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RESEARCH ARTICLE
Daily oral consumption of hydrolyzed type 1
collagen is chondroprotective and anti-
inflammatory in murine posttraumatic
osteoarthritis
Qurratul-Ain Dar
1,2☯
, Eric M. Schott
1,3☯
, Sarah E. Catheline
1,3☯
, Robert D. Maynard
1,3
,
Zhaoyang Liu
1
, Fadia Kamal
1,4
, Christopher W. Farnsworth
1,3
, John P. Ketz
1,4
, Robert
A. Mooney
1,3
, Matthew J. Hilton
5
, Jennifer H. Jonason
1,4
, Janne Prawitt
6
, Michael
J. Zuscik
1,4
*
1Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York,
United States of America, 2Department of Biology, University of Rochester, Rochester, New York, United
States of America, 3Department of Pathology and Laboratory Medicine, University of Rochester Medical
Center, Rochester, New York, United States of America, 4Department of Orthopaedics & Rehabilitation,
University of Rochester Medical Center, Rochester, New York, United States of America, 5Orthopaedic
Cellular, Developmental & Genome Laboratories, Duke University, Durham, North Carolina, United States of
America, 6Rousselot BVBA, Gent, Belgium
☯These authors contributed equally to this work.
*michael_zuscik@urmc.rochester.edu
Abstract
Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying
therapies. Thus, strategies that offer chondroprotective or regenerative capability represent
a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1)
preparation has been reported to reduce pain in human OA and support a positive influence
on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we
examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA).
In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supple-
ment of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various
time points post-injury, hydroxyproline (hProline) assays were performed on blood samples
to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were
performed, including histomorphometry, OARSI and synovial scoring, immunohistochemis-
try and mRNA expression studies. Confirming ingestion of the supplements, serum hProline
levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented
mice, chondroprotective effects were observed in injured knee joints, with dose-dependent
increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks
post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with
reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also
displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting
an anti-inflammatory effect. These findings establish that in the context of murine knee
PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chon-
drocytes, and anti-inflammatory. While the underlying mechanism driving these effects is
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 1 / 24
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OPEN ACCESS
Citation: Dar Q-A, Schott EM, Catheline SE,
Maynard RD, Liu Z, Kamal F, et al. (2017) Daily oral
consumption of hydrolyzed type 1 collagen is
chondroprotective and anti-inflammatory in murine
posttraumatic osteoarthritis. PLoS ONE 12(4):
e0174705. https://doi.org/10.1371/journal.
pone.0174705
Editor: Sudha Agarwal, Ohio State University,
UNITED STATES
Received: August 22, 2016
Accepted: March 14, 2017
Published: April 6, 2017
Copyright: ©2017 Dar et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by Rousselot
BVBA contract number 057066 (MJZ, study
design, data collection, data analysis, data
interpretation, writing), NIH/NIAMS 5P50
AR054041-5471 (MJZ, study design, data
interpretation, writing), NIH/NIAMS 5R01
AR063071 (MJH, study design), NIH/NIAMS 5T32
AR053459 (data collection, data analysis, data
yet to be determined, these findings provide the first tissue and cellular level information
explaining the already published evidence of symptom relief supported by hCol1 in human
knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the
context of PTOA.
Introduction
Osteoarthritis (OA) is one of the most prevalent diseases in the world, with recent estimates
projecting that >250 million people are afflicted globally [1]. In the US, OA afflicts 35 million
people [2], with diarthrodial and spinal OA being the most prevalent disease, surpassing the
next top four medical disorders combined (heart, pulmonary, mental health and diabetic con-
ditions) [3]. In a recent analysis, global medical costs for lower extremity OA exceed $350 bil-
lion/year, with the reduced quality of life and physical function of OA patients exerting an
additional hidden economic impact that surpasses $50 billion/year [4]. Despite these statistics,
there are no disease modifying therapies available, with management of OA patients consisting
only of symptom palliation [5,6] via nonsteroidal anti-inflammatory drugs, intraarticular
injection of corticosteroids or hyaluronic acid, narcotic-based analgesia including opioids, and
joint arthroplasty. Thus, the development of therapeutic strategies that offer protective and/or
regenerative capability is a critical unmet need and central pursuit in the OA field.
OA is a joint disease of multifactorial etiology characterized by degeneration and loss of
articular cartilage and meniscus, subchondral bone sclerosis, osteophyte formation, and syno-
vial hyperplasia [7–9]. Etiologic complexity and ‘whole organ’ involvement of multiple tissues
within the OA joint during the degenerative process represent significant challenges in the
development of disease modifying therapeutic strategies. Over the past two decades, more
than a dozen human clinical trials have been performed to test candidate disease modifying
OA drugs (DMOADs), none of which have emerged to be accepted as a bona fide therapeutic
agent [10,11]. This list includes orally administered nutraceutical agents comprised of carti-
lage matrix components that are widely marketed as joint protective. In the case of chondroitin
sulfate and glucosamine, the two most widely available and top selling formulations, mixed
clinical trial results leave open the question of their efficacy [12].
An agent with a reported positive influence on chondrocyte function and potential disease
modifying capability in OA is hydrolyzed type 1 collagen (referred to in this report as hCol1).
hCol1 is a mixture of type I collagen peptides of different molecular weights that are generated
via enzymatic digestion of type I collagen extracted from animal connective tissues. The pep-
tide mixture, which contains an abundance of hydroxyproline, proline and glycine, is absorbed
dose-dependently following bolus oral delivery [13], with a series of di- and tri-peptides peak-
ing in the circulation within one hour after consumption in humans [14]. hCol1 is considered
safe as an oral supplement [15], and when consumed daily, beneficial effects have been
observed in bone [16] and skin [17,18]. Recent data suggesting that hCol1 has chondrogenic
effects in vitro [19] is consistent with the growing consensus that collagen derivatives may be
chondroprotective in OA [20] and aligns with recent clinical evidence of its symptom-relieving
effects in human subjects suffering from knee joint pain [21]. Overall, based on this body of
data, in this study we posed the hypothesis that daily oral supplementation with hCol1 will
have protective effects in joints undergoing the OA degenerative process. To address this
hypothesis and characterize the joint protective effect(s) of hCol1, we conducted a preclinical
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 2 / 24
interpretation), and NIH/NIAMS Core Center Grant
1P30 AR069655 (data collection).
Competing interests: A research contract was
struck with Rousselot BVBA that provided partial
financial support for the research that was
performed in this study. Co-author Dr. Janne
Prawitt, employee of Rousselot BVBA is a scientific
contributor to the work, as delineated in the CRediT
& Author Contributions section in the submission
system. Prior to completion of peer review, a
provisional patent was filed protecting intellectual
property related to findings presented in this
report. The research contract between Rousselot
BVBA and the University of Rochester does not
alter our adherence to PLOS ONE policies on
sharing data and materials; i.e., there are no
restrictions on sharing of data and/or materials.
study examining the impact of this agent, at the tissue and cellular level, in an established
mouse model of posttraumatic knee OA (PTOA).
Materials and methods
Animals
All handling of mice and in vivo experimental procedures performed in studies reported here
were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC)
at the University of Rochester (protocol number UCAR-2005-226R). Male C57BL/6J mice
were purchased from Jackson Laboratories and were housed individually in micro-isolator
cages on a 12 hour light/dark cycle. Male mice were used in this study due to a faster and more
temporally predictable progression of degeneration that has been documented in models of
PTOA [22]. Mice had ad libitum access to standard chow and fresh water, and were supple-
mented with hCol1 of bovine origin and a mean molecular weight of 2000 Da (Peptan
1
B2000, Rousselot) using a method previously described to deliver daily doses of estradiol [23].
Briefly, hCol1 was incorporated into hazelnut cream such that 150mg of the mixture would
deliver either low dose (LD, 3.8mg) or high dose (HD, 38mg) hCol1 when completely con-
sumed. The HD is the body weight adjusted mouse equivalent to the 7.4g/day recommended
human dose. At the beginning of the experimental time line (Fig 1C), 12 week old mice were
presented with an autoclavable ceramic tile loaded with a 150mg aliquot of hazelnut cream
vehicle, LD hCol1 or HD hCol1 (Fig 1A). These experimental supplements were provided
daily at the same time (in the morning), and once trained, the mice consumed the entire pro-
vided amount within 2 minutes (Fig 1B). Modeling the daily consumption regimen suggested
for lifelong joint health in humans, mice were fed the supplements daily during the entire
experimental protocol until collection of experimental endpoints (Fig 1D).
To initiate PTOA, we employed a method developed and routinely used by our group
known as meniscal-ligamentous injury (MLI) [24–27]. Briefly, mice in the experimental proto-
col (at 17 weeks of age) were anesthetized via intraperitoneal injection of 60mg/kg ketamine
and 4mg/kg xylazine and after creating a 3mm incision over the anteromedial aspect of the
right knee joint, the medial collateral ligament of the right knee was transected and a segment
of the anterior horn of the medial meniscus was excised. This injury leads to detectable PTOA
joint changes by 4 weeks post-injury and progresses over 4 months [24], similar to that seen in
the DMM model of posttraumatic OA [28]. The contralateral limb provided a sham control,
with the surgery consisting of only the incision (no joint structures were manipulated). Mice
were provided buprenorphine analgesia (0.5mg/kg) at the time of surgery and every 12 hours
for 3 days.
Tissue fixation and histology preparation
A previously established systematic approach to preparation, sectioning and visualizing mouse
knee joint articular cartilage was employed for all tissue-based assays [24]. At the time of har-
vest (3 or 12 weeks post-MLI), mice were sacrificed using an AMVA-approved method and
the knee joints were dissected with the femur and tibia intact to maintain joint structure. Tis-
sues were fixed in 4% paraformaldehyde at 4˚C for 72 hours, decalcified in 5% formic acid for
10 days, processed using a microwave processor, and embedded in paraffin. Tissue blocks
were then serially sectioned in the midsagittal plane through the medial compartment of the
joint. A series of 5μm thick sections were cut at three distinct levels within the medial compart-
ment, mounted on positively-charged glass slides, baked at 60˚C overnight, de-paraffinized in
xylene, and rehydrated in decreasing concentrations of ethanol. To support study of tissue
structure via histomorphometry and to accommodate OARSI and Synovial scoring methods,
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 3 / 24
Fig 1. Effective bolus delivery of hCol1 and experimental timeline. Hazelnut cream was used as a
vehicle to deliver daily bolus doses of hCol1 to mice such that a delivery of a 150mg mixture provided a daily
bolus dose of either 3.8mg (LD) or 38mg (HD) hCol1 (Control = hazelnut cream alone). Experimental mixtures
were placed on autoclavable ceramic tiles (A) and presented to individually housemice (B) at the same time
daily. After 5–7 days of training with vehicle alone, mice consumed the full amount presented within 2 minutes.
Panel (C) depicts the experimental timeline. Mice were presented hazelnut cream daily in the bolus feeding
regimen for a 1 week training period (blue line), and then Control, LD and HD daily supplements were initiated
and continued for the remainder of the experiment (green line). After 4 weeks of supplementation, MLI (right
knee) and Sham (left knee) surgery was performed (t = 0), followed by tissue harvests at 3 weeks and 12
weeks post-surgery. (D) To confirm successful delivery of hCol1, serum hProline levels were quantified via
ELISA. Serum samples collected 1 week before (-1) and 2 weeks after surgery were harvested 3 hours after
the mice consumed supplements (left graph). Serum samples collected 3 and 12 weeks after surgery were
harvested 1 hour after consumption of the supplements (right graph). Symbols () represent the hProline level
in the serum of individual mice. Bars represent the average hProline level for each experimental group (±
SEM, N = 6). Significant differences between groups were identified via two-way ANOVA with a Tukey’s
multiple comparisons post-test (*p<0.05, **p<0.01 compared to Control).
https://doi.org/10.1371/journal.pone.0174705.g001
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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mounted sections were stained with either Toluidine Blue/Fast Green (0.04%/0.02%), Alcian
Blue Hematoxylin/Orange G or Safranin O/Fast Green (1%/0.02%) using optimized protocols
established by the Histology, Biochemistry and Molecular Imaging Core in the Center for
Musculoskeletal Research at the University of Rochester. Unstained sections were used for the
various molecular and cellular analyses described below.
Cartilage histomorphometry
Two different approaches were used to quantify cartilage content. An automated method to
quantify tissue architecture in murine allograft healing [29] was modified and utilized to deter-
mine total cartilage area (uncalcified plus calcified) using Toluidine Blue/Fast Green-stained
sections. After scanning to high resolution digital files using an Olympus VS120 Virtual Slide
Microscope/Slide Scanner system, histologic images were analyzed using a software-based
application that was developed to automatically distinguish between bone and cartilage based
on the contrasting stain differential between these tissues (Toluidine Blue-stained cartilage
and Fast Green-stained subchondral bone). Experimental image files were serially analyzed to
quantify total cartilage area on the tibial plateau and femoral condyle, with the application
returning information about each area separately. To compliment this, we also performed
manual histomorphometry using the OsteoMetrics System as we have previously published
[24,26]. Briefly, Safranin O/Fast Green stained sections were individually viewed using an
Olympus BH2 light microscope interfaced with the OsteoMetrics System via a digital camera.
OsteoMeasureXP software facilitated quantification of uncalcified cartilage, calcified cartilage
and chondrocyte populations. Articular cartilage areas examined on the tibial plateau and fem-
oral condyle were defined to be between the anterior and posterior horns of the meniscus,
using a region of interest of defined size for all sections that were analyzed.
OARSI and synovial scoring
Regarding cartilage, semi-quantitative histopathologic grading was performed using a scoring
system that has been established by the OARSI histopathology initiative as the standard
method for grading of mouse cartilage degeneration [30]. Based on this system, cartilage grad-
ing was carried out using Alcian Blue Hematoxylin/Orange G-stained joint sections using the
following scale: 0 = normal cartilage, 0.5 = loss of proteoglycan stain without cartilage damage,
1 = mild superficial fibrillation, 2 = fibrillation and/or clefting extending below the superficial
zone, 3 = mild (<25%) loss of cartilage, 4 = moderate (25–50%) loss of cartilage, 5 = severe
(50–75%) loss of non-calcified cartilage, and 6 = eburnation with >75% loss of cartilage. Syno-
vial scores were also obtained from each joint section, with the score reporting on the degree
of synovial hyperplasia (i.e. thickness and cellularity of the synovial membrane) as we have
previously performed [31]. Briefly, a subjective scoring system of 0 to 2 was employed: 0 = a
synovial lining that is several (2–3) cell layers thick or <10μm thick (normal), 1 = synovial
thickening with a lining cell layer between 5 and 10 cells thick or between 10μm and 20μm
thick, and 2 = severe thickening of the synovial lining >10 cells and/or >20μm thick. OARSI
and synovial scoring was performed by four blinded observers (QAD, EMS, RAM and MJZ)
and observer agreement for each score was evaluated in pairs via calculation of a weighted
kappa coefficient, using Fleiss-Cohen weights, as we have described [24]. The average pairwise
coefficient was 0.90, indicative of strong agreement between the observers. The four scores
(OARSI and synovium) for each section were averaged and the data from each group of mice
were combined.
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 5 / 24
Molecular analysis of tissues
Mouse knee joint sections evaluated by immunohistochemistry were treated as follows:
Endogenous peroxidases were quenched with BLOXALL (Vector) for 10 minutes followed by
treatment with 3% hydrogen peroxide for 20 min, and a 1:20 dilution of normal goat serum
for 30 min. Slides were incubated overnight at 4˚C with a rabbit anti-mouse TNF polyclonal
(1:200; Abcam #ab6671) or a rabbit anti-human Ki-67 monoclonal antibody (1:200; Abcam
#ab66155). For TNF detection, slides were then rinsed with phosphate-buffered saline contain-
ing 0.5% Tween 20, and incubated for 30 minutes at room temperature with a biotinylated
goat anti-rabbit IgG (1:200; Vector). Antibody binding to TNF was detected following applica-
tion of ABC reagent from Vectastain Elite ABC Kit (Vector) for 30 minutes, with a 5 minute
application of ImmPACT DAB Peroxidase (HRP) Substrate (Vector) for 5 minutes. Nuclei
were counterstained for 20 seconds with Mayer’s Hematoxylin (Biocare Medical). For detec-
tion of Ki-67, after overnight incubation with primary antibody, slides were rinsed with phos-
phate-buffered saline and incubated for 30 minutes at room temperature with Alexa Fluor
488-conjugated goat anti-rabbit IgG (1:250; Invitrogen #ab150077). Apoptotic chondrocytes
were identified using the TUNEL In Situ Cell Death Detection Kit (Roche, #11684795910) as
instructed by the manufacturer. In the case of Ki-67 and TUNEL, nuclei were counterstained
with DAPI and experimental fluorescence was imaged using a Zeiss Axioskop 40 BF/DF/Fluo-
rescence Microscope with a SPOT RT3 Color/Slider Camera.
qRTPCR analysis of synovial TNF expression
At sacrifice, synovial capsules were harvested from experimental joints with the aid of surgical
loops. Recovered synovial tissue was stored at -80˚C until extraction of total mRNA. mRNA
was isolated from individual capsules using the Qiagen RNeasy Fibrous Tissue mini kit (Qia-
gen) using the manufacturer’s instructions. One μg of total RNA was used to synthesize cDNA
using the iScript cDNA Synthesis Kit (BioRad). The abundance of mouse β-actin,Tnf,Il1β,
Mmp13 and Prg4 was then assessed by qRTPCR using SYBR Green Real Time PCR Master
Mix (Qiagen). Reactions were carried out using a Rotor Gene 6000 PCR machine. Forward
and reverse primer sequences for each of these gene transcripts are reported in Table 1.
Statistical analyses
For all histomorphometry-based analysis of cartilage architecture, including cartilage area and
chondrocyte population studies, one-way ANOVA with a Tukey’s multiple comparisons post-
test was performed. After confirming blinded observer agreement via calculation of weighted
kappa coefficients, OARSI and synovial scoring studies were analyzed using a Kruskal-Wallis
Test with a Dunn’s multiple comparisons post-test. These analyses and all graphing of the data
were carried out using Graphpad Prism software. Differences between groups were considered
significant when a p-value <0.05 was achieved.
Table 1. qRTPCR primer sequences.
Transcript Forward Primer Reverse Primer
Tnf 5’-CTCTTCTGTCTACTGAACTTCGGG-3’ 5’-GAGAAGATGATCTGAGTGTGAGGG-3’
Il1β5’-CACAGCAGCACATCAACAAG-3’ 5’-GTGCTCATGTCCTCATCCTG-3’
Mmp13 5’-AAGATGTGGAGTGCCTGATG-3’ 5’-AAGGCCTTCTCCACTTCAGA-3’
Prg4 5’-AGTGCTGTCCTGATTTCAAGAG-3’ 5’-GGTGATTTGGGTGAGCGTTTGGTA-3’
β-actin 5’-TGTTACCAACTGGGACGACA-3’ 5’-CTGGGTCATCTTTTCACGGT-3’
https://doi.org/10.1371/journal.pone.0174705.t001
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 6 / 24
Results
Strategy for bolus delivery of hCol1
Daily bolus delivery of hCol1 over the course of the 16 week experimental time line (Fig 1C)
was used to investigate joint protective effects that we hypothesize are supported by long term
daily consumption of hCol1 to support joint health. To support the daily bolus delivery strat-
egy, hCol1 was incorporated into hazelnut cream (Nutella
1
) such that a 150mg amount of the
experimental mixtures would support delivery of 3.8mg (LD) or 38mg (HD) of hCol1. Once
prepared, 150mg aliquots of Control (hazelnut cream vehicle alone), LD and HD hCol1 were
deposited onto sterile ceramic tiles (Fig 1A) and presented to each individually housed mouse
at the same time in the morning daily (Fig 1B). Mice typically consumed the entire supplement
within 2 minutes, supporting bolus delivery. To confirm successful absorption of the hCol1
supplements into the circulation, blood samples were collected from mice 1 week before and 2,
3, and 12 weeks after surgery (sham or MLI, Fig 1C). For the first two time points, the blood
draw was collected 3 hours after consumption of the supplements, while the second two time
points involved a blood draw 1 hour after consumption. As expected, serum levels of hProline
were increased following delivery of the hCol1 supplements. When collected 3 hours after
bolus delivery, only the HD hCol1 group displayed a significant increase in serum hProline
levels (Fig 1D). Comparatively, when blood draws were collected 45 minutes after bolus deliv-
ery, both LD and HD groups displayed significant increases in serum hProline levels, with
apparent dose dependency (Fig 1E). These results confirm that the bolus delivery regimen was
effective, with increased serum levels of hProline establishing increased absorption of hCol1
following both LD and HD hCol1 supplementation.
Impact of hCol1 supplements on cartilage structure
Initial analysis of the knee joint articular cartilage involved automated histomorphometry to
determine total cartilage area in sagittal sections stained with Toluidine Blue and Fast Green.
Stained sections were digitized to images and an ‘Articular Cartilage Application’ was devel-
oped on the Visiopharm Platform to streamline the determination of total cartilage area on the
femoral condyle and tibial plateau. As we have seen previously in the MLI model of PTOA
[24–27], the main cartilage changes played out on the tibial plateau, where there was signifi-
cant total cartilage loss detectable at both 3 weeks (Fig 2B) and 12 weeks (Fig 2A and 2C) post
injury. No changes in femoral condyle articular cartilage area were detected at either 3 or 12
weeks after injury or in any of the experimental groups (data not shown). Suggesting possible
chondroprotective effects of hCol1 supplementation, total tibial cartilage area loss 3 weeks
after MLI was prevented in the LD hCol1 group, with the HD group trending toward improve-
ment but missing significance (Fig 2B). At the 12 weeks post-MLI time point, hCol1 was dose-
dependently protective, with total tibial cartilage area loss mitigated significantly in the HD
group, and the LD group trending toward significance (Fig 2C). It should be noted that there
were no discernable effects of hCol1 supplementation on baseline (Sham-operated) articular
cartilage area (data not shown). Overall, these results provide the first indication that dietary
supplementation with hCol1 could be protective in the development and progression of
PTOA.
While the automated histomorphometry approach was useful in identifying protective
effects of hCol1 in PTOA, the underlying changes in cartilage architecture and cellularity that
contributed to the overall preservation of total tibial cartilage were not discernable using this
method. Thus, additional analyses were performed on Safranin O/Fast Green- or Alcian Blue
Hematoxylin/Orange G-stained sections using a manual histomorphometry approach
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 7 / 24
Fig 2. Cartilage loss following MLI in hCol1-fed mice is reduced. Panel (A) presents an array of
representative 40x Toluidine Blue/Fast Green stained sagittal sections from the medial compartment of sham
and MLI joints 12 weeks post-injury under various treatment conditions (control = vehicle, LD = 3.8mg hCol1/
day, HD = 38mg hCol1/day). Joint structures are labeled (F = femur, M = meniscus, T = tibia) and the black
scale bar depicts 100μm. Total tibial cartilage area was determined in these representative sections as well as
a series of similarly stained serial sections from all experimental joints at both 3 weeks (B) and 12 weeks (C)
post-MLI using an automated approach (Visiopharm System). Symbols () represent the average tibial
cartilage area of 3 sections/joint. Bars represent the average tibial cartilage area for each experimental group
(±SEM, N = 6). Significant differences between experimental groups were identified via one-way ANOVA with
a Tukey’s multiple comparisons post-test (*p<0.05, **p<0.01 compared to Control Sham;
x
p<0.05,
xx
p<0.01
compared to Control MLI).
https://doi.org/10.1371/journal.pone.0174705.g002
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 8 / 24
(Osteomeasure system) as well as standardized OARSI cartilage scoring. At 3 weeks post-MLI,
there were observable differences in cartilage architecture between experimental groups,
including apparent preservation of tibial uncalcified cartilage and cellularity in this zone in
mice supplemented with hCol1. Consistent with the automated analysis (Fig 2B), reduction of
tibial uncalcified cartilage area following MLI was mitigated significantly by hCol1 supplemen-
tation, with the LD group showing significant effects and the HD trending in that direction
(Fig 3B). Regarding calcified cartilage area (tide mark to osteochondral junction), MLI had no
effect and neither did hCol1 supplementation (Fig 3C). Loss of chondrocytes from the tibial
uncalcified zone following MLI appeared to be reduced in LD and HD hCol1 fed animals (Fig
3A), but this trend failed to achieve statistical significance (Fig 3D). Suggesting that hCol1 sup-
ports chondrocyte proteoglycan matrix production at the 3 week time point post-MLI, the
number and percentage of Safranin O-positive chondrocyte lacunae were significantly
increased in LD hCol1-supplemented mice, with the HD group trending in that direction (Fig
3E and 3F). Finally, the impact of MLI on the joint cartilage 3 weeks post injury was significant
based on OARSI scoring, but the generalized analysis of cartilage content using this scoring
system was not sensitive enough to identify any improvement in the hCol1 supplemented
groups (Fig 3G). It should be noted that there were no significant differences between experi-
mental groups in other structural analyses performed, including uncalcified and calcified carti-
lage area on the femoral condyles and the number of hypertrophic chondrocytes, and in any
zone of the cartilage on the femoral condyles and tibial plateaus (data not shown). Also of
note, the apparent inability of HD hCol1 to match the efficacy of LD hCol1 in preserving
uncalcified cartilage and Safranin O-positive chondrocyte populations at 3 weeks post-MLI
may be due to two outlier samples in the HD group that caused a large error (Fig 2B, 2E & 2F).
A similar set of manual histomorphometric analyses were performed in Safranin O/Fast
Green-stained sagittal joint sections harvested 12 weeks post-MLI. At this time point, hCol1
appeared to provide more significant protection than at 3 weeks, with representative sections
depicting apparent preservation of articular cartilage area and cellularity (Fig 4A). Use of
Osteomeasure again facilitated quantification of key cartilage structural elements, with clear
hCol1 dose-dependent preservation of uncalcified tibial cartilage following MLI (Fig 4B).
Complimenting this effect was a protection against MLI-induced expansion of the tibial calci-
fied cartilage zone, with joints from HD hCol1-treated mice showing a significant reduction in
calcified cartilage area (Fig 4C). Regarding cellularity, MLI-induced nearly complete loss of
chondrocytes in the uncalcified tibial cartilage by 12 weeks, with hCol1 dose dependently pro-
tecting against this effect (Fig 4D). Remarkably, chondrocyte number in the HD hCol1 group
was not significantly different from sham operated control group, indicating substantial effi-
cacy of the supplement in preserving cartilage cellularity. Regarding the number and percent-
age of Safranin O-positive chondrocyte lacunae in the tibial uncalcified cartilage, the nearly
complete loss of cells producing proteoglycan matrix 12 weeks post-MLI was fully rescued in
mice supplemented with hCol1 (Fig 4E and 4F). In fact, suggestive of a chondroregenerative
effect, both LD and HD hCol1 groups displayed both a greater number and greater percentage
of Safranin O-stained lacunae in tibia uncalcified cartilage compared to Control Sham joints
(Fig 4E and 4F). While, as expected, the OARSI score was significantly increased 12 weeks
post-MLI, hCol1 supplemented mice only showed modest improvement of the score, with the
LD group trending toward significance (Fig 4G). As discussed for analytics performed on
joints at the 3 week time point, it should be noted that there were no significant differences
between experimental groups in uncalcified and calcified cartilage area on the femoral con-
dyles, and in the number of hypertrophic chondrocytes in any zone of the cartilage on the fem-
oral condyles and tibial plateaus (data not shown).
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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Fig 3. hCol1 is chondroprotective in the early stages of murine PTOA. Panel (A) presents an array of representative 40x Safranin O/
Fast Green stained sagittal sections (40x) from the medial compartment of sham and MLI joints 3 weeks post-injury under various treatment
conditions (control = vehicle, LD = 3.8mg hCol1/day, HD = 38mg hCol1/day). Joint structures are labeled(F = femur, M = meniscus, T = tibia)
and the black box denotes the area shown in the zoomed images, where the tidemarks are denoted with a yellow dashed line. Black scale
bars depict 100μm. Cartilage architecture was evaluated using the Osteomeasure System to determine the tibial uncalcified cartilage area
(B), tibial calcified cartilage area (C), the number of chondrocytes in the tibial uncalcified cartilage (D), and the number (E) and percentage (F)
of Safranin-O positive (SafO
+
) chondrocytes in the tibial uncalcified cartilage. OARSI scoring of the sections analyzed by histomorphometry
was also performed (G). For histomorphometry and cell counting, symbols () represent the average measurement made from 3 sections/
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 10 / 24
PTOA-induced upregulation of MMP13 is ameliorated by hCol1
An important step in cartilage degeneration is the aberrant transition of articular chondro-
cytes into the hypertrophic state, an event marked by the expression of catabolic enzymes
such as MMP13, and the marker of terminal hypertrophy, Type 10 Collagen (ColX). Accord-
ingly, it is possible that the chondroprotective action of hCol1 documented in Figs 2–4is
associated with an inhibition of chondrocyte hypertrophy. To address this question, immu-
nohistochemistry was performed to evaluate MMP13 and ColX protein levels in the articular
cartilage. As expected, and indicative of chondrocyte hypertrophy, Control MLI mice had
elevated levels of both MMP13 and ColX in the uncalcified cartilage compared to Control
Sham mice at the 3 week time point (Fig 5A and 5B). Mice supplemented with HD hCol1
were protected from the MLI induced MMP13 staining in the uncalcified cartilage, suggest-
ing that preservation of cartilage architecture in this group could in part be due to inhibion
of matrix degeneration (Fig 5A). In contrast, ColX staining was not reduced in MLI mice
supplemented with hCol1, indicating that hCol1 did not impact terminal chondrocyte hyper-
trophy (Fig 5B). Overall, no differences were observed in the calcified cartilage, as chondro-
cytes in this zone were all actively producing similar levels of both MMP13 and ColX (Fig 5A
and 5B). It should be noted that at the 12 week time point, MLI joints from control-supple-
mented mice had lost most of the uncalcified cartilage and associated chondrocytes, making
comparisons with the treated groups uninformative (data not shown). Together, these data
suggest that mice supplemented with the HD hCol1 may have been protected from cartilage
degeneration in part due to reduced MMP13 production by chondrocytes residing in the
uncalcified cartilage.
Impact of hCol1 supplements on articular chondrocyte life cycle
The remarkable ability of hCol1 supplementation to protect against chondrocyte loss in
murine PTOA (Fig 4D) could be a key driver of its chondroprotective effect by setting the
stage for persisting chondrocytes to produce matrix molecules (Fig 4E and 4F). To determine
if the protection against MLI-induced chondrocyte loss was due to stimulation of proliferation
or inhibition of apoptosis, representative tissue sections were analyzed via PCNA and TUNEL
staining. While there was no detectable difference in the number of proliferative chondrocytes
between experimental groups at 3 weeks post-MLI based on PCNA immunodetection (data
not shown), MLI-induction of broad chondrocyte apoptosis was reduced 3 weeks post-MLI in
mice supplemented with hCol1 (Fig 6). Representative TUNEL-stained sections showed a
marked reduction in the number of apoptotic chondrocytes in superficial-to-middle layer car-
tilage zones (zoom panels in Fig 6). It should be noted that analysis was not informative at the
12 week time point because MLI joints in the control-supplemented mice were TUNEL-nega-
tive due to broad apoptotic loss of chondrocytes that had occurred prior to the harvest. Overall,
these data suggest that the significantly larger number of chondrocytes seen in the uncalcified
cartilage of hCol1 supplemented mice at 12 weeks post-MLI (Fig 4D) could be due to protec-
tion from apoptosis earlier in the disease process.
joint. For OARSI Scoring, symbols () represent the average score for each joint based on scoring of 3 sections/joint by four observers. Bars
in all graphs represent the average for each experimental group (±SEM, N = 6). Significant differences between experimental groups in the
histomorphometry data (B-F) were identified via one-way ANOVA with a Tukey’s multiple comparisons post-test (*p<0.05, **p<0.01,
***p<0.001 compared to Control Sham;
x
p<0.05,
xx
p<0.01 compared to Control MLI). Significant differences between experimental groups
in the OARSI data (G) were identified via a Kruskal-Wallis Test with a Dunn’s multiple comparisons post-test (*p<0.05, compared to Control
Sham).
https://doi.org/10.1371/journal.pone.0174705.g003
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 11 / 24
Fig 4. hCol1 protects against cartilage loss in mid to late stage murine PTOA. Panel (A) presents an array of representative 40x
Safranin O/Fast Green stained sagittal sections from the medial compartment of sham and MLI joints 12 weeks post-injury under various
treatment conditions (control = vehicle, LD = 3.8mg hCol1/day, HD = 38mg hCol1/day). Joint structures are labeled (F = femur,
M = meniscus, T = tibia) and the tidemarks are denoted with a yellow dashed line in the zoomed images. Black scale bars depict 100μm.
Cartilage architecture was evaluated using the Osteomeasure System to determine the tibial uncalcified cartilage area(B), the tibial calcified
cartilage (C), the number of chondrocytes in the tibial uncalcified cartilage (D), and the number (E) and percentage(F) of Safranin-O positive
(SafO
+
) chondrocytes in the tibial uncalcified cartilage. OARSI scoring of the sections analyzed by histomorphometry was also performed
(G). For histomorphometry and cell counting, symbols () represent the average measurement made from 3 sections/joint. For OARSI
Scoring, symbols () represent the average score for each joint based on scoring of 3 sections/joint by four observers. Bars in all graphs
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 12 / 24
represent the average for each experimental group (±SEM, N = 6). Significant differences between experimental groups in the
histomorphometry data (B-F) were identified via one-way ANOVA with a Tukey’s multiple comparisons post-test (*p<0.05, **p<0.01,
***p<0.001 compared to Control Sham;
x
p<0.05,
xx
p<0.01 compared to Control MLI, N = 6). Significant differences between experimental
groups in the OARSI data (G) were identified via a Kruskal-Wallis Test with a Dunn’s multiple comparisons post-test (*p<0.05, **p<0.01
compared to Control Sham).
https://doi.org/10.1371/journal.pone.0174705.g004
Fig 5. hCol1 reduces MMP13 levels in articular cartilage of mice following MLI. 3 weeks post-injury (Sham or MLI),
knee joints were harvested from mice and hypertrophic chondrocytes were analyzed by immunohistochemistry ofMMP13
and ColX. Representative sagittal sections depict (A) MMP13 and (B) ColX stained chondrocytes (brown) with cell nuclei
counterstained with hematoxylin (blue). Yellow dasted lines highlight the tide mark, separating calcified cartilage from
uncalcified cartilage. Joint structures are labeled (F = femur, M = meniscus), and the black scale bar depicts 100μm.
https://doi.org/10.1371/journal.pone.0174705.g005
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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Oral delivery of hCol1 protects against PTOA-induced synovial change
The marked preservation of matrix-producing chondrocytes post-MLI in hCol1 supplemented
mice begged for subsequent investigation of the impact of hCol1 on inflammatory synovial
changes that are known to contribute to chondrocyte death following joint injury [32,33].
Comparison of representative Safranin O/Fast Green stained sagittal knee joint sections
revealed an apparent reduction in the severity of synovial hyperplasia in mice provided the
hCol1 supplements. At both 3 and 12 weeks post-MLI, synovial thickness was increased com-
pared to Control Sham joints, while mice provided hCol1 were protected from MLI-induced
hyperplastic synovial change (Fig 7A). Synovial phenotypes were further investigated using a
synovial scoring method, revealing significant effects in mice provided hCol1 supplements
that were consistent with the representative histology. As expected, MLI induced a significant
Fig 6. Chondrocyte apoptosis post-MLI is reduced in hCol1-supplemented mice. Joints were harvested
from mice 3 weeks post-injury (Sham or MLI) and apoptotic cells were identified via TUNEL staining.
Representative 100x sagittal sections (right column of panels) show the overall scope of cellular apoptosis
(green), with all cell nuclei DAPI labeled (blue). The yellow dashed lines depict the articular cartilage surface
and the red dashed lines outline the anterior and posterior horns of the meniscus. The region demarcated with
the white box is magnified in the right column (zoom). White scale bars depict 100μm.
https://doi.org/10.1371/journal.pone.0174705.g006
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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Fig 7. Synovial hyperplasia is reduced in mice supplemented with hCol1. (A) Tissue sections stained
with Safranin O/Fast Green were used to examine the synovium. Representative 40x sagittal sections from
Sham and MLI joints of mice supplemented with Control (vehicle, hazelnut cream), LD hCol1 or HD hCol1 that
were harvested at 3 and 12 weeks post-injury are depicted. Joint structures are labeled (F = femur, M =
meniscus, T = tibia), and synovial membranes are demarcated with black arrows. The red line highlights the
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 15 / 24
increase in synovial score (i.e. more robust synovial hyperplasia) at both 3 weeks (Fig 7B) and
12 weeks (Fig 7C) post-MLI. At 3 weeks, synovial scores trended toward a reduction (improve-
ment) in the MLI groups that were supplemented with hCol1 (Fig 7B). Comparatively, at 12
weeks post-MLI, the synovium in mice from the HD hCol1 group was significantly less hyper-
plastic, suggesting that hCol1 supplements effectively reduced synovial/joint inflammation
that is present in PTOA.
Among several cytokines implicated in diarthrodial joint degeneration, TNF is estab-
lished as a central inflammatory mediator that is present in synovial fluid and upregulated
in hyperplastic synovium in PTOA, particularly in the early post-trauma time frame [34,
35]. To investigate the impact of hCol1 supplementation on TNF expression post-MLI, both
immunohistochemical staining and mRNA analysis of synovial membranes were per-
formed. Representative immunostained sagittal sections revealed increased TNF expression
at both 3 and 12 weeks post-MLI, with mice from the HD hCol1 cohort substantially pro-
tected from this effect (Fig 8A). This was confirmed via quantitative analysis of Tnf mRNA
levels in synovial membranes harvested from the various experimental groups. Specifically,
3 weeks post-injury, both LD and HD hCol1 supplementation significantly reduced Tnf lev-
els in synovium from both the sham and MLI cohorts (Fig 8B). While the overall synovial
level of Tnf was lower 12 weeks post-injury, hCol1 still effectively reduced synovial Tnf in
MLI joints, with the HD group achieving statistical significance (Fig 8C). It should be noted
that while the synovial expression of other genes, including Il1β,Prg4 and Mmp13, was
increased following injury, particularly at 12 weeks post-MLI, hCol1 supplementation did
not have a significant impact on expression level (data not shown). Overall, these findings
suggest that dietary supplementation with hCol1 reduces synovial hyperplasia and mitigates
synovial Tnf expression in both early and mid-stage PTOA.
Discussion
In this study, experiments were carried out to investigate the impact of orally delivered hCol1
on the joint degenerative process in a mouse model of PTOA. Close examination of cartilage
architecture, chondrocyte populations and synovial change revealed significant chondropro-
tective and anti-inflammatory effects in injured joints at early and mid-stages of OA degenera-
tion. These novel findings support the hypothesis that daily oral consumption of hCol1 is joint
protective and disease modifying in OA joints.
The concept that oral consumption of matrix components from cartilage and connective
tissue may be effective in treating arthritic conditions has been debated since the mid 1980s.
Among these agents, the most widely recognized, heavily studied, and broadly available are
glucosamine and chondroitin sulfate, with published evidence suggesting that they are joint
protective in OA based on work in animal models [36] and humans [37–40]. Fueling the effi-
cacy debate, the widely cited Glucosamine/Chondroitin Sulfate Arthritis Intervention Trial
known as GAIT concluded that these agents are not symptom-relieving or disease modifying
in human OA [41]. Despite these and other conflicting reports, and without definitive
thickness of hyperplastic synovium in the Control MLI section and the black scale bar depicts 100μm. A
synovial scoring method was also employed to quantify synovial hyperplasia at both 3 weeks (B) and 12
weeks (C) post-injury. Symbols () represent the average Synovial Score for each joint based on scoring of 3
sections/joint by four observers. Bars represent the average Synovial Score per experimental group
(±standard deviation, N = 5–8 joints). Significant differences between experimental groups were identified via
a Kruskal-Wallis Test with a Dunn’s multiple comparisons post-test (*p<0.05, **p<0.01, ***p<0.001
compared to Control Sham).
https://doi.org/10.1371/journal.pone.0174705.g007
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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preclinical study of joint tissue effects, glucosamine and chondroitin sulfate have been contin-
ued to be widely marketed as nutraceuticals that are joint protective and symptom relieving in
various types of arthritis [42–44]. Study and use of these molecules has also opened the door to
the development of other molecules of similar origin that likewise are thought to impact joint
health, including collagens in their native state or as hydrolysates.
In the case of collagen-based nutraceuticals, numerous formulations have been developed
which are marketed as dietary supplements or as additives or ingredients in the food industry.
They are gaining popularity due to their documented positive effects on skin and bone.
Fig 8. Post-injury upregulation of TNF in the synovium is reducedin mice supplemented with hCol1. (A)
Representative TNF immunostained sagittal sections (100x) from Sham and MLI joints of mice supplemented
with Control (vehicle, hazelnut cream), LD hCol1 or HD hCol1 that were harvested at 3 and 12 weeks post-injury
are shown. Joint structures are labeled (F = femur, M = meniscus, T = tibia), synovial membranes are
demarcated with red arrows, and brown staining of the tissue indicates intensity and location of TNF expression.
The black scale bar depicts 100μm. mRNA was purified from synovial tissue collected from a separate cohort of
similarly-treated mice at 3 weeks (B) and 12 weeks (C) post-injury. qRTPCR was performed to quantify Tnf
expression level. Symbols () represent the Tnf level in each synovial sample and bars represent the average
Tnf level for each experimental group (±SEM, N = 6). Significant differences between groups were identified via
one-way ANOVA with a Tukey’s multiple comparisons post-test (*p<0.05 compared to Control Sham,
xx
p<0.01
compared to Control MLI).
https://doi.org/10.1371/journal.pone.0174705.g008
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
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Specifically, collagen peptides enhance skin hydration and elasticity and stimulate the produc-
tion of dermal matrix [17,45]. In the skeleton, collagen peptides have been shown to increase
bone strength and mineral density, supporting their potential disease modifying capability in
osteoporosis [16,46,47]. Since collagen is a major component of the matrix in key joint struc-
tures including the articular cartilage, collagen peptides have also been proposed as potential
disease modifiers in arthritic conditions [20]. Early work sparking this idea reported that oral
administration of native type 2 collagen was effective at mitigating arthritic symptoms in ani-
mal models of rheumatoid arthritis (RA) [48,49], with a human clinical trial demonstrating
analogous protection in RA patients orally consuming chicken type 2 collagen [50]. A series of
reports further substantiate this idea, demonstrating that various preparations of orally admin-
istered type 2 collagen effectively ameliorate collagen-induced arthritis in mice [51], in obese
arthritic dogs [36], and in equine OA [52]. Remarkably, they have also been shown to have
therapeutic efficacy in managing symptoms in human OA [53]. Similarly, oral supplementa-
tion with a type 1 collagen hydrolysate has been shown to structurally improve the thickness of
articular cartilage in OA via gadolinium-enhanced MRI [54], suggesting potential disease-
modifying capability of this agent. hCol1, a specific collagen-based product marketed by Rous-
selot under the trade name Peptan
1
, has also recently been shown by Jiang et al. to reduce
joint pain in a prospective, randomized, double blind, placebo-controlled clinical trial in
elderly women with mild to moderate knee OA [21]. This study, which reported improved
Western Ontario and McMaster Universities Arthritis Index (WOMAC) and Lysholm scores
in subjects consuming 8gm Peptan per day for 6 months, establishes the idea that hCol1 sup-
ports joint health and reduces joint pain during early to mid-stage OA. Given that little is
known about the biological action of hCol1 at the tissue and cellular level, we executed experi-
ments to elucidate its impact on joint structure in a preclinical model of PTOA.
An experimental protocol was carried out in this study that involved daily oral delivery of
hCol1 to mice that were induced to develop PTOA via surgical administration of a meniscal-
ligamentous injury (Fig 1). Two doses of hCol1 were chosen, with the higher dose (38mg/day)
set to be the body weight equivalent to the human dose (7.4g/day) that was employed in the
Jiang et al. clinical study mentioned above [21]. To investigate the hypothesized joint-protec-
tive impact of long term use, mice were supplemented with hCol1 for a one month pre-treat-
ment period, followed by administration of MLI surgery and subsequent assessment of joints.
Structural and cellular evaluation of the articular cartilage of sham and injured knee joints
using both automated and manual histomorphometric methods revealed that while there were
no discernable cartilage effects in the sham/normal joints, at early and later time points in the
development of OA, mice administered hCol1 were chondroprotected, particularly at 12
weeks post-injury. Uncalcified tibial articular cartilage was preserved and the articular chon-
drocyte population was maintained (Figs 2,3and 4), possibly via reduction of MMP13 and
inhibition of chondrocyte apoptosis (Figs 5and 6, respectively). Regarding apoptosis specifi-
cally, its inhibition was particularly apparent on the femoral condyles (Fig 6), which correlated
with the complete protection from cartilage loss in this region of the joint (Fig 4). Interestingly,
chondrocyte populations were not only preserved, but the number of cells actively producing
Safranin O stained proteoglycan matrix was increased significantly in the hCol1-treated
groups at both time points. These results parallel published work suggesting chondrogenic and
cartilage matrix anabolic effects of hCol1 in adipose tissue derived stem cells [19], and the ana-
bolic effects of type 1 collagen hydrolysate and prolyl-hydroxyproline [55,56] and collagen
peptide fragments [57] on chondrocyte matrix production.
In addition to the remarkable cartilage and chondrocyte effects observed in the degenerat-
ing knee of mice supplemented with hCol1, significant changes were also observed in the syno-
vium. While there were no discernable synovial effects of the supplements in sham operated
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 18 / 24
joints, synovial hyperplasia was evident at both early and late time points following injury
(3 and 12 weeks respectively), with semi-quantitative scoring revealing significant protection
from this hyperplasia in mice supplemented with hCol1 (Fig 7). This protection was accompa-
nied by reduced TNF protein and mRNA expression, suggesting an anti-inflammatory effect
of hCol1 on the OA joint (Fig 8). This protection seemed to be restricted to TNF; synovial
mRNA expression of other cytokines and catabolic factors known to participate in articular
cartilage degeneration, including Il1b,Mmp13 and ADAMTS5, were not affected in the hCol1
supplemented groups (data not shown). While hCol1 and other similar collagen-based supple-
ments have not been shown to protect against the production of inflammatory mediators in
joint tissue, there is evidence of anti-inflammatory effects of type 1 collagen hydrolysate
and peptides in cardiovascular disease [58] and in ulcerative colitis [59]. Of note, the anti-
inflammatory effects of oral hCol1 in the synovium described in this report parallel the anti-
inflammatory effects that have been reported for glucosamine, chondroitin sulfate and glycos-
aminoglycans (reviewed in [44,60]). Overall, when considered in conjunction with the
observed cartilage structure modifications, daily oral supplementation with hCol1 has signifi-
cant potential as a cartilage protective and anti-inflammatory agent in the treatment of OA.
The detailed analysis of joint structure that was completed in this study represents a novel
attempt to understand the influence of a dietary supplement on OA joint degeneration specifi-
cally. The numerous soft tissue matrix-based oral supplements and nutraceuticals described
above, including collagen-based agents, glucosamine, glycosaminoglycan, and chondroitin/
keratin sulfate, have been studied in vitro (chondrocyte and synovial cultures) and/or in ani-
mal and human OA with pain assessment representing a primary measure of efficacy. Missing
from many of these studies is the direct examination of the impact of these agents on cartilage
architecture, chondrocyte populations and synovial status in animal models of disease. To
definitively be classified as disease modifying, these structural analyses must be performed to
establish tissue-level impact; it is for this reason that a detailed study of joint structure was pur-
sued in this investigation. The positive effects of hCol1 on articular cartilage architecture and
synovial hyperplasia that are presented in this report not only provide a structural basis for the
mitigation of pain that has been reported in OA patients [21], but they also provide a strong
rationale to further evaluate hCol1 effects on joint structure in the context of human disease.
An open question that is yet to be answered for hCol1 and other nutraceuticals that are pur-
ported to be joint protective, including type 2 collagen-based preparations, glucosamine and
chondroitin sulfate, relates to their mechanism of action. The dominant hypothesis posits that
these agents, or their digested fragments, are absorbed across the intestinal lumen, enter the
blood stream and have direct effects on articular chondrocytes or other cells in the joint. Sup-
porting this general idea, regarding collagen peptides specifically, hydroxyproline-containing
di- and tri-peptides are detectable in the human serum after oral consumption [13,61], with
90% of consumed peptides absorbed within 6 hours in mice [62]. Consistent with this, we
detected a significant dose dependent surge in serum hydroxyproline levels in mice that were
supplemented with hCol1 (Fig 1). Serum hydroxyproline was highest when blood was sampled
within 1 hour of consumption (Fig 1E), although the surge was still detectable at 3 hours in the
high dose hCol1 group (Fig 1D). Once in the serum, these peptide fragments presumably have
access to all organ systems, with one report confirming distribution of a radiolabeled prepara-
tion to skeletal elements, muscle, skin and cartilage in rats [63]. Another study performed in
rats provides autoradiographic evidence for the presence of radiolabeled proline-hydroxypro-
line dipeptide in articular cartilage and synovium within 30 minutes of ingestion [64]. As men-
tioned earlier in this report, there are also in vitro data indicating that various types of collagen
peptides can directly affect chondrocyte function [19,45,55,57], supporting the hypothesis
that absorbed collagen peptide fragments could directly influence cell behavior in the joint.
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 19 / 24
While this line of reasoning lays out a basis for orally ingested collagen peptides or cartilage
matrix molecules to impact joint tissues, the nature of the peptides/compounds that actually
make it to the joint is not known, and the pathway of action, whether it be in the chondrocyte,
synoviocyte, or other cell in the joint, can only be speculated. Current effort in the field aims at
addressing these questions.
Conclusions
The efficacy of nutraceuticals comprised of cartilage matrix components in supporting joint
health and protecting against OA progression has been tested and debated over the past two
decades. Dietary supplements containing chondroitin and glucosamine are marketed world-
wide and have been evaluated in human OA clinical trials, with mixed results. With both in
vitro and anecdotal animal and human data suggesting that oral consumption of hCol1 could
represent a strategy for supporting joint health, a preclinical study was executed to definitively
determine the impact of this agent on the cartilage degenerative process that plays out in a
murine model of PTOA. Results reported here suggest that daily consumption of hCol1 pro-
tects against cartilage loss and stimulates the production of proteoglycan by chondrocytes in
injured joints. Articular chondrocyte number is also increased in the joints of mice from the
hCol1 groups, possibly due to an inhibition of apoptosis in these cells. Trauma-related synovial
hyperplasia is also reduced in supplemented mice, and this effect occurs in conjunction with
reduced synovial TNF expression. Overall, these results suggest that hCol1 is chondroprotec-
tive 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.
Acknowledgments
The authors wish to acknowledge the outstanding technical contributions of Sarah Mack and
Kathy Maltby of the Histology, Biochemistry and Molecular Imaging Core in the Center for
Musculoskeletal Research at the University of Rochester Medical Center. We also thank Brit-
tany Garrison for assistance in the husbandry of mice during the execution of the experimental
protocol, and Dr. Longze Zhang for his expertise developing automated histomorphometric
methods to assess cartilage area.
Author Contributions
Conceptualization: JPK RAM MJH JHJ JP MJZ.
Data curation: Q-AD EMS SEC RDM ZL FK CWF.
Formal analysis: Q-AD EMS SEC RDM ZL FK CWF RAM JHJ MJZ.
Funding acquisition: MJH JP MJZ.
Investigation: Q-AD EMS SEC RDM ZL FK CWF.
Project administration: JPK RAM MJH JHJ JP MJZ.
Supervision: JPK RAM MJH JHJ JP MJZ.
Validation: JPK RAM MJH JHJ MJZ.
Visualization: MJZ.
Writing – original draft: MJZ.
Hydrolyzed type 1 collagen protects against posttraumatic osteoarthritis in mice
PLOS ONE | https://doi.org/10.1371/journal.pone.0174705 April 6, 2017 20 / 24
Writing – review & editing: Q-AD EMS SEC RDM ZL FK CWF JPK RAM MJH JHJ JP MJZ.
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