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Oral hydrolyzed type 1 collagen induces chondroregeneration and inhibits synovial inflammation in murine posttraumatic osteoarthritis

      Purpose: Osteoarthritis (OA) is a degenerative joint disease that culminates with the irreversible loss of articular cartilage. Currently there is no disease modifying therapy, with management restricted to palliation. Thus, therapeutic strategies that offer chondroprotective or regenerative capability are needed. Nutraceutical products consisting of cartilage matrix components have been marketed as effective in maintaining joint health with little supporting data. Recently, a hydrolyzed type 1 collagen preparation manufactured by Rousselot (Peptan) has been reported to support a positive influence on chondrocyte function. Based on data suggesting chondrogenic effects of Peptan in vitro and clinical evidence of its symptom-relieving effects in subjects suffering from joint pain when consumed daily, we tested the hypothesis that Peptan is joint-protective in the context of murine posttraumatic OA.
      Methods: Animals: Male C57BL/6 J mice (7 wks old) were placed on a control, low dose (LD, 3.8 mg Peptan), or high dose (HD, 38 mg Peptan, 7.4 g human dose equivalent) dietary supplement that was administered in Nutella at the same time daily. After 5 wks of feeding, meniscal-ligamentous injury (MLI) surgery was performed on the right knee (left knee serving as contralateral sham). Mice were continued on the supplement for 3 or 12 wks. Serum Harvest & Hydroxyproline Assay: Serum samples were obtained via submandibular bleeding within 60 min of administering supplement and hydroxyproline levels were measured using a kit (BioVision). RT-qPCR: mRNA was isolated from knee joint capsules, cDNA was synthesized, and gene expression was quantified using SYBR Green RT-PCR Master Mix (Qiagen). Histology: Mice were perfusion fixed with 4% PFA and knee joints were post-fixed for 72 hrs, decalcified in formic acid for 10 days, and embedded in paraffin. Sagittal sections were cut from the medial knee joint compartment and stained to visualize articular cartilage and synovium. Joints were examined via OARSI scoring, histomorphometry and immunohistochemistry. Statistics: Values represent means ± SEM and two-way ANOVA with a Bonferroni-Dunn multiple comparison test was performed to identify significance between groups (p < 0.05, N = 6).
      Results: Hydroxyproline (hP) levels were elevated in Peptan-fed mice: Daily delivery of Peptan in Nutella supported bolus delivery of the experimental dietary supplement, evidenced by significant and dose-dependent increases in serum hP levels 60 min after feeding, with hP being an amino acid specific to collagen. Peptan-fed mice display chondroregeneration post-MLI: Total and uncalcified tibial cartilage area was increased at 3 & 12 wks post-MLI (Fig. 1A). Significant increases in chondrocyte number (Fig. 1C) and chondrocyte pericellular proteoglycan deposition (Fig. 1D) were also observed in mice fed Peptan. All tissue effects were dose-dependent (compare LD and HD in Fig. 1A–D). Peptan-fed mice have reduced synovial hyperplasia and TNF expression: Using a semi-quantitative grading system, synovial hyperplasia was reduced at 3 & 12 weeks post-MLI. Correlated with this, synovial TNF levels were also reduced at both time points based on IHC and RT-qPCR analyses.
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      Fig. 1. Safranin O/Fast Green staining of knee joints 12 weeks after MLI reveals enhanced pericellular proteoglycan content and thicker articular cartilage on the tibial plateau in LD and HD Peptan-treated groups (A). Histomorphometry revealed significant positive effects of Peptan on tibial uncalcified cartilage area (B), total cell (chondrocyte) counts in the tibial uncalcified cartilage (C) as well as an increased percentage (D) of Safranin O+ cells. x,* = p < 0.05, xxx,*** = p < 0.001, 2 way ANOVA & Bonferroni-Dunn multiple comparison, N=6; x or xxx denotes differences between sham and MLI within each group.