Table 1.

Synthesis of preclinical studies on collagen derivatives in cartilage repair

First author (year)	Country of study center	Objective of the study	Animal model/sample size	Intervention/control	Admin. route/dosage	Source of collagen/molecular weight
In vitro studies
Boonmaleerat (2018) [41]	Thailand	To investigate the effects of different sizes of fish CH on both physiological and pathological conditions	Porcine articular cartilage explants/3 explant pieces, 10 mg each, cultured in a 24-well culture plate	1) Fish collagen hydrolysate (FCH) with a MW of < 3 kDa 2) FCH with a MW of 3–10 kDa 3) FCH with a MW > 10 kDa vs. Control (DMEM)	100 μg/ml of each collagen fraction (in physiological and pathological conditions)	Fish (skin and scales of tilapia fish [Oreochromis niloticus]) / 1) < 3 kDa 2) 3–10 kDa 3) > 10 kDa
Furuzawa-Carballeda (2009) [42]	Mexico	To evaluate the effect of polymerized-collagen in co-cultures of cartilage and synovial tissue obtained from patients with knee OA	Synovium and cartilage from five patients with knee OA 60 explants (2 × 15 active and control)	1% dialyzed polymerized-collagen vs. No treatment (absence of 1% dialyzed polymerized-collagen)	0.6 µg ml−1 of collagen	Porcine (porcine type I dermal collagen)/NA
Schadow (2013) [43]	Germany	To evaluate for the first time whether different bovine CH preparations indeed modulate the metabolism of collagen and proteoglycans from human OA cartilage explants and determined the chemical composition of oligopeptides representing collagen fragments	Articular cartilage of OA patients/ Collagen biosynthesis experiments: N = 12; Cartilage degradation experiments: N = 5; N = 6	Collagen hydrolysates (3 different preparations – RDH; RDH-N; CH-Alpha® –) / Untreated explants (controls)	0–10 mg/ml collagen hydrolysates	Bovine origin/ 1) RDH (3,500 Da) 2) RDH-N (3,250 Da) 3) CH-Alpha® (3,300 Da)
Schadow (2017) [44]	Germany	To analyze commercially available fish and porcine CHs. Specifically, to examine whether these CHs: (a) modulate the synthesis of type II collagen and inhibit the loss of proteoglycan from human OA knee cartilage; (b) affect IL-6 trans-signaling and the aggrecanases and MMPs that mediate the degradation of cartilage in OA; and (c) determine their peptide composition	Articular cartilage of OA patients/ Collagen biosynthesis experiments: n = 4–5 Cartilage degradation experiments: n = 6	Collagen hydrolysates (3 different preparations : Mobiforte®; FGH; FGH-N) / Untreated controls from the same joint	0–10 mg/ml CH	Porcine origin/ Mobiforte® (3.120 Da) Fish origin/ Peptan® F 2000 (2.000 Da) Fish origin/ Peptan® F 5000 (5.000 Da)
In vivo studies
Bagi (2017) [25]	USA	To test the ability of undenatured native chicken type II collagen administered orally at the time of cartilage injury imposed by PMMT to prevent the excessive deterioration and improve the healing of articular cartilage	Lewis rats/ 1) PMMT/ UC-II (Active): 10 2) PMMT/ vehicle: 10 3) Naïve rats (intact control): 10 4) Sham surgery: 10	Undenatured native type II collagen (UC-II) vs 1) Vehicle treatment; 2) Intact control 3) Sham surgery control (all with no treatment)	Oral/ 0.66 mg/kg/day	Chicken/NA
Dai (2018) [45]	China	To develop a novel type II collagen that is cost-effective, non-immunogenic, and might have an anti-inflammatory effect on OA. (…). Finally, rat models with surgery-induced OA were used to evaluate the OA-relieving effect of SCII to mimic the minimally invasive therapy in clinics	Sprague–Dawley (SD) rats/ 24 male adult rats divided into 4 groups (3 with induced OA, 1 sham group)	Squid collagen type II (for two ACLT + pMMx groups – different dosages) vs Phosphate buffered saline (sham group and ACLT + pMMx group)	Intra-articular injection/ SCII 3 mg/ml or SCII 10 mg/ml, (100 μl/joint cavity) once a week, for 5 weeks	Squid cartilage/110 kDa
Dai (2018) [26]	China	To evaluate a newly developed squid type II collagen (SCII) for repairing OA-induced cartilage lesions	Male SD rats/18 SD rats, 12 of which received ACLT + pMMx surgery to induce OA (the other 6 rats received sham surgery)	Squid collagen type II (ACLT + pMMx + SCII group) vs Phosphate buffered saline (for sham surgery group and ACLT + pMMx group)	Intra-articular injection/ 10 mg/ml SCII (100 μl/joint cavity), once a week, for 5 weeks	Squid cartilage/ NA
Dar (2017) [46]	USA	To examine the impact of orally administered hydrolyzed type 1 collagen (hCol1) in a model of posttraumatic OA (PTOA)	Male C57BL/6J mice/4 groups of 6 mice each (2 active and 2 control groups)	Hydrolyzed type 1 collagen (incorporated into hazelnut cream) vs Hazelnut cream vehicle alone (Nutella®)	Oral/ Low dose (LD, 3.8 mg) or high dose (HD, 38 mg) hCol1, once daily	Bovine/ 2000 Da
Di Cesare Mannelli (2013) [47]	Italy	To evaluate the role of low doses of native type II collagen in the rat model of OA induced by sodium monoiodoacetate (MIA)	Male Sprague–Dawley rats/ 4 groups of 12 animals each	Native type II collagen vs CMC (vehicle solution)	Oral/ 1, 3, or 10 mg kg−1 collagen daily	Porcine
Hashida (2003) [48]	Japan	To investigate the effects of orally administered collagen and glucosamine on cartilage repair	Rabbits N = 27; divided into two groups of 2- and 3-week experimental duration G1: 12 G2: 15 Each group was divided into sub-groups of 3 rabbits	Water-soluble collagen (S-collagen) vs 1) D-glucosamine 2) S-collagen + D-glucosamine (CoG group) 3) Control 4) glycine (only for the 3 weeks group)	Oral/ 8 ml of S-collagen per day (2.4 g as collagen content). The CoG group had 8 ml of S-collagen and 1 g of d-glucosamine every day	Cocks comb (degraded by proteinase to various sizes of peptide)/500, 1000, and 10,000 Da
Isaka (2017) [49]	Japan	To investigate the effect of CP on articular cartilage in OA by measuring levels of serum biomarkers for type II collagen, (…), as well as the histopathological changes occurring, in experimental ACLT model rats. Additionally, the effect of CP on type II collagen degradation and MMP-13 expression was investigated using immunohistochemical staining	Sprague–Dawley rats/ Total: 28, 7 per group, 4 groups 1) Control group 2) Sham - operated group 3) ACLT group without CP 4) ACLT group with CP	Collagen peptides (in standard laboratory diet) vs. Standard laboratory diet without collagen peptides (for control, sham- operated and ACLT without CP groups)	Oral/ 5.0–6.7 g/kg/day CP (determined based on the body weight and food intake)	NA/NA
Nakatani (2009) [50]	Japan	To examine the protective effect of CH and its specific dipeptide, Pro-Hyp (prolyl-hydroxyproline), in relation to primary degenerative articular cartilage of mice	Mice (Ten-week-old male C57BL/6J mice)/ 4 groups of six mice each (total: 24):	Collagen hydrolysate (CH contained approximately 6% Pro-Hyp) in diet/ vs 1) Standard diet 2) Gluten (in diet) 3) Pro-Hyp (in diet)	Oral/ 5 g porcine Skin gelatin/100 g of diet	Porcine skin gelatin/ Average molecular weight of 5000 Da
Naraoka (2013) [51]	Japan	First, to examine the therapeutic effects of collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with hyaluronan (HA)	Female Japanese white rabbits/ Four groups of 18 rabbits each (total: 72)	Collagen tripeptide (Ctp) vs 1) Normal saline 2) HA 3) Ctp + HA	Intra-articular injection/ 3.0 mg/ml Ctp dissolved in saline; or 3.0 mg/ml Ctp/HA in which Ctp was dissolved in HA solution	From gelatin derived from pig skin containing type I and type III collagen/ NA
Ohara (2010) [52]	Japan	To study the effects of Pro-Hyp on glycosaminoglycans synthesis using in vitro cultured synovium cells and oral ingestion of collagen hydrolysates in a guinea pig model of OA	Dunkin-Hartley guinea pigs/ G1: 12 (Distilled water) G2: 12 (fish scale type I CH) G3: 10 (porcine skin type I CH)	Fish scale type I collagen hydrolysates & Porcine skin type I collagen hydrolysates vs. Distilled water	Oral/ 0.84 g/kg/day (5 days per week)	Fish scale & porcine skin/ NA
Ohnishi (2013) [53]	Japan	To investigate the correlation between OA severity and concentration of serum biomarkers such as keratan sulfate (KS), hyaluronic acid (HA), and chondroitin sulfate (CS) 846 epitope. To research the effect of glucosamine and fish collagen peptide (FCP) on ACLT in rabbits. To assess the correlations between serum biomarkers and histological findings	Rabbits (female Japanese albino)/ 4 groups of 3 rabbits each	Fish collagen peptides (FCP) vs. 1) Tap water (control) 2) Glucosamine 3) FCP and glucosamine	Oral/ 1.0 g of powdered FCP/day or 1.0 g of FCP and 1.0 g of glucosamine daily	Fish collagen peptides (FCP) from skins of Gadiformes species/ Average molecular weight of 3000 Da
Xu (2007) [54]	China	To explore therapeutic effects of oral chicken collagen type II (CCII) on rat OA and analyze concomitant changes in the level of MMP-13, MMP-9, cathepsin K and their mRNA level as well as tissue inhibitor of matrix metalloproteinase (TIMP)1 mRNA level in articular cartilage of osteoarthritic rats	Wistar rats/ Total: 132 G1: 36 (OA rats—placebo) G2: 24 (Collagen 20 µg/day) G3: 24 (Collagen 80 µg/day) G4: 24 (Excipient) G5: 24 (Rats without OA—placebo)	Chicken collagen type II (CCII) vs. 1) Placebo (saline solution) 2) Excipient (0.25% mannitol)	Oral/ 20 µg/day (0.002% CCII solution 1 ml) or 80 µg/day (0.008% CCII solution 1 ml)	Chicken/ NA

First author (year)	Brand name (manufacturer)	Funding source or sponsor of the study	Treatment or study Duration	Authors' conclusion	Aeras of uncertainty/future directions of research, as suggested by the authors of the publications
In vitro studies
Boonmaleerat (2018) [41]	NA/ (Hainan Huayan Biotech Co., Ltd., Haikou, China)	Thailand Center of Excellence for Innovation in Chemistry (PERCH-CIC) for the financial support	28 days	All three fractions FCH had no effect on cartilage metabolism in physiological condition, but small and medium fractions had adverse effect on cartilage in pathological condition. Taken together, various sizes of FCH showed different effects on cartilage metabolism. Therefore, different sizes of FCH play different roles on cartilage metabolism, especially in the pathological condition	The different sizes of FCH had different effects on gene and protein expression in both physiological and pathological studies on the chondrocyte metabolism. Their ability to induce cellular signaling is an interesting area for investigation
Furuzawa-Carballeda (2009) [42]	NA	Supported by grants from The National Council of Science and Technology	7 days	The addition of polymerized-type I collagen to cartilage and synovial tissue co-cultures induced up-regulation of chondrocytes proliferation and cartilage extracellular matrix proteins production (COMP, type II collagen and proteoglycans) as well as an anti-inflammatory cytokine (IL-10) and the down-modulation of pro-inflammatory cytokines (IL-1β and TNF-α). It is possible that this mechanism might contribute to induce tissue regeneration and down-regulation of inflammation in OA	NA
Schadow (2013) [43]	RDH, Peptan™ B 5000 & RDH-N, Peptan™ B 2000 (Rousselot SAS, Puteaux, France); CH-Alpha® (Gelita Health Products GmbH, Eberbach, Germany)	Supported in part by a grant of the DRB-Foundation. No additional external funding received for this study	Collagen biosynthesis: 24 h Cartilage degradation in the absence of 5.0 ng/ml IL-1β: 6 days; In the presence of IL-1β and NO: 3 days	Our study has clearly elaborated for the first time that CHs from various sources differ significantly with respect to both their chemical composition of oligopeptides representing collagen fragments as well as their effects on human articular cartilage. Since marked effects on human chondrocytes were observed, depending on the CHs preparation investigated, our in vitro study indicates that CHs used as nutraceuticals might be either ineffective or even detrimental to OA cartilage	Metabolized collagen fragments or other collagen hydrolysate preparations might contain therapeutically useful peptides. Thus, their biomedical properties have to be studied thoroughly both in vitro and in animal as well as clinical trials before being applied as safe and effective nutraceuticals in patients
Schadow (2017) [44]	Mobiforte® (Astrid Twardy GmbH, Unterföhring, Germany) FGH, Peptan® F 5000; & FGH-N, Peptan® F 2000 (Rousselot SAS, Puteaux, France)	Supported in part by a grant from the DRB foundation, European Structural Funds Grant 26,220,220,005 and VEGA Agency Grant 2/0145/17	Collagen synthesis: 24 h Cartilage degradation: 6 days	Based on our current and earlier [Schadow, 2013] in vitro findings, we conclude that: (a) the term “collagen hydrolysate” is the generic name of a heterogeneous group of nonfibrillating collagenous peptide mixtures; and (b) CHs do not stimulate type II collagen biosynthesis in human articular cartilage. Further, due to the high variability in peptide composition between CH preparations, no effect can be extrapolated from a CH to another mixture. Thus, each orally administered CH preparation must be carefully analyzed in vitro and in vivo regarding pleiotropic effects before this peptide mixture can be attested to be an effective and safe nutraceutical for patients	The distinct activities on OA cartilage are attributed to the differences of the peptide composition, and the extent to which a single oligopeptide or a combination of oligopeptides, aggregates, and metabolized peptides contributes in vivo to the major components of joint tissues remains to be determined
In vivo studies
Bagi (2017) [25]	UC-II® (InterHealth, Benicia, CA)	Supported by Pfizer Consumer Healthcare	8 weeks	Study results demonstrate that a clinically relevant daily dose of UC-II when applied immediately after injury can improve the mechanical function of the injured knee and prevent excessive deterioration of articular cartilage	Better address joint functionality and impact of disuse and load bearing on cartilage metabolism, use of radiolabeled compound to assess metabolism and tissue distribution of UC-II and use of adequate immunological, histo-chemical and molecular methods to address some of the lingering questions regarding mechanism of action of “slow-acting” product such as UC-II
Dai (2018) [45]	NA (The authors, using Cartilage of Peru squid provided by Shanghai Fisheries (The authors Research Institute)	Supported by grants from National Natural Science Foundation of China, and Shanghai Sci-Tech Committee Foundation	5 weeks	These data suggested that the newly developed SCII could not only avoid the immunogenic risks of collagen derived from terrestrial animals, but more importantly, provide new choice for the control and treatment of OA	NA
Dai (2018) [26]	NA (The authors, using cartilage of Peru squid provided by Shanghai Fisheries Research Institute)	Supported by grants from National Natural Science Foundation of China, Shanghai Sci-Tech Committee Foundation-Pujiang Program, and National Key Research and Development Program of China	5 weeks	Our findings show that SCII immunomodulates M2 activation of macrophages to skew the local OA microenvironment towards a pro-chondrogenic atmosphere, and promotes cartilage repair under inflammatory condition. It shows great potential for SCII to be a novel biomaterial for cartilage repair in OA	NA
Dar (2017) [46]	Peptan® B2000, Rousselot	Supported by Rousselot BVBA, and grants from NIH/NIAMS	16 weeks	Overall, these results suggest that hCol1 is chondroprotective and anti-inflammatory in posttraumatic OA, setting the stage for further mechanistic study and evaluation of joint structural modifications and potential disease-modifying effects in a human clinical trial	An open question that is yet to be answered for hCol1 and other nutraceuticals that are purported to be joint protective, including type 2 collagen-based preparations, glucosamine and chondroitin sulfate, relates to their mechanism of action
Di Cesare Mannelli (2013) [47]	NA (Bioiberica, Spain)	Funded by the Italian Ministry of Instruction, University and Research and by the University of Florence	13 days	These results describe the preclinical efficacy of low dosages of native type II collagen as pain reliever by a mechanism that involves a protective effect on cartilage	This evidence highlights the interest for further investigation about the mechanism of low dose collagen and its relevance in osteoarthritis therapy
Hashida (2003) [48]	(Nippon Ham Packers, CO., Tokyo)	NA	2 and 3 weeks	GlcN and the collagen peptides were effective not only for cartilage damage repair, but also for increasing normal cartilage proteoglycan and glycosamino-glycan content. These effects were not observed for the administration of D-glucose, and only a partial effect was observed for glycine, which is the main component amino acid of the collagen peptides. Simultaneous administration of collagen and GlcN gave a fairly enhanced healing on restoration of cartilage injuries	Further investigation is required to determine the effect of each amino acid of collagen on cartilage repair
Isaka (2017) [49]	NA/ (Nitta Gelatin, Inc., Osaka, Japan)	Supported in part by a grant from the Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science and Technology, Japan	8 weeks	The results of the present study suggest that CP has the potential to exert chondroprotective action on OA by inhibiting MMP-13 expression and type II collagen degeneration	Further studies investigating the components of CP, such as Pro-Hyp, are required to elucidate the detailed mechanism behind the beneficial effect of CP on joint health
Nakatani (2009) [50]	NA/ (Nitta Gelatin; Osaka, Japan)	NA	3 weeks	This study implies that the bioactive peptide, Pro-Hyp, is derived not only from collagen in living tissues but also from dietary supplements such as gelatin, and that it functions in target tissues	Further studies are necessary to investigate the effects of CH supplements. Also, understanding the mechanism of action of CH on chondrocyte differentiation would provide a rational basis for the development of chondroprotective therapies for damaged joints
Naraoka (2013) [51]	NA/ (Central Research Institute, Jellice Corp.; Sendai, Japan)	NA	15 weeks	Periodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be exerted by promotion of type II collagen synthesis predominantly	Further examination is needed to determine the optimal dose, frequency and duration of Ctp injection therapy for the OA knee, based on in vitro studies and several additional in vivo studies
Ohara (2010) [52]	NA/ (Nitta Gelatin, Osaka, Japan)	NA	4 weeks	These results suggest that food-derived Hyp-containing peptides can affect the PGs and morphological changes associated with osteoarthritic cartilage, which might be mediated by stimulation of hyaluronic acid production in the synovium	NA
Ohnishi (2013) [53]	NA/ (Yaizu Suisankagaku Industry Co., Ltd., Shizuoka, Japan)	NA	4 weeks	From our present results, oral administration of FCP and/or glucosamine effectively controlled cartilage degradation in an ACLT model. Estimation of various biomarkers for arthritis will be useful for assessing the progression of cartilaginous degradation; in this present study, the detected levels of HA and CS 846 correlated with the histological findings, suggesting that estimation of HA and CS 846 might be useful for monitoring OA progression. However, there were no significant differences between the control and treatment group concentrations	These results indicate that there are individual differences for each biomarker; therefore, a longer-term experiment should be conducted to evaluate the significance of the biomarkers in the ACLT model. Our results indicate the possibility that the measured concentrations of biomarkers can be used in addition to histological findings to evaluate cartilage injury
Xu (2007) [54]	NA/ (Shanghai institute of herbal biology, Shanghai, China)	NA	8 weeks	Oral CCII reduced articular cartilage degradation of osteoarthritic rats and may probably be a potent drug candidate for OA treatment	The exact mechanism by which CCII reduced the OA cartilage damage remains uncertain and cannot be answered within the scope of this study

ACLT anterior cruciate ligament transection, Admin. administration, CH collagen hydrolysate, CMC 1% carboxymethylcellulose sodium salt, CP collagen peptides, GlcN d-glucosamine, MMP matrix metalloproteinase, MW molecular weight, NA information not available, OA osteoarthritis, PMMT partial medial meniscectomy tear, pMMx partial medial meniscectomy, SCII squid type II collagen