (A47503, Thomas Scientific). The stained skeletons were stored in glycerol.

Von Kossa staining

Von Kossa staining was performed with 1% silver nitrate solution (LC227501, Fisher Scientific) in a glass Coplin jar placed under ultraviolet light for 20–30 min. 54 Unreacted silver was washed with 5% sodium thiosulfate (01525, Chem-Impex). The slides were dehydrated through a graded alcohol series and mounted with permanent mounting medium.

Immunofluorescence microscopy

Tibial sections with a thickness of 6 μm were gently rinsed with PBS and incubated with proteinase K (20 μg·mL −1 , D3001-2-5, Zymo Research) for 10 min at room temperature. Subsequently, the sections were blocked in 5% normal serum (10000 C, Thermo Fisher Scientific) in PBS-T (0.4% Triton X-100 in PBS) or incubated with antibodies against type II collagen (1:100, ab34712, Abcam), Sox2 (1:200, 66411-1-Ig, Proteintech), Oct4 (1:200, 11263-1-AP, Proteintech) and CD31 (1:100, sc-81158, Santa Cruz Biotechnology) in blocking buffer at 4 °C overnight. The tissue sections were washed three times with PBS and then incubated with Alexa Fluor 488-conjugated anti-rabbit (1:200, A11008, Invitrogen) and Alexa Fluor 647-conjugated anti-mouse (1:200, A-21236, Invitrogen) secondary antibodies for 1 h at room temperature. The coverslips were mounted with FluoroShield (F6057, Sigma–Aldrich).

To quantify the percentage of tdTomato + cells, multiple fields of z-stacked photographs were randomly captured. At least 30 images were measured. The percentage of tdTomato + cells was calculated from the ratio of tdTomato + cells to total cells observed in each compartment and each sample (five sections were collected from each sample). Six mice from each group were evaluated. The assessments were independently performed by two authors who were blinded to the groups. 55 The average percentage of tdTomato + cells in each sample was pooled and calculated by two authors. The average tdTomato + percentage in each group was obtained from a pool of six mice and calculated.

PEGASOS passive immersion and blood vessel staining

PEGASOS passive immersion and blood vessel staining were performed as reported previously. 56 Briefly, before transcardial perfusion, the mice were anesthetized with an intraperitoneal injection of a combination of xylazine and ketamine (10–12.5 mg·kg −1 xylazine; 80–100 mg·kg −1 body weight ketamine). Subsequently, 50 mL of ice-cold heparin PBS (10 U·mL −1 heparin sodium in 0.01 mol·L −1 PBS) was injected transcardially to wash out the blood, and 50 mL of 4% paraformaldehyde (PFA) in 0.01 mol·L −1 PBS (pH 7.4) was infused transcardially for fixation.

To clear the calvarial bones, the samples were fixed in 4% PFA at room temperature for 12 h and then immersed in 0.5 mol·L −1 EDTA (pH 7.0) at 37 °C in a shaker for two days. The samples were then decolorized with Quadrol decolorization solution for one day at 37 °C in a shaker. The samples were subsequently transferred into 1.5-mL Eppendorf tubes containing blocking solution composed of 10% dimethyl sulfoxide, 0.5% IgePal630 and 1X casein buffer in 1 mL of 0.01 mol·L −1 PBS overnight blocking at room temperature. After blocking, the samples were stained with CD-31 primary antibodies in blocking solution for three days at 4 °C on a shaker, then washed at least three times with PBS at room temperature for one day. The samples were incubated with freshly prepared secondary antibodies diluted with blocking solution for another three days at 4 °C on a shaker and washed again for half a day, then incubated with 30%, 50%, and 70% gradient tB delipidation solutions for 4 h with each solution and then with tB-PEG for two days for dehydration. The samples were subsequently immersed in BB-PEG medium at 37 °C for half a day for clearing. Images were acquired using an Sp8 confocal microscope (Leica) with a 25X lens. 3-D reconstruction images were generated using Imaris 9.0 (Bitplane). Stacked images were generated using the “volume rendering” function. Optical slices were obtained using the “orthoslicer” function, and 3-D images were generated using the “snapshot” function.

Micro-CT analysis

A quantitative analysis of the gross bone morphology and microarchitecture was performed by micro-CT (Micro-CT 35, Scanco Medical AG, Brüttisellen, Switzerland) at Penn Center for Musculoskeletal Disorders (PCMD), University of Pennsylvania. Briefly, femurs from 4-week-old Col2-creERT and Col2-creERT; DTA +/+ mice were fixed and scanned, and three-dimensional images were reconstituted from the scans. The cross-sectional scans were analyzed to evaluate the changes in the femur. The ROIs were then compiled into 3D datasets using a Gaussian filter (sigma = 1.2, support = 2) to reduce noise and converted to binary images with a fixed grayscale threshold of 316. The trabecular bone architecture between the distal femoral metaphysis and midshaft was assessed. Two hundred (200) slices (2 mm) above the highest point of the GP were contoured for trabecular bone analysis of the BV·TV −1 , Tb.Th, Tb.N, Tb.Sp.

Cell culture

Cells were isolated from the calvarial bone of newborns as described previously. 44 Briefly, calvarial bone was dissected from newborns, subjected to sequential collagenase type II (2 mg·mL −1 ) (17101015, Gibco) and trypsin (SH3004202, GE Healthcare) digestion for 30 min and cut into tiny pieces. The minced bones were treated with collagenase type II and trypsin again and then plated in tissue culture dishes. The cells that migrated from the bone pieces were passaged and used for further studies.

BMSCs were isolated as previously described. 44 Briefly, femurs and tibias were dissected from 4-week-old Col2-cre;tdTomato mice. The bones were cut on both ends with sterile blades (22079690, Fisher Scientific). The bone marrow was flushed with complete α-MEM using 23-gauge needle syringes. The cells were then transferred for flow cytometry sorting and cell culture.

GP chondrocytes were isolated as previously described. 43 Briefly, distal epiphyses of the femurs from 4-week-old Col2-cre;tdTomato were manually dislodged, and the attached soft tissues and woven bones were carefully removed. Dissected epiphyses were incubated in 3 mL of Hank’s balanced salt solution (HBSS, H6648, Sigma) at 37 °C for 60 min in a shaking incubator. The AC and secondary ossification centers were subsequently removed. The dissected GPs were minced using a disposable scalpel (22079690, Fisher Scientific) and further incubated with Liberase TM at 37 °C for 60 min in a shaking incubator. The cells were mechanically triturated using an 18-gauge needle and a 1-mL Luer-Lok syringe and filtered through a 70-µm cell strainer into a 50-mL tube on ice to obtain a single-cell suspension. The cells were pelleted and resuspended in the appropriate medium for subsequent analyses.

AC chondrocytes were isolated as previously described. 25 Briefly, 4-week-old Col2-cre;tdTomato mice were euthanized at P28. The AC from the femoral heads was isolated by first thoroughly removing the soft tissue and bone and then incubating the sample with collagenase type IV (LS004188, Worthington Biochemical Corp) (3 mg·mL −1 ) for 45 min at 37 °C. Cartilage pieces were obtained and incubated in 0.5 mg·mL −1 collagenase type IV solution overnight at 37 °C. The cells were then filtered through a 40-μm cell strainer, collected and obtained as a single-cell suspension. The cells were pelleted and resuspended in the appropriate medium for subsequent analyses.

Flow cytometry

Cells were resuspended in 100 mL of cell staining buffer (420201, BioLegend), incubated with anti-CD16/32 antibody for 20 min on ice to block Fc receptors, and stained with fluorochrome-conjugated or isotype control antibodies on ice for 20 min. To identify MSCs, the cells were incubated with anti-CD31-APC (102419, BioLegend, 1:100) antibody and then stained with biotin-conjugated antibodies. After washing with staining medium, the cells were incubated with streptavidin-brilliant violet 421 TM (BioLegend, 1:500). Flow cytometry analysis was performed using a five-laser BD LSR Fortessa (Ex. 405/488/561/640 nm) and FACSDiva software. The acquired raw data were further analyzed using FlowJo software (Tree Star). Representative plots of at least three independent biological samples are shown in the figure.

CFU-F assay and in vitro differentiation

The following procedures were modified from a previous report. 57 For CFU-F assays, freshly prepared unfractionated bone marrow single-cell suspensions were plated at a density of ~10 4 cells per cm 2 (0.21 mL of culture medium) in 100-mm dishes containing DMEM supplemented with 20% qualified FBS, 10 mol·L −1 Y-27632 (TOCRIS) and 1% penicillin/streptomycin. For CFU-F assays with sorted cells, tdTomato + cells were sorted and directly seeded into culture at a density of 10 cells per cm 2 in 6-well plates to ensure that colonies would form at clonal density and could thus be counted. The cultures were incubated at 37 °C in a humidified atmosphere with 5% O 2 and 10% CO 2 for 7–10 days. The CFU-F colonies were counted after 7–10 days of culture.

For the in vitro differentiation assay, tdTomato + cells were sorted, seeded into each well of 48-well plates and cultured for 14 days. Adipocyte, chondrocyte and osteoblastic differentiation was induced with different differentiation media and detected by staining with oil red O (O0625–25G, Sigma–Aldrich), Alcian blue (26385–01, Electron Microscopy Sciences) and Alizarin red (A47503, Thomas Scientific), respectively, as performed previously. 58 , 59

Bone fracture

Closed femoral fractures with intramedullary nail fixation were created in 10-week-old mice (Col2-creERT;tdTomato or Col2-creERT;DTA +/+ ;tdTomato) as previously described. 25 Briefly, closed fractures were generated using a three-point blunt guillotine driven by a dropped weight, which created a uniform transverse fracture of the femur. The fractured femurs were harvested at 12 weeks of age for analysis. Three independent experiments with six ( n  = 6) mice per condition were performed.

Scratch-wound assay

Scratch wound assays were performed as previously described. 60 The same numbers of cells from the calvarial bone of Col2-cre;tdTomato and Col2-cre;DTA +/+ ;tdTomato mice were seeded in six-well plates. The monolayer cells were scratched and then cultivated under normal conditions. For each group, the migration distances at 0, 12, 24, and 72 h after scratching were measured.

Blood vessel tube formation assay

Mouse tdTomato + and tdTomato − cells were isolated from the calvarial bone and cartilage of Col2-cre;tdTomato mice and depleted of erythrocytes by hypotonic lysis. tdTomato + and tdTomato − cells were isolated by cell sorting using a BD FACS ARIA II sorter, and the purity of the sorted cells was higher than 95%. The cells were resuspended (1.5 × 10 6 cells per mL) in endothelial growth medium (EGM-2) supplemented with EGF, hydrocortisone, VEGF, fibroblast growth factor-B, heparin, insulin-like growth factor, gentamicin, and 5% heat-inactivated fetal bovine serum (CC3162, Lonza).

To evaluate the effect of tdTomato + and tdTomato + cells on tube formation capacity, tdTomato + and tdTomato − cells from mouse calvarial bone and cartilage were plated on fibrin gel (Millicell, Cat. MMA130, Millipore)-coated culture plates. Cells were plated at 3 × 10 5 cells per well, and specific medium was added to each group. Tube formation was monitored for up to 12 hours, and a photographic record at 20x magnification was obtained.

All the data are presented as the means ± s.ds. The Shapiro–Wilk test for normality and Bartlett’s test for variance were performed to determine the appropriate statistical tests for the data analyses. Comparisons between two groups were assessed by Student’s t test, and comparisons among groups of samples were assessed by one-way ANOVA followed by Tukey’s multiple comparison test. The number of animals and the number of experimental repetitions are presented in the figure legends. GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA) was used for these analyses. (* P  < 0.05, ** P  < 0.01, *** P  < 0.000 1, NS = not statistically significant.)

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Acknowledgements

Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and the National Institute on Aging, part of the National Institutes of Health, under Award Numbers DE023105, AR066101 and AG048388 and Department of Defense office of the Congressionally Directed Medical Research Programs (CDMRP), under Award Number of RA210159 to SY. The research was sponsored by the Shanghai Sailing Program (21YF1436400) and National Natural Science Foundation of China (82102608) to XL. XL was also supported by the China Scholarship Council (CSC) Grant #201706260178. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors acknowledge the Penn Center for Musculoskeletal Disorders for providing access to the Micro-CT instrument (NIH/NIAMS P30 AR069619). We acknowledge the contributions of Dr. Qiang Fu and Song Guo’ to Supplementary Figs 9 , 10 .

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Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

Xinhua Li, Shuting Yang, Gongsheng Yuan & Shuying Yang

Department of Orthopedics, Shanghai General Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, 200080, P. R. China

Department of Spinal Surgery, East Hospital, Tongji University, School of Medicine, Shanghai, 200120, China

Department of Restorative Sciences, College of Dentistry, Texas A&M University, Dallas, TX, USA

Dian Jing & Hu Zhao

Department of Orthopedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

The Penn Center for Musculoskeletal Disorders, School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

Shuying Yang

Center for Innovation & Precision Dentistry, School of Dental Medicine, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA

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Contributions

XL, GY, DJ and SY performed the experiments, interpreted the data and wrote the initial draft of the manuscript. STY managed mouse colonies and assisted with the experiments. XL and SY conceived and supervised the study and wrote the manuscript. LQ and HZ provided critical suggestions, reagents and technical assistance during the study.

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Correspondence to Shuying Yang .

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Li, X., Yang, S., Yuan, G. et al. Type II collagen-positive progenitors are important stem cells in controlling skeletal development and vascular formation. Bone Res 10 , 46 (2022). https://doi.org/10.1038/s41413-022-00214-z

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Poly-L-Lactic Acid Fillers Improved Dermal Collagen Synthesis by Modulating M2 Macrophage Polarization in Aged Animal Skin

Affiliations.

• 1 Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon 21999, Republic of Korea.
• 2 Doctorbom Clinic, Seoul 06614, Republic of Korea.
• 3 Department of Anatomy & Cell Biology, Gachon University College of Medicine, Incheon 21936, Republic of Korea.
• 4 SACCI Bio Co., Seoul 1007, Republic of Korea.
• 5 Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical Center, Gachon University, Incheon 21565, Republic of Korea.
• 6 Department of Health Sciences and Technology, Gachon Advanced Institute for Health & Sciences and Technology (GAIHST), Gachon University, Incheon 21999, Republic of Korea.
• PMID: 37174720
• PMCID: PMC10177436
• DOI: 10.3390/cells12091320

Poly-L-lactic acid (PLLA) fillers correct cutaneous volume loss by stimulating fibroblasts to synthesize collagen and by augmenting the volume. PLLA triggers the macrophage-induced activation of fibroblasts that secrete transforming growth factor-β (TGF-β). However, whether M2 macrophage polarization is involved in PLLA-induced collagen synthesis via fibroblast activation in aged skin is not known. Therefore, we evaluated the effect of PLLA on dermal collagen synthesis via M2 polarization in an H 2 O 2 -induced cellular senescence model and aged animal skin. H 2 O 2 -treated macrophages had increased expression levels of the M1 marker CD80 and decreased expression levels of the M2 marker CD163, which were reversed by PLLA. The expression levels of interleukin (IL)-4 and IL-13, which mediate M2 polarization, were decreased in H 2 O 2 -treated macrophages and increased upon the PLLA treatment. CD163, IL-4, and IL-13 expression levels were decreased in aged skin, but increased after the PLLA treatment. The expression levels of TGF-β, pSMAD2/SMAD2, connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA), collagen type 1A1 (COL1A1), and COL3A1 were also decreased in aged skin, but increased after the PLLA treatment. Moreover, PLLA upregulated phosphatidylinositol 3-kinase p85α (PI3-kinase p85α)/protein kinase B (AKT) signaling, leading to fibroblast proliferation. PLLA decreased the expression of matrix metalloproteinase (MMP) 2 and MMP3, which destroy collagen and elastin fibers in aged skin. The amount of collagen and elastin fibers in aged skin increased following the PLLA treatment. In conclusion, PLLA causes M2 polarization by increasing IL-4 and IL-13 levels and upregulating TGF-β expression and collagen synthesis in aged skin.

Keywords: M2 polarization; collagen and elastin synthesis; poly-L-lactic acid; senescence.

Publication types

• Research Support, Non-U.S. Gov't
• Collagen / metabolism
• Elastin* / metabolism
• Hydrogen Peroxide / metabolism
• Hydrogen Peroxide / pharmacology
• Interleukin-13 / metabolism
• Interleukin-4* / metabolism
• Macrophages / metabolism
• Transforming Growth Factor beta / metabolism
• poly(lactide)
• Interleukin-4
• Hydrogen Peroxide
• Interleukin-13
• Transforming Growth Factor beta

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8 Health Benefits of Collagen

From skin and bones to gut health and more, here’s how the trendy supplement may boost your health and well-being.

Who would have thought that a substance found in bones and skin could become the need-to-have supplement? We’re talking  collagen .

“Collagen is a type of protein that plays an important role in building and supporting many tissues, from bones and cartilage to skin, hair, eyes, and the digestive system,” says Sonya Angelone, RDN, who practices in San Francisco.

When you take a  collagen supplement  or eat foods rich in collagen, you are typically consuming collagen that comes from an animal, says Ryanne Lachman, RDN, an integrative and functional nutritionist in Cleveland. (Vegan forms of collagen, however, are now available.)

Collagen peptides are often sold in powdered or capsule form, and collagen can also be  consumed in bone broth .

As with any supplement, collagen comes with some potential side effects.

RELATED:  6 Supplements for Glowy Skin and Gorgeous Hair

Aside from that, a universal downside to collagen supplements is that they add an extra expense to your grocery bill. Prices can range from about $25 for a jar of the powdered form to $50 or more for a bottle of 90 capsules.

If you’re going to invest in taking collagen supplements, it’s important to know the bevy of possible benefits collagen supplements can provide. Read on.

1. Supplements Help Replace What’s Naturally Lost Through Aging

2. collagen is an easy-to-digest source of protein.

Your body works hard to digest protein from sources like  chicken  or beef, and some people may find they deal with digestive symptoms like burping or stomach pain after a meal, explains Lachman. But collagen supplements are hydrolyzed, meaning the collagen is broken down, a process that makes it easier for your body to digest. Collagen supplements (particularly the powders) may be a more comfortable way to get protein into your diet, she says. The process of hydrolyzing also allows collagen peptides to dissolve in water, which makes it relatively simple to use them in everyday foods (like water or smoothies).

3. Collagen Smooths Wrinkles and Boosts Elasticity in Skin

RELATED:  20 Ways to Preserve — and Boost — Collagen in Your Face

4. Collagen May Relieve Joint Aches and Pains

Joint pain can make it difficult to exercise, which can knock you off track in meeting your fitness goals. A collagen supplement may help you stay the course. “There is some evidence that collagen can be great for supporting connective tissues and improving joint pain after exercise,” says Angelone.

5. Oral Supplements May Promote Gut Health

In inflammatory digestive conditions, such as inflammatory bowel disease (IBD), there is a “gut healing” theory about collagen. “Some research finds that collagen levels are decreased in patients with these conditions. By taking collagen, you would help correct a deficiency,” says Lachman.

Since collagen may contribute to these symptoms in some folks, it’s important to keep track of how you feel when taking it and make adjustments to how much, when, or if you take it as needed.

RELATED:  10 Foods to Help Beat Rheumatoid Arthritis Inflammation

6. Collagen May Buoy Your Heart Health

Researchers think that the collagen may fortify blood vessel walls to reduce the risk of artery disease.

7. Peptides Can Keep Bones Healthy

RELATED:  3 Natural Ways to Build Bones and Prevent Osteoporosis

8. Collagen May Be Useful in Joint Disease

Editorial sources and fact-checking.

Everyday Health follows strict sourcing guidelines to ensure the accuracy of its content, outlined in our editorial policy . We use only trustworthy sources, including peer-reviewed studies, board-certified medical experts, patients with lived experience, and information from top institutions.

• Cooperman T. Are There Side Effects From Collagen Supplements? ConsumerLab.com. April 23, 2021.
• Collagen. Harvard T.H. Chan School of Public Health. May 2021.
• Collagen. Cleveland Clinic. May 23, 2022.
• Choi FD et al. Oral Collagen Supplementation: A Systematic Review of Dermatological Applications. Journal of Drugs in Dermatology . January 2019.
• Dewi DAR et al. Exploring the Impact of Hydrolyzed Collagen Oral Supplementation on Skin Rejuvenation: A Systematic Review and Meta-Analysis. Cureus . December 2023.
• Zdzieblik D et al. Improvement of Activity-Related Knee Joint Discomfort Following Supplementation of Specific Collagen Peptides. Applied Physiology, Nutrition, and Metabolism . June 2017.
• van Haaften WT et al. Misbalance in Type III Collagen Formation/Degradation as a Novel Serological Biomarker for Penetrating (Montreal B3) Crohn's Disease. Alimentary Pharmacology and Therapeutics . May 2017.
• Koutroubakis IE et al. Serum Laminin and Collagen IV in Inflammatory Bowel Disease. Journal of Clinical Pathology . November 2023.
• Abrahams M et al. Effect of a Daily Collagen Peptide Supplement on Digestive Symptoms in Healthy Women: 2-Phase Mixed Methods Study. JMIR Formative Research . May 2022.
• Tomosugi N et al. Effect of Collagen Tripeptide on Atherosclerosis in Healthy Humans. Journal of Atherosclerosis and Thrombosis . May 2017.
• Arteriosclerosis/Atherosclerosis. Mayo Clinic. July 2022.
• What Women Need to Know. Bone Health & Osteoporosis Foundation.
• König D et al. Specific Collagen Peptides Improve Bone Mineral Density and Bone Markers in Postmenopausal Women — a Randomized Controlled Study. Nutrients . January 2018.
• Mohebbi R et al. Exercise Training and Bone Mineral Density in Postmenopausal Women: An Updated Systematic Review and Meta-Analysis of Intervention Studies With Emphasis on Potential Moderators. Osteoporosis International . February 2023.
• León-López A et al. Hydrolyzed Collagen — Sources and Applications. Molecules . November 2019.
• Bakilan F et al. Effects of Native Type II Collagen Treatment on Knee Osteoarthritis: A Randomized Controlled Trial. Eurasian Journal of Medicine . June 2016.
• Cooperman T. Are There Side Effects From Collagen Supplements?  ConsumerLab.com . April 23, 2021.
• Collagen.  Harvard T.H. Chan School of Public Health . May 2021.
• Collagen. Cleveland Clinic . May 23, 2022.
• Choi FD et al. Oral Collagen Supplementation: A Systematic Review of Dermatological Applications.  Journal of Drugs in Dermatology . January 2019.
• Zdzieblik D et al. Improvement of Activity-Related Knee Joint Discomfort Following Supplementation of Specific Collagen Peptides.  Applied Physiology, Nutrition, and Metabolism . June 2017.
• Koutroubakis IE et al. Serum Laminin and Collagen IV in Inflammatory Bowel Disease. Journal of Clinical Pathology . November 2003.
• Tomosugi N et al. Effect of Collagen Tripeptide on Atherosclerosis in Healthy Humans. Journal of  Atherosclerosis and Thrombosis . May 1, 2017.
• Arteriosclerosis / Atherosclerosis: Symptoms and Causes.  Mayo Clinic . June 1, 2022.
• What Women Need to Know. Bone Health & Osteoporosis Foundation .
• König D et al. Specific Collagen Peptides Improve Bone Mineral Density and Bone Markers in Postmenopausal Women — a Randomized Controlled Study.  Nutrients . January 2018.
• Bakilan F et al. Effects of Native Type II Collagen Treatment on Knee Osteoarthritis: A Randomized Controlled Trial.  Eurasian Journal of Medicine . June 2016.
• Metabolism and Weight Loss: How You Burn Calories.  Mayo Clinic . October 8, 2022.

Experts share the 16 best collagen supplements of 2024, plus their benefits

M ove over personalized supplements and multivitamins, ditch the protein powder, and wave bye-bye to your skincare routine. It’s time to run for the collagen.

If you’ve spent time on social media in recent years, you’ve probably noticed more people pushing collagen products – like Jennifer Aniston’s favorite Vital Proteins Collagen Peptides, Amazon’s ELEMIS Pro-Collagen Cleansing Balm, or ultimate luxury wellness brand, Thorne’s Collagen Plus.

So what is this new skincare spectacle? A crazy fad or science-backed cure for sagging or stretching skin? Collagen is the most abundant structural protein in animals, providing skin with structure and strengthening bones, according to Healthline. Humans produce collagen naturally, but as we age, collagen begins breaking down and it becomes more difficult to make more.

• Best Overall: Hum Collagen Love
• Best Powder: Vital Proteins Collagen Peptides 
• Best for Gut Health: Ancient Nutrition Collagen Protein
• Best for Joint Health: Clean Simple Eats unflavored collagen powder
• Best for Hair and Nails: Thorne Collagen Plus
• Best Vegetarian: Move & Glow Collagen Pills for Women & Men
• Best Gummies: Olly Collagen Rings
• Best Amazon reviews: Vital Proteins Collagen Gummies
• Best Budget-Friendly: Youtheory Collagen
• Best Value: California Gold Nutrition, Hydrolyzed Collagen Peptides + Vitamin C

While there are plenty of collagen-rich foods to add to your diet, including bone broth and pork skin, some may decide to integrate collagen supplements and powders into their routine for a quicker method of replenishing the lost protein. Most collagen supplements are hydrolyzed, meaning the collagen has been broken down so it absorbs more easily.

A comprehensive review of 26 studies by the National Library of Medicine  found that most women who took one to 12 grams of collagen per day for four to 12 weeks experienced improved skin elasticity and hydration. Other studies cited by Healthline indicate that collagen could also help prevent bone loss, improve gut health, and strengthen hair and nails.

Before adding any new supplements to your routine, it’s always recommended that you consult with your healthcare professional.

With this array of potential benefits, The Post decided to reach out to Regan La Testa , an IPEC-certified Nutritional Coach, Personal Trainer, and Hormone Specialist, who also happens to have firsthand experience with a variety of collagen brands.

• Enriched with Vitamins C, E, Biotin Pomegranate, Acerola, Camu Camu, Asparagus, Acai, Okra 
• free of artificial fillers, binders, and ingredients
• Eu Natural partners with Vitamin Angels to provide vitamins to women and children
• Pill form not ideal for all people
• Unclear how many grams of collagen are in each serving

Form: Capsules

Flavor: Unflavored

“This capsule is a great alternative for vegetarians, as it is made from eggshells and not from animals. This makes it more effective than vegan products. It contains Type I, V and X of collagen as well as hyaluronic acid, biotin, and a superfood blend for added benefits,” La Testa explained. Given the variety of collagen types, Move & Glow is not just focused on hair, skin, and nails, but also could improve joint pain and mobility.

“The brand also seems to be very focused on no fillers, being gluten-free, non-dairy, keto and paleo friendly…One other amazing thing about the company is that for every bottle purchased, a year of vitamins is given to a child in need. All ingredients are sourced in the USA,” La Testa noted. Unfortunately, it is unclear exactly how many grams of collagen are contained in the product.

• Includes Vitamin C and Hyaluronic Acid
• Free of artificial sweeteners
• Third-party tested
• Requires three capsules per serving

La Testa has recommended this collagen supplement based on the combination of ingredients.

“It contains Vitamin C and Hyaluronic Acid, which combined with collagen are believed to be a great way to support skin health and anti-aging with hydration and minimizing the appearance of wrinkles,” she shared. Another great draw is the lack of fillers — “It is free of artificial sweeteners, colors, flavors and preservatives, which is a plus! It has also been 3rd party tested!  All in all, I think this is a decent product and appears to be transparent. It may be a bit costly.” Hum Nutrition hits each requirement when it comes to collagen, but beware that you’ll need to take three capsules per serving for the full benefits (may not be ideal for those who are pill-averse).

• Paleo-friendly
• Dissolves into hot and cold liquids
• High amount of collagen per serving
• No scoop is provided, so requires careful measuring

Form: Powder

This popular celeb-approved brand hits all the marks for La Testa. “I have personally tried Vital Proteins Collagen Peptides powder. It is flavorless can be mixed in any liquid, and has Vitamin C and Hyaluronic Acid,” she pointed out.

While La Testa preferred the original Vital Proteins formula, the new advanced edition does offer a plentiful scoop of collagen — 20 grams per serving to be exact, and it is sourced from grass-fed, pasture-raised bovine, making it a wonderful paleo-friendly option. The one downside to this brand is that you’re going to have to carefully measure it out yourself. The new packaging does not contain a scoop.

• Combinations of protein, Vitamin C, and probiotics for a more holistic approach to health
• Multiple flavor options available including unflavored
• Good amount of collagen per serving
• Some reviewers say that the packaging is only about half full which is slightly misleading
• Other brands may dissolve better

Flavor: Unflavored, Cucumber Lime, Strawberry Lemonade, Vanilla, more

“I am currently using Ancient Nutrition Collagen Protein,” La Testa said. “It is a flavorless powder that contains ten types of protein, nine grams of protein and ten grams of collagen per scoop,” she noted. This combination collagen powder also contains Vitamin C and soil-based organism (SBO) probiotics, which La Testa says help promote good gut health.

Ancient Nutrition’s collagen powder is made with proprietary fermented eggshell collagen, which is said to minimize exercise-induced discomfort. The fermentation process also allows your body to absorb the ingredients better. A few reviewers mentioned that this brand has a more gritty texture than others, however the plentiful array of flavors makes up for it.

• Sourced with types I, II, and III of bovine
• Some mixed reviews on the intensity of the flavors

Flavor: Unflavored, Peach Mango, Blue Hawaii, Cucumber Lime, more

“I just purchased Clean Simple Eats unflavored collagen powder,” said La Testa. She has yet to start taking it, but she likes the serving size and bovine sources. “It contains nine grams of collagen protein per serving, it is third-party tested and is bovine sourced with Types I, II, and III and claims to support skin, hair, nails and joint health. I’m looking forward to starting to use this product!”

Reviewers have shared that this formula is quite effective — some noting that it has aided in their post-workout recovery and others suggesting it has helped with joint pain. There are some mixed reviews on the flavors, with some mentioning that it’s a bit on the sweet and sour side, so you should be prepared for a stronger kick to your smoothie.

• Derived from bovine (grass-fed and hormone-free cows)
• Gluten-free
• Higher amount of collagen per serving
• Mixed opinions on stevia used to sweeten

Flavor: Passion berry flavored

“This product is focused on improving skin, hair, and nails. Thorne is a reputable supplement company which is great and the product is gluten-free which I love. The collagen is sourced from cows that are grass-fed and hormone-free. Also pluses,” La Testa shared. 

“It contains 13 grams of collagen per serving so it is on the higher end and contains only Types I and III of collagen, which is not unusual.” For La Testa, the only big downside to this brand is that it only comes in one flavor: berry passion. The formula uses stevia for sweetness, which is not ideal for all people. “It may also be cost-prohibitive for people who are looking to take collagen but are on a budget,” La Testa added.

• 20 grams of collagen per serving
• Available in a variety of sizes
• No added sugar
• Some reviewers say it has more carbs then they would like to consumer per serving

Bulletproof offers a range of science-backed supplements. Personally, I can’t get enough of the vanilla collagen protein bars , which make for an incredible on-the-go snack. But for those seeking the basics, we suggest the Bulletproof collagen peptide formula, which is packed with 20 grams of collagen per serving. I love the variety of sizes this brand offers — including single-dosed packets.

• Good option for those who are resistant to capsules or powders
• Delicious, sweet, peach flavor
• Easy to chew
• Less collagen than other brands (2.5 grams)
• Exposure to heat or sunlight may lead to melting

Form: Gummies

For a reliable drugstore option, we recommend going to Olly. While this brand does not contain quite as much collagen as La Testa recommended (it only offers 2.5 grams per serving), it could be an ideal choice for someone who prefers getting their nutrients through delicious, and easy-to-chew gummies. The brand is “clinically tested,” and most reviewers agree that as far as flavor goes, Olly has nailed it. The Peach Bellini flavor is like candy. The gummies are also gluten-free and use no artificial colors.

• Made without gluten, dairy, and soy
• Free of synthetic colors and artificial flavors
• Smooth, easy-to-chew texture
• Sweet flavor
• Four gummies are required per serving
• Lower amount of collagen (2.5 grams)
• Vegetable oil and sugar

Form: Gummy

Flavor: Grape

Vital Proteins has also made their collagen blend accessible in a gummy — what’s not to love? These decadent bites have received rave reviews on Amazon, with many people saying that they noticed differences in their hair and nails within weeks of use.

Some people love the gentle grape flavor and soft texture, but there are a few mixed reviews on the sweetness level. Most people agree that the real draw of these gummies is that they are gluten, dairy, and soy-free as well as free of synthetic colors and artificial flavors. On the downside, each serving only provides a 2.5-gram dose of collagen, which is certainly on the lower side.

• Blended with Vitamin C
• Dairy, gluten, and soy-free
• Many reviewers agree these are effective when used correctly
• A lower dose of collagen (six grams)

For collagen focused particularly on improving skin, hair, and nails, opt for a formula with type I and III collagen. This advanced formula is also blended with Vitamin C for easy absorption, and reviewers say their skin was glowing after consecutive use. Perhaps our favorite part about this supplement, is that it is available for under $10. Collagen can be expensive, so for those looking to save, we recommend going with Youththeory.

• Contains Vitamin C
• Long-lasting supply (250 tablets in each container)
• Many reviewers noticed improvements in their skin
• Ingredient testing transparency
• Lower levels of collagen (three grams)
• Contains some sodium
• Must take three tablets twice a day

Editor’s Note: This item will be back in stock on April 26, 2024. Add your email to be notified when it’s available.

For shoppers who are very weary of what they’re putting in their bodies, it’s important to find a brand that discloses its testing practices. California Gold Nutrition utilizes third-party testing on its health products and provides key findings that are helpful for consumers — like microbial analyses and heavy metal analyses.

This hydrolyzed collagen also comes with a 100% GOLD guarantee, meaning you can get your money back if you’re unsatisfied with the product for any reason. The one downside of this formula is that you’ll need to take six tablets a day for the best results. For those who don’t enjoy taking pills, this may not be the best option.

• Type I and III collagen peptides
• Easily dissolves into liquid
• 2 scoops are required to get the full 20 grams of collagen, which means you’ll go through it faster

One of our editor’s love this brand of collagen powder, swearing she sees a noticeable difference when she stops taking it.

“I always thought collagen wasn’t worth the hype or money,” she says. “However, I’ve become a convert. I used it for several months and didn’t think much of it. It was when I stopped taking it that I noticed a huge difference in how my skin looked. If you’re curious, it wasn’t great. Now I take it everyday. My skin looks plumper. I’m also a runner, and it’s supposed to help with joint health, which is important to me.”

• Produced from grass-fed cows
• Award winning
• Developed to take with other NEEDED supplements, which increases overall cost

This collagen protein is geared towards mothers-to-be. However, many of us like our collagen without hormones, from pasture-fed cows. Third-party tested and winner of the Purity Award , this supplement from NEEDED is something we’d be more than happy to add to a cup of cocoa or shake. It’s flavorless, so it can be mixed with practically anything — even a bowl of ice cream or yogurt.

• Uses hydrolyzed peptides
• 19 amino acids
• Scoop is on the smaller side

We’re seriously impressed with the reviews on this collagen powder. It doesn’t change the taste or consistency of whatever you mix it with, and a single scoop gives 10 gorgeous grams of collagen. Best of all, in a small survey conducted by Live Conscious with 695 people, 85% noticed a positive different in their hair, while 87% saw an improvement in mobility.

• Only need 1-2 tablespoons per day
• Available in the flavors mango, and pineapple
• Taste may be too rich for some with a sensitive pallet

Not into powder or pills? No problem. Spoiled Child and its liquid collagen has your back. All it takes is a tablespoon (or two) per day, and you’ve got your dose in. Featuring more than 7,000 reviews, this bestseller comes in the tropical flavor pineapple, as well as mango, and can still be mixed in water, juice or a fruit-based smoothie. While some customers noted they haven’t seen a visible difference since taking it, the website has some amazing before and afters from people who have.

• Also has vitamin C, E and biotin
• Delicious chocolate flavor
• According to some reviews, the unflavored option has an unpleasant aftertaste — try the chocolate instead!

Craving something sweet? Maybe this collagen powder from mindbodygreen will nip that craving. Is it exactly the same as a candy bar? Nope, but it also has ingredients that are way better for you, including anti-inflammatory turmeric. The cocoa used is organic, and so is the monk fruit used to sweeten the powder. Although the reviews for the unflavored version of this supplement aren’t great, as far as the chocolate is concerned, folks like it.

As a certified nutritional coach, La Testa specializes in supporting mothers and women over 35 who are working towards taking control of their health, and increasing their energy, and confidence inside and out.

“Collagen, a protein that is responsible for healthy joints and skin elasticity, is located in your muscles, blood, and bones. Our bodies make collagen naturally from amino acids but to do so, it needs: proline, vitamin C; copper, glycine, and zinc. These all come from eating a healthy, well-balanced diet,” shared La Testa.

“As supplements, collagen comes in a digestible form called collagen peptides or hydrolyzed collagen,” explained La Testa. “Collagen supplements are made from the tissues of fish, cows, pigs or chicken. While there are vegan collagen supplements, made from bacteria and yeast, it is unclear whether this type of collagen provides the same possible benefits as collagen made from animal sources.”

“Collagen supplement companies assert that taking collagen supplements provides many benefits to the body. Evidence is still out as to whether this is accurate,’ revealed La testa. She prefers to say that collagen may play a role in strengthening skin, lowering inflammation, preventing bone loss, boosting muscle mass, improving gut health, strengthening hair and nails, and maintaining brain health.

La Testa has shared that collagen can be taken in the form of a powder, capsule, liquid, or gummies: “Based on my research and understanding, it is unclear which form is the most effective. Most of the debate I’ve seen is between powder and liquid forms. I personally use powder because it can easily be mixed with other things. I know people who swear by liquid forms that are daily dose ‘packs’ drunk straight from the package.”

“Generally it is recommended to take collagen one time daily, between 2.5 and 15 grams. Most products I’ve seen or used are approximately 10 grams per scoop of powder,” she noted.

“I prefer supplements that are 3rd party tested because there is no federal oversight of nutritional supplements. Additionally, collagen supplements that contain Types I, II, III, V, and X of collagen may be ideal,” said La Testa. She tries to avoid products with fillers. She also notes that some people have preferences when it comes to flavors or unflavored options.

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• v.4; 2021 Dec

Collagen type II: From biosynthesis to advanced biomaterials for cartilage engineering

a Regenerative, Modular & Developmental Engineering Laboratory (REMODEL) and Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway (NUI Galway), Galway, Ireland

SH Korntner

b Teagasc Research Centre, Ashtown, Ireland

DI Zeugolis

c Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research and School of Mechanical & Materials Engineering, University College Dublin (UCD), Dublin, Ireland

• • Collagen type II is the major constituent of cartilage tissue.
• • Collagen type I devices in cartilage engineering are associated with suboptimal functional therapeutic outcomes.
• • In vitro and preclinical data clearly illustrate the potential of collagen type II as building block in cartilage engineering approaches.

Collagen type II is the major constituent of cartilage tissue. Yet, cartilage engineering approaches are primarily based on collagen type I devices that are associated with suboptimal functional therapeutic outcomes. Herein, we briefly describe cartilage's development and cellular and extracellular composition and organisation. We also provide an overview of collagen type II biosynthesis and purification protocols from tissues of terrestrial and marine species and recombinant systems. We then advocate the use of collagen type II as a building block in cartilage engineering approaches, based on safety, efficiency and efficacy data that have been derived over the years from numerous in vitro and in vivo studies.

1. Introduction

Articular cartilage is a specialised connective tissue of the joints [ 1 , 2 ]. Hyaline cartilage provides a smooth and lubricated surface for articulation and facilitates the transmission of loads with low frictional coefficient [ 3 , 4 ]. As articular cartilage lacks blood vessels, lymphatics and nerves, it has limited capacity for intrinsic healing and repair [ 5 , 6 ]. Articular cartilage injuries are frequently caused by sports and recreational activities and, if left untreated, articular cartilage lesions form fibrocartilage and lead to osteoarthritis (OA).

OA is a whole joint disease, involving structural alterations in the hyaline articular cartilage, subchondral bone, ligaments, capsule synovium and periarticular muscles [7] . OA affects over 250 million people worldwide [8] and financially drains healthcare systems. For example, medical costs account for 1.0% to 2.5% of gross domestic product of high-income countries [9] and in USA alone, the annual insurer spending for OA-related medical care is estimated to be US$ 185.5 billion [10] . OA, due to the imbalance between the repair and the damage of joint cartilage, leads to structural destruction and failure of the synovial joint [11] . The pathogenesis of OA involves compositional changes and structural / integrity losses of cartilage [12] . Initially, disruption, caused by physical forces, happens at the cartilage surface and is followed by the expansion of the calcified zone into the radial zone. Then, the hypertrophic chondrocytes synthesise extracellular matrix (ECM) degradation products and proinflammatory mediators. Subsequently, the bone turnover is increased in the subchondral bone and vascular invasion takes place towards the cartilage region [13] . Clinically, the knee, hip, hand, spine and foot are the most common sites of OA, followed by the wrists, shoulders and ankles [14] . OA incidents gradually increase over the years, due to the combined effects of ageing, obesity and heavy work activities [15] , [16] , [17] . In fact, by 2032, the proportion of the population aged >45 with any doctor-diagnosed OA is estimated to increase from 26.6% to 29.5% [18] . OA is more prevalent in women than in men, with female-to-male ratio ranging from 1.5 to 4.0 [19] . Exercise, weight loss (in the case of overweight patients) and walking aids are widely recommended to improve daily activities of OA patients [ 20 , 21 ]. Pharmacological agents [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , surgical procedures [30] , [31] , [32] , [33] and advanced therapy medicinal products [34] , [35] , [36] , [37] have shown variable degree of efficiency and effectiveness, considering their complexity, cost of goods and regulatory hurdles. To this end, biomaterial-based therapies are continuously gaining pace, especially for large defects [38] , [39] , [40] , [41] .

Herein, we provide a brief description of cartilage's development, cellular and extracellular composition and organisation and then focus on collagen type II biosynthesis, extraction protocols, scaffold fabrication and in vitro, in vivo and clinical data, in light of recent studies that demonstrate the inability of collagen type I scaffolds to yield functional therapeutic outcomes [ 42 , 43 ].

2. Cartilage development, cellular and extracellular composition and organisation

Cartilage morphogenesis [44] , [45] , [46] , [47] , [48] is a complex and well-orchestrated sequence of numerous intracellular and extracellular spatiotemporal events, responsible for the tissue composition and structure ( Fig. 1 ). Articular cartilage and long bones are formed by endochondral ossification that is initiated from the lateral growth plate that contains mesenchymal stem cells that secrete hyaluronan and collagen type I. As these stem cells move towards the centre of the limb, they aggregate; stop proliferating and expressing collagen type I; and start expressing N-cadherin, tenascin-C and cell adhesion molecules. Formation of tight aggregates marks the start of the condensation processes that involves stem cell aggregation and increased hyaluronidase activity that in turn decreases hyaluronan and cell movement and increases cell-cell interactions. These increased cell-cell interactions trigger signalling pathways responsible for the initiation of chondrogenic differentiation. An array of small proteoglycans (PGs, e.g. versican, perlecan), growth factors (e.g. fibroblast growth factors, FGF; bone morphogenetic proteins, BMPs; transforming growth factor-β, TGF-β), transcription factors (e.g. Sox5, Sox6, Sox9), signalling molecules (e.g. sonic hedgehog, Indian hedgehog) and ECM molecules (e.g. matrilins, fibronectin) contribute in chondrogenic differentiation and cartilage formation. Subsequently, the cells cease the expression of adhesion molecules, resume proliferation via action of growth hormone, parathyroid hormone-related peptide and insulin-like growth factor-1 (IGF-1), initiate ECM synthesis (e.g. collagen types II, IX, X and XI; aggrecan; decorin; annexin II, V and VI; tenascins; thrombospondins; cartilage oligomeric matrix protein) and decrease production of fibronectin. A series of maturation steps then takes place for the differentiation of committed chondrocytes to pre-hypertrophic, hypertrophic and matrix-mineralising chondrocytes. Hypertrophic chondrocytes increase in size, start to synthesise calcified matrix rich in collagen type X and alkaline phosphatase, synthesise an array of terminal differentiation molecules (e.g. matrix metalloproteinase-13; Runx2, Runx3, BMP-6, BMP-2, BMP-7, aggrecan, hyaluronan) and cease to synthesise others (e.g. Sox9, collagen type II).

Sequential stem cell differentiation to chondrocytes and associated ECM changes (A). Chondrocyte secreted ECM and interactions (B). Chondrocyte cellular (left) and extracellular (right) organisation (C).

Articular cartilage contains a highly specialised cell population, the chondrocytes, which is responsible for the production, organisation and maintenance of the cartilage ECM [ 49 , 50 ]. It also contains a small number of mesenchymal progenitor cells, the number of which increases in osteoarthritic articular cartilage [ 51 , 52 ]. A diverse range of collagen types are encountered in cartilage with distinct functions [53] , [54] , [55] , [56] . For example, collagen type II is the predominant component of the cartilage ECM, forms a fibrillar network primarily responsible for the mechanical integrity of the tissue and plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development and OA pathogenesis [ 57 , 58 ]. Loss of collagen type II has been shown to accelerate chondrocyte hypertrophy and OA progression, through the BMP-SMAD1 pathway [59] . Collagen type III is extensively crosslinked to collagen type II and regulates collagen fibrillar structure and biomechanics in cartilage tissue [ 60 , 61 ]. Collagen type VI is expressed in both healthy and OA cartilage tissues, is the major component of the chondrocyte pericellular matrix and enhances cartilage regeneration via stimulation of chondrocyte proliferation [ 62 , 63 ]. Collagen type IX covalently crosslinks to collagen type II with the collagenous (called COL3) and the non-collagenous (called NC4) domains of the molecules projecting at periodic distances away from the surface of the fibril. These projections allow it to interact with numerous components of cartilage tissue (e.g. cartilage oligomeric protein, heparin, fibromodulin), ultimately stabilising and organising the fibrillar collagen network in cartilage [64] , [65] , [66] , [67] , [68] . Collagen type X is a short chain, non-fibril-forming collagen, primarily synthesised by hypertrophic chondrocytes, that enables endochondral ossification by regulating matrix mineralisation and is essential for mesenchymal stem cell cartilage formation and endochondral ossification [69] , [70] , [71] , [72] , [73] . Collagen type XI interacts with various cartilage components (e.g. collagen type II, collagen type IX, perlecan, heparan sulphate) to form a meshwork that provides cartilage matrix stabilisation, mechanical resilience and homeostasis. The ratio of collagen type XI to collagen type II regulates fibre diameter, with thick fibres having more collagen type II [74] , [75] , [76] , [77] . Articular cartilage also contains a variety of PGs (e.g. aggrecan, decorin, biglycan and fibromodulin) and glycosaminoglycans (GAGs, e.g. chondroitin sulphate, keratan sulphate and hyaluronan) that represent ∼ 10% of tissue dry weight, subject to age and disease state. PGs and GAGs play significant role in both normal tissue function and arthritis manifestation and progression [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] , [89] . Indeed, physiological PGs and GAGs synthesis and composition contribute towards normal cartilage function and properties; control the release and protect against proteolysis of bounded cytokines, chemokines and growth factors; and modulate various signalling cascades that facilitate cell attachment and motility and cell-cell and cell-ECM interactions. For example, under physiological conditions, the major PG found in cartilage is aggrecan that interacts with hyaluronan to occupy the interfibrillar space of the cartilage ECM and provide cartilage with its osmotic properties to resist compressive loads [90] . On the other hand, in arthritis, PGs and GAGs are significantly degraded by matrix metalloproteinases and their breakdown products are released into synovial fluid, eliciting an inflammatory response [91] , [92] , [93] . Overall, ECM synthesis and degradation are regulated by the change of chondrocyte proliferation and metabolism under normal and OA conditions [94] , [95] , [96] , [97] , [98] ; the effect of hormones [99] , [100] , [101] and growth factors [102] , [103] , [104] ; aging [105] , [106] , [107] ; oxygen tension [108] , [109] , [110] , [111] ; and mechanical loading [112] , [113] , [114] , [115] , [116] .

Structurally speaking, articular cartilage is divided into the superficial, transitional, radial and calcified zones from the joint surface to the subchondral bone, with distinct composition and architectural features [117] , [118] , [119] , [120] . The superficial zone is the gliding surface of the joint; contains high concentration of collagen and low concentration of PGs; and adjoins a layer of elongated chondrocytes organised parallel to the articular surface. The transitional zone contains collagen fibres larger than those in the superficial zone, which are arranged randomly within this zone; is composed of spheroid-shaped chondrocytes; and has higher concentration of PGs compared to the superficial zone. The radial zone contains the largest collagen fibres, which are organised in a columnar pattern, perpendicularly to the joint surface; has the lowest concentration of chondrocytes; and has high PG content. Between the radial and the calcified zone, there is a wavy and irregular line, termed tidemark, that prevents the collagen fibres from being sheared of anchorage to the calcified zone. The calcified zone separates the radial zone of cartilage from subchondral bone ensuring a cohesive connection between them; has no PGs; and contains spheroid-shaped chondrocytes, which present a hypertrophic phenotype and synthesise collagen type X.

3. Collagen type II biosynthesis, extraction and synthesis via recombinant technologies

Collagen type II exhibits a triple stranded, coiled rod-like structure, is expressed as a homotrimer (i.e. [a1(II)] 3 ) and is synthesised exclusively by chondrocytes [ 121 , 122 ]. During synthesis, the procollagen chains undergo proline and lysine hydroxylation by prolyl-3-hydroxylase, prolyl-4-hydroxylase and lysyl hydroxylase [123] . The modification of proline and lysine hydroxylation requires ascorbic acid, iron and 2-oxo-glutarate. These steps occur prior to the formation of the triple helical structure as hydroxyproline is critical for the stabilisation of the collagen triple helix. Prolyl-4-hydroxylase triggers protein disulphide-isomerase activity, which leads to the formation of the collagen triple helix in the endoplasmic reticulum, as the association of collagen chains requires correct disulphide bond formation in the C-propeptide region of procollagen. Lysyl hydroxylase catalyses the hydroxylation of proline and lysine at both helical and non-helical regions of procollagen polypeptide chains. Moreover, some of the hydroxylysine residues then undergo glycosylation mediated by hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase [ 124 , 125 ]. Procollagen processing and crosslinking occurs in the extracellular space. The C-terminal and N-terminal non-helical propeptides of secreted procollagen molecules are removed by procollagen C-proteinases and members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases (N-proteinases). Consequently, the removal of propeptides results in a decreased solubility of procollagen molecules, which assemble into a collagen triple helical structure with a higher organisation. Subsequent intra- and inter- molecular crosslinking enables the formation of insoluble and closely packed fibrils of 50 nm in diameter, able to withhold mechanical loads [ 126 , 127 ].

Over the years, various tissues have been used and extraction protocols have been proposed to obtain collagen type II, albeit with variable degree of efficiency with respect to purity, from terrestrial and marine species. From the terrestrial species, bovine [ 128 , 129 ], porcine [ 130 , 131 ] and chicken [ 132 , 133 ] tissues are preferred for collagen type II extraction. It is interesting to note that it has been suggested that collagen type II retains memory of the tissue that is extracted from, with articular cartilage derived collagen type II to be more effective than auricular and tracheal cartilage derived collagen type II in inducing chondrogenic differentiation of human stem cells [131] . With respect to marine species, squid [134] , jellyfish [ 135 , 136 ], amur sturgeon [137] , hoki [138] and chondrichthyes (e.g. sharks [139] , [140] , [141] , skates [142] , rays [143] ), a diverse group of cartilaginous fish that lack true bone and exhibit a skeleton solely comprised of unmineralized cartilage, have been used for collagen type II extraction. In general, high yield, pure collagen type II preparations are produced by acid solubilisation, pepsin digestion and repeated salt precipitation / acid solubilisation and finally dialysis methods ( Fig. 2 ). Considering though the antibiotic usage in animal breeding, religious tenets and the potential for interspecies disease transmission [144] , [145] , [146] , recombinant collagen technologies have been developed for biomedical applications [147] , [148] , [149] . In this frontier, cells [150] , [151] , [152] , yeast [153] and baculovirus-silkworm [154] systems have been used to express procollagen type II. Compared with the yeast expression system, the insect cell expression system has lower background interference and facilitates post-translational processing and modification [ 154 , 155 ]. Nonetheless, recombinant technologies are still of low yield and are primarily utilised for niche biomedicine areas [156] .

Collagen type II extraction flow chart from cartilage tissues (the detailed protocol can be found here [ 131 , 143 ]) and electrophoretic mobility of collagen type I and collagen type II (the detailed protocol can be found here [223] ). Notes: As the same protocol is used to extract collagen type I [224] , [225] , [226] , attention should be paid during dissection to remove all not cartilaginous tissues. Tissue to acetic acid / pepsin solution ratio: 1:1 g/l. Tissue to pepsin ratio: 10:1 w/w. High activity pepsin (e.g. 3200-4500 units per mg protein) is recommended. Sieve: approximately 1,000 μm in diameter. Filter mesh: 100 μm in diameter. Centrifugation details: 20 min, 8000 rpm, < 8 °C. After the second NaCl precipitation, dissolution is conducted in minimum amount of acetic acid in order to produce a high in concentration collagen type II solution. All experiments are conducted at 4-8 ºC to avoid collagen denaturation.

4. Collagen type II scaffolds in cartilage engineering

Mammalian, marine and recombinant collagen type II-based hydrogels and sponges (primary scaffold conformations used in cartilage engineering) have been shown to both maintain and induce chondrogenic phenotype in vitro ( Table 1 ). To enhance mechanical integrity, crosslinking (e.g. poly(ethylene glycol) ether tetrasuccinimidyl glutarate [157] , carbodiimide [158] , [159] , [160] , genipin [161] ) and/or blending with other polymers (e.g. polyvinyl alcohol [162] , poly(L-lactide) and poly(lactide-co-glycolide) [ 163 , 164 ], chitosan [165] ) is traditionally employed. Although structural configurational differences between collagen type II hydrogels and sponges have been shown to induce different chondrocyte response (with respect to morphology, proliferation and gene expression), in the end, both scaffolds have been shown to stimulate comparable chondrogenesis [166] . It is also worth noting that collagen type II electrospun scaffolds have also been developed [167] , [168] , [169] , but the unavoidable denaturation of collagen prior or during the process [ 170 , 171 ] has restricted their use. Advances in engineering (e.g. bioprinted collagen type II hydrogels with cell density gradient [172] , alginate / collagen type II microbeads [173] , hyaluronic acid / collagen type II microspheres [174] ) and/or functionalisation technologies (e.g. chondroitin sulphate [ 175 , 176 ], hyaluronic acid [158] , glycosaminoglycan [177] ) have made available elegant collagen type II scaffolds with enhanced in vitro chondrogenic potential. In preclinical setting, collagen type II scaffolds (with / without functional molecules and/or with / without cells) have been shown to stimulate hyaline neocartilage formation in chondral and osteochondral defects of a diverse range of animal species ( Table 2 ).

Indicative examples of mammalian, marine and recombinant collagen type II scaffolds that have been shown to maintain and/or induce chondrogenic phenotype in vitro . Abbreviations: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-sulfo-N-hydroxy-succinimide: EDC-NHS; Adipose derived stem cells: ADSCs; Bone marrow stem cells: BMSCs; Cartilage oligomeric matrix protein: COMP; Chondroitin sulphate: CS; Dehydrothermal: DHT; Extracellular matrix: ECM; Fibroblast growth. factor: FGF; Glycosaminoglycan: GAG; Hyaluronan: HA; Insulin-like growth factor: IGF; Matrix metalloproteinase: MMP; Proteoglycan: PG; Sex-determining region Y-type box transcription factor 9: SOX9; Ultraviolet irradiation: UV.

Indicative examples of mammalian, marine and recombinant collagen type II scaffolds in preclinical models. Abbreviations: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-sulfo-N-hydroxy-succinimide: EDC-NHS; Adipose derived stem cells: ADSCs; Chondroitin sulphate: CS; Dehydrothermal: DHT; Bone marrow stem cells: BMSCs; Glycosaminoglycan: GAG; Osteoarthritis: OA; Ultraviolet irradiation: UV

Despite all the available data-to-date that have comprehensively shown the importance of collagen type II in chondrogenic induction or maintenance and in cartilage repair and regeneration, numerous studies still utilise collagen type I in cartilage engineering [178] , [179] , [180] , [181] . This is surprising, as the clear superiority of collagen type II over collagen type I in cartilage engineering has been well-documented in the literature, possibly due to biochemical signals (i.e. the lack of collagen type I and the presence of collagen type II and other bounded cartilage-specific constituents) [131] . In in vitro setting, for example, collagen type II, as opposed to collagen type I, scaffolds have been shown to maintain round chondrocyte morphology and to significantly increase DNA and collagen type II and GAG synthesis [ 182 , 183 ]. Collagen type II, as opposed to collagen type I, scaffolds have also been shown to more effectively induce chondrogenic induction of adipose derived stem cells [184] and bone marrow stem cells [ 175 , 185 ], as judged by round cell morphology (via the integrin β1-mediated Rho A/Rock signalling pathway [184] ), upregulation of chondrogenic genes (e.g. collagen type II, collagen type X, aggrecan, COMP, SOX6, SOX9) and increased synthesis of cartilage matrix (e.g. collagen type II, PG, GAG). It is also worth noting that increased collagen type II, as opposed to collagen type I, scaffolds induced differentiation of adipose derived stem cells to nucleus pulposus cells, as judged by increased SOX9, aggrecan and collagen type II gene expression; increased sulphated PG synthesis; expression of KRT19 marker; and increased phosphorylated Smad3 expression [186] . These in vitro observations were also verified in preclinical models. For example, in cartilage defects in the femurs of rabbits, 13 weeks post implantation, collagen type I / collagen type II hydrogels showed statistically higher cartilage repair score than either collagen type I alone hydrogels (both loaded with bone marrows stem cells) or empty defect controls (pure collagen type II hydrogels were not used) [187] . In full-thickness defects in the femoral trochlea of adolescent rabbits, although collagen type I scaffolds induced higher than collagen type II scaffolds cell migration into the defect, the collagen type II scaffolds more effectively than collagen type I scaffolds directed invaded cells towards chondrocyte phenotype and 12 weeks post implantation, the cartilage contours in defects with collagen type I scaffolds were repaired with fibro-cartilage tissue, whilst defects treated with collagen type II scaffolds, although the original contour was not completely restored in all animals, showed an increase in the amount of superficial cartilage-like tissue [188] . In the trochlea grooves of the knees of dogs, 15 weeks post implantation, groups treated with collagen type II scaffolds and chondrocytes showed the greatest total amount of reparative tissue and the tissue at subchondral region of defects was positive for collagen type II, GAGs and PGs [189] . In a 4-month-old domestic pig full-thickness cartilage lesion model, 4 months post operation, a human recombinant collagen type II / polylactide scaffold most frequently formed hyaline cartilage than the spontaneous healing group and a collagen type I / collagen type III scaffold [190] .

In the commercial arena, it is interesting to note that, to the best of our knowledge, only a handful of companies provide high purity collagen type II (e.g. porcine articular cartilage derived collagen type II, Symatese) and that no single collagen type II-based device is available, whilst numerous collagen type I devices are available for cartilage engineering (e.g. Chondro-Gide®, Geistlich Pharma AG; Novocart® Basic, TETEC AG; MeRG®, Bioteck Srl), despite the fact that collagen type I scaffolds have failed to demonstrate efficiency in healing of human osteochondral [43] and large cartilage [42] defects. This limited technology transfer of collagen type II can be attributed to two main reasons. Firstly, we believe that commercialisation of collagen type II-based devices has been compromised by early studies that showed native collagen type II from human, chick, murine and bovine cartilage to induce inflammatory arthritis in rats [191] , [192] , [193] and in non-human primates [194] ; and antibodies of native and denatured collagen type II to be present in patients with early rheumatoid arthritis and chronic gouty arthritis [195] , [196] , [197] . It is worth noting though that effectively crosslinked collagen type II does not induce arthritis in rats [198] and studies have demonstrated collagen type II devices to promote efficient defect filling and hyaline neocartilage formation. For example, 15 weeks post operation, defects in trochlear grooves of adult dogs (that were treated with microfracture, microfracture with collagen type II scaffold and collagen type II scaffold loaded chondrocytes) resulted in 56% to 86% total defect filling, with the microfracture with collagen type II scaffold treatment group inducing the highest defect filling capacity [199] . When collagen type II scaffold were seeded for 4 weeks with chondrocytes (after 3 weeks of monolayer expansion) and then implanted in a canine trochlear groove defect model, 15 weeks post implantation, although the repaired tissue formed had significantly lower compressive stiffness than the native cartilage, the total defect filling ranged from 70% to 100%, with hyaline cartilage accounting for 42 ± 10% of the defect area [200] . The second issue that may be responsible for the limited use of collagen type II in medical device development is the difficulty in producing high amounts of high purity and high yield, all in comparison to collagen type I, collagen type II preparations. Obviously, the main reason behind this is the articular cartilage tissue availability, in comparison to skin, for example, tissue. Having said that, a typical cartilage defect is a lot smaller than a typical skin defect and therefore extraction of collagen type II constitutes a value for money proposition.

5. Conclusions

Cartilage injuries and pathophysiologies continuously increase and financially drain healthcare systems worldwide. In the quest of the optimal building block for cartilage engineering scaffolds, collagen type II has been overlooked due to either outdated data or economic drivers, despite being the most abundant extracellular matrix component of cartilage. This review clearly illustrates the beneficial effects of collagen type II in cartilage engineering and urges the adaptation of a more rational and biomimetic approach in designing biomaterial-based therapeutic strategies for functional cartilage repair and regeneration.

Conflict of Interest

The authors declare no conflict of interest.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work forms part of the Teagasc Walsh Fellowship (grant agreement No. 2014045) and the ReValueProtein Research Project (grant agreement No. 11/F/043) supported by the Department of Agriculture, Food and the Marine under the National Development Plan 2007-2013 funded by the Irish Government. This publication has emanated from research supported by grants from Science Foundation Ireland (SFI) under grant numbers 15/CDA/3629 and 19/FFP/6982 and Science Foundation Ireland (SFI) and European Regional Development Fund (ERDF) under grant number 13/RC/2073_2. This work has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme, grant agreement No. 866126.

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The EMA says it is analysing about 150 reports of possible cases of self-injury and suicidal thoughts . This doesn’t mean the medicines caused these effects, however, only that people reported these experiences after starting to take them. “More work is needed to determine if a causal link exists,” says Michael Schwartz at the University of Washington in Seattle.

A spokesperson at Novo Nordisk, the manufacturer of liraglutide and the semaglutide drugs Ozempic and Wegovy, told New Scientist : “GLP-1 receptor agonists have been used to treat type 2 diabetes for more than 15 years and for treatment of obesity for eight years. The safety data collected from large clinical trial programmes and post marketing surveillance have not demonstrated a causal association between semaglutide or liraglutide and suicidal and self-harming thoughts.”

Need a listening ear? UK Samaritans : 116123; US 988 Suicide & Crisis Lifeline : 988; hotlines in other countries.

Do these drugs help with addictions?

Perhaps. There have been many anecdotal reports of people taking these medicines for diabetes or weight control who lose their urge to drink alcohol or see waning of other habits that could be described as “behavioural addictions”, such as compulsive shopping.

This is supported by research in animals that found the GLP-1 analogues lower consumption of alcohol and addictive drugs. A small trial in people has hinted at a similar effect from a GLP-1 analogue called exenatide, which reduced heavy drinking, but only in people who were obese .

The explanation could be connected with the way the drugs act on the brain to reduce food cravings, but the exact mechanism is unclear. We are only at the beginning of understanding how these treatments could support people with alcohol and drug additions, says Daniel Drucker at Sinai Health in Toronto, Canada. “The clinical trial data is not yet in to substantiate the anecdotes.”

What happens to a user’s weight long-term?

In an aforementioned study, taking semaglutide once a week led to people losing about 15 per cent of their body weight, compared with 2 per cent for those taking a placebo. Carried out in 2022, this two-year trial looked at people who were obese or overweight, but didn’t have diabetes.

In most countries, Wegovy has been approved for use for two years, but if people stop taking the drug, they generally regain the lost weight – two-thirds of it after one year, according to one trial .

But in 2024, Donna Ryan at the Pennington Biomedical Research Center in Louisiana and her colleagues found that taking the same dose of semaglutide once a week for four years led to reductions in weight that were maintained for the entire period.

The team compared more than 17,000 adults in 41 countries who were obese or overweight and didn’t have diabetes, but did have a form of heart disease. Among those taking semaglutide, weight loss continued to week 65 and was sustained over the trial , with the participants’ losing on average 10.2 per cent of their body weight, compared with 1.5 per cent in the placebo group.

Beyond Wegovy: Could the next wave of weight-loss drugs end obesity?

“Our long-term analysis of semaglutide establishes that clinically relevant weight loss can be sustained for up to four years in a geographically and racially diverse population of adults with overweight and obesity but not diabetes,” said Ryan in a statement.

It has been suggested that using semaglutide for weight loss needs to be a long-term approach. “The weight loss is only sustained for as long as the drug is taken because as soon as you stop the drug, all of those physiological processes that are trying to get your body weight back up kick in again,” says Cork.

The results have led to calls to extend Wegovy’s treatment to at least four years. Under the current two-year approval, there will probably be demand from people for doctors to continue prescribing the drug “off-label”. “I think there’ll be a big push to try to change those guidelines,” says Cork. Ozempic can be prescribed long-term for type 2 diabetes because the condition is usually life-long.

Do these drugs reduce the risk of heart-related events?

It has been suggested that by bringing people’s weight towards a healthier range, these medications may inadvertently reduce the risk of heart-related events, such as heart attacks and strokes.

Based on the same dataset used by Ryan and her colleagues, a team at University College London examined the relationship between a change in weight and cardiovascular outcomes. Presenting their results at the European Congress on Obesity in Venice, Italy, the researchers found that semaglutide led to a 20 per cent reduction in such events, irrespective of people’s starting weight or the amount they lost.

“Our findings show that the magnitude of this treatment effect with semaglutide is independent of the amount of weight lost, suggesting that the drug has other actions which lower cardiovascular risk beyond reducing unhealthy body fat,” said team member John Deanfield in a statement. “These alternative mechanisms may include positive impacts on blood sugar, blood pressure, or inflammation, as well as direct effects on the heart muscle and blood vessels, or a combination of one or more of these.”

The results suggest that semaglutide could reduce the risk of heart-related events among people who are just mildly overweight. However, the study wasn’t designed to measure its effect as a preventative treatment. Further research is also required among an even more racially diverse group of people.

• weight loss

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