Extracellular Fixed Charge Density and Osmotic Effects on Chondrocyte Proteoglycan Replenishment after Enzymatic Digestion of Cartilage Explants

INTRODUCTION Chondrocytes maintain a balance between catabolic and anabolic activities in order to provide cartilage tissue with its characteristic concentrations of extracellular matrix molecules such as glycosaminoglycans (GAGs) and collagen. Disruption of this balance may have implications for disease progression and loss of tissue properties. As demonstrated in a recent study [1], when the native GAG environment is depleted through enzymatic digestion, chondrocytes will replenish these molecules, returning to native concentrations after 13 days in culture. The mechanisms underlying this replenishment are unclear, including how the chondrocyte senses extracellular matrix (ECM) loss and then recovery of native concentrations. One possibility is through a feedback loop that is moleculeand receptormediated or one that responds to charge or osmotic effects from the highlynegatively charged GAG molecules and the salt environment that they create. Our previous work has demonstrated that isolated chondrocyte GAG production is not down-regulated by culture osmolarity levels found in the native tissue [2]. Because cellular interactions with non-GAG components of the ECM may play an important regulatory role in cell biosynthesis, in this study we build upon our previous work by examining in situ chondrocyte GAG production after enzymatic depletion in explant culture, in the presence of media of native fixed charge density (FCD) or osmolarity values. METHODS Full-depth explants were harvested from the femoral condyle of immature bovine knees, separated from the bony interfacial region, and cultured in CDM (DMEM + dexamethasone + ascorbic acid + ITS) overnight. The samples were then weighed sterilely and subjected to one of two serial enzymatic digestions at 37 °C to remove tissue GAGs. Digestion A (DIG A) consisted of treatment with 0.125 U/mL chondrotinase ABC (CABC, Sigma) with protease inhibitor (PI) cocktail (Roche) for 15 hours, followed by 5 U/mL streptomyces hyaluronidase (HA) with PI cocktail for 24 hours. Digestion B (DIG B) consisted of treatment with 0.125 U/mL CABC with PI cocktail for 12 hours, followed by 0.5 mg/mL trypsin for 3 hours and 5 U/mL HA with PI cocktail for 24 hours. Live/dead staining was performed to confirm cell viability after digestion. Digested samples were maintained for 2 weeks in 4 recovery media conditions: 397 mOsm CDM prepared with chondroitin sulfate-A (CS) (Sigma), 300 mOsm CDM (300), 400 mOsm CDM prepared with sucrose (400S), and 400 mOsm CDM prepared with NaCl (400N). CS concentration (5% w/v) was chosen to recreate the native fixed charge density environment based on previously obtained values for native GAG concentrations through the depth of the tissue; this concentration yielded a media osmolarity near 400 mOsm. 300 and 400 mOsm media osmolarity values were chosen based on previous modeling of tissue osmolarity [2]. The former represents a subphysiologic value, while the latter represents a similar or higher value than found through the majority of the tissue depth (hypothesized to have negative feedback potential). The 400 mOsm media was prepared with the two osmolytes to distinguish between the contributions of osmotic and charge effects to tissue recovery results. Before and after digestion, and at days 7 (d7) and 14 (d14) of recovery, samples were harvested to quantify GAG and collagen content by DMMB and OHP assays, respectively. One-way ANOVA analysis was performed to determine significant effect (p<0.05) of the two digestions on tissue GAG and collagen content. Student’s t-tests were performed to determine significant (p<0.05) differences of each media recovery condition with 1) pre-digest values and 2) d0 post-respective-digest values. Two-way ANOVA was run on d7 and d14 GAG and collagen data for each digestion protocol for factors of time in culture (2 levels) and media condition (4 levels) to determine significance of the main effect of each of the factors and interaction between the factors. If a factor had a significant effect with ANOVA, Tukey HSD post-hoc analysis was run to determine significant (p<0.05) differences between each media-day group. RESULTS Live/dead staining of explants before and after digestion showed similar amounts of live cells throughout the tissue. One-way ANOVA returned no significant effect of digestion on explant collagen content. Twoway ANOVA on d4 and d14 data returned no significant overall effect of day or media condition on collagen content and no significant interaction between the variables for either digest condition. One-way ANOVA returned a significant effect of digestion on GAG content. DIG A did not significantly decrease GAG content (5.7 vs. 7.0% pre-digest, p=0.13), while DIG B did (0.3% vs. 7.0% pre-digest, p<0.001) (Fig. 1). Two-way ANOVA returned significant effects for day and media condition on GAG content for both digest conditions. At d7 after DIG A, explants cultured in CS media exhibited higher GAG content than the other media conditions, though the effect was not significant, while there was no effect of media osmolarity alone. By d14 after DIG A, explants cultured in CS media had GAG levels significantly higher than pre-digest values. Both CS and 400N media conditions also showed significantly higher GAG levels at d14 than post-DIG A and respective media condition d7 values. Similarly, at d7 after DIG B, there was no difference between media of 300 and 400 mOsm, and here CS media exhibited a significant increase in GAG from post-digest levels and significantly higher GAG than 300 and 400S groups. By day 14, though, all media groups had achieved significantly higher GAG levels than d0 postdigest values, and there was no significance between CS and other media groups.