Advances in the Regulation of Periostin for Osteoarthritic Cartilage Repair Applications

Figures

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  Figure 1. Increased expression of Postn in human OA cartilage. (A) Immunohistochemical analysis of human OA knee cartilage identified increased cell-associated POSTN in lesional areas when compared to normal cartilage (back arrows). OA knee cartilage, obtained from four donors ranging from 35 to 70 years of age and undergoing total knee arthroplasty, was sectioned and stained for POSTN with Vectastain reagents (Vector Laboratories, Burlingame, CA, USA). (B) Quantitation of periostin positively expressing chondrocytes in superficial and deep cartilage zones. Two-way ANOVA; comparison between normal and OA cartilages; **** p < 0.0001; * p < 0.05; n = 4.

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  Figure 2. LN suppresses the upregulation of POSTN in human OA osteochondral explants: (a) Human OA osteochondral explants were treated with LN (100 µg/mL) or PBS (CTL) for 14 days. Changes in expression were determined by POSTN staining (arrows); (b) immunohistochemical analysis of POSTN expression in human OA samples, where the stains were divided into three categories: cells unstained with POSTN, cells with minimal Postn staining, and cells with saturated POSTN staining. Means ± SDs; n = 4 donors; chi-squared test.

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  Figure 3. Effect of LN on POSTN expression in primary human osteoarthritis chondrocytes. (A) Freshly isolated human osteoarthritis (OA) chondrocytes were cultured as micro-pellets and treated for 6 days with LN at 1 µg/mL and 100 µg/mL. Quantitative real-time PCR was used to measure POSTN mRNA expression levels, normalized against GAPDH. (B) Representative western blot analysis showing the effects of the LN treatments (at 1 µg/mL and 100 µg/mL) on POSTN protein levels over a 3-day treatment period. GAPDH was used as the loading control. (C) Densitometric analysis of POSTN protein levels from western blots (panel B), normalized to GAPDH. Statistical significance is indicated as * p < 0.05; ** p < 0.01; and **** p < 0.0001 compared to the untreated control group.

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  Figure 4. LN suppresses the upregulation of POSTN in a rabbit model of OA. Skeletally mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) of their left femorotibial joints to induce joint degeneration typical of OA. Beginning at 3 weeks postoperatively, and every three weeks thereafter for 12 weeks, either saline (1 mL) or sLN (100 µg in 1 mL of saline) was injected intraarticularly into the operated knee. Six additional rabbits underwent sham surgery but without ACLT or postoperative injections: (A) POSTN expression, as determined using immunostaining at 12 weeks, was significantly higher in the ACLT rabbits’ knee cartilage when compared with sham knees; (B) the immunohistochemical analysis of the POSTN expression in the rabbit model of OA, where the stains were divided into three categories: cells unstained with POSTN, cells with minimal POSTN staining, and cells with saturated POSTN staining. Statistical significance was assessed using a chi-squared test (p < 0.00001).

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  Figure 5. LN decreases POSTN signaling in human OA chondrocytes: (A) western blots demonstrating the inhibition of periostin-induced increases in β-catenin (β-Cat) accumulation by LN; (B) densitometry of blots presented in (A). ANOVA; post hoc Dunnett’s test; *** p < 0.0001; n = 3.

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  Figure 6. LN regulates periostin-induced gene expression in human OA chondrocytes. Chondrocyte pellets were treated with Link N (at 1 or 100 µg/mL) or PBS (CTL) for 6 days in the absence or presence of periostin (20 μg/mL). Gene expression was measured using qPCR. Means ± SDs; n = 3 donors; ANOVA; post hoc Dunnett’s multiple comparison test; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 in comparison with the control.

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  Figure 7. LN interacts with POSTN and induces dissociation: (A) The peptide docking of the LN to POSTN (crystal structure: 5yjg) was determined using the CABS–dock web server. The model was created using PyMOL (Schrodinger, LLC). POSTN residues known to be important in its dimerization are shown to interact with LN. (B) The immunoprecipitation (IP) of the LN with POSTN. Biotinylated LN or biotinylated scrambled LN (SC) was attached to Avidin-labelled agarose beads and then incubated with POSTN. Western blotting was performed to identify POSTN–LN interactions. Lane 1: CTL (PBS) with POSTN; lane 2: IP of LN with POSTN; lane 3: SC with POSTN. (C) POSTN incubated with LN dissociates dimer/oligomer formation.