Platelet-derived growth factor-coated decellularized meniscus scaffold for integrative healing of meniscus tears

Abstract

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration. Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-β (PDGFRβ) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks. Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRβ expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF. In conclusion, PDGF-BB-coated scaffold increased PDGFRβ expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears.

Statement of significance: Meniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use. We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells. The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties. This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

Keywords: Cell migration; Decellularization; Heparin; Meniscus; PDGF.

Figure 1.

PDGF-BB release kinetics from DMS. PDGF-BB was conjugated to DMS or heparin coated DMS and DMS was cultured at 37 °C for up to 16 days. Supernatants were collected at the indicated time points and analyzed for PDGF-BB by ELISA. Results are from 3 separate experiments. Data are shown as mean +/− standard error (SE).

Figure 2.

Cell migration in injured meniscus explants cultured with inserted DMS. DAPI stained sections of explants cultured for 2 weeks (n=3-6 per group, 10x). a. Native non-injured meniscus. b. Injured meniscus cultured without DMS. c. Injured meniscus cultured with DMS. d. Injured meniscus cultured with DMS-Hep-PDGF-BB. e. Graph with numbers of cells at the borderline. f. Graph with total cell numbers in the explant. Data represent the mean of 6-8 values from 3 separate experiments.

Figure 3.

PDGFRβ positive cells in injured meniscus explants. Anti-PDGFRβ stained sections of explants cultured for 2 weeks (n=3-6 per group, 40x). a. Native non-injured meniscus. b. Injured meniscus cultured without DMS. c. Injured meniscus cultured with DMS. d. Injured meniscus cultured with DMS-Hep-PDGF-BB. e. Graph with numbers of migrated cells. Data represent the mean of 6-8 values from 3 separate experiments.

Figure 4.

Migratory and proliferating cells in injured meniscus explants. DAPI, β–actin and Ki67 stained sections of explants cultured for 2 weeks (n=3-6 per group). a-d. Injured meniscus cultured without DMS: (a) Merged; (b) DAPI; (c) β–actin; (d) Ki67. e-h. Injured meniscus cultured with DMS: (e) Merged; (f) DAPI; (g) β–actin (h) Ki67. i-l. Injured meniscus cultured with DMS-Hep-PDGF-BB: (i) Merged; (j) DAPI; (k) β–actin; (1) Ki67. m. β–actin staining of injured meniscus cultured with DMS-Hep-PDGF-BB. The elongated actin subunits (lamellipodia) are strongly stained lamella and are indicative of cell migration. Non-migrating cells show negative actin staining or short actin structures. The area marked by a yellow square in panel k is shown at higher magnification in panel m. n. Graph with % lamellipodium positive cells. o. Graph with the ratio of cell diameter (long/short diameter). p. Graph with Ki67 positive cells. a-l: 20x; m: 60x

Figure 5.

ECM formation in the injured meniscus explants. a-b. Safranin-O staining: Injured meniscus cultured with DMS for 2 and 4 weeks. c-d. Safranin-O staining: Injured meniscus cultured with DMS-Hep-PDGF-BB for 2 and 4 weeks. e-f. Picrosirius red staining: Injured meniscus cultured with DMS for 2 and 4 weeks. g-h. Picrosirius red staining: Injured meniscus cultured with DMS-Hep-PDGF-BB for 2 and 4 weeks. i-k. Differential interference contrast (DIC) imaging: (i) Injured meniscus without DMS; (j) with DMS; (k) with DMS-Hep-PDGF-BB. l-m. Safranin-O positive stained area (% of total area) and integration % between DMS and explant assessed by pircrosirius red staining and shown as % integrated interface of total interface area.

Figure 6.

Mechanical properties of the injured meniscus explants cultured with DMS. Injured explants were inserted with DMS or DMS-Hep-PDGF-BB and cultured for 2 and 4 weeks. Tensile properties were measured by pulling to failure. Data represent the mean +/− standard error (SE) of 7-10 values from 3 separate experiments.