Effectiveness of BMP-2 and PDGF-BB Adsorption onto a Collagen/Collagen-Magnesium-Hydroxyapatite Scaffold in Weight-Bearing and Non-Weight-Bearing Osteochondral Defect Bone Repair: In Vitro, Ex Vivo and In Vivo Evaluation

Abstract

Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.

Keywords: animal model; biocompatible materials; bone morphogenetic proteins; osteochondral lesion; platelet-derived growth factor; regenerative medicine; tissue engineering; weight-bearing.

Figure 1

SEM images of (A) the cartilaginous layer (collagen) and (B) the bone layer (60% collagen and 40% magnesium-hydroxyapatite).

Figure 2

Bone morphogenetic protein 2 (BMP-2) and platelet-derived growth factor-BB (PDGF-BB) released from the different layers of the Col/Col-Mg-HAp scaffold. (A) In vitro release of BMP-2 from the different layers of Col/Col-Mg-HAp scaffold over a 14-day period. (B) In vitro release of PDGF-BB from the different layers of the Col/Col-Mg-HAp scaffold over a 7-day period. The release of BMP-2 and PDGF-BB is presented as the released dose at each time point. Data points indicate the mean ± SD of 3 samples per time point.

Figure 3

Adsorption of BMP-2 or PDGF-BB might improve the cell recruitment property ex vivo. (A) Representative images of the 3-week constructs stained with H&E. Scale bars indicate 1 mm and 100 µm, respectively. Magnified images showed cell infiltration at the periphery (black square), in the centre (red square) of the collagen-only layer, and in the Col-Mg-HAp layer (blue square) of the scaffold. Black arrows indicate infiltrated cells. (B) The number of cells infiltrated into the scaffolds. Each bar indicates the mean ± SD of 4 samples per condition. * p < 0.05 analysed by a Kruskal–Wallis test.

Figure 4

The effect of BMP-2 or PDGF-BB adsorption onto Col/Col-Mg-HAp scaffolds on osteochondral defect repair in a semi-orthotopic model in vivo. (A) Representative images of the 4-week repair constructs stained with Safranin O. Scale bars indicate 1 mm. Black arrows indicate cartilage-like tissue. Red arrows indicate blood vessels. (B) The percentage of the defect filled with osteochondral tissue (%) in the osteochondral defects at 4 weeks. (C) Representative images of the 8-week repair constructs stained with Safranin O, and the 8-week CT images. The best, average, and worst repaired samples are presented based on osteochondral tissue volume (%). Scale bars indicate 1 mm. Red arrows indicate blood vessels. (D) The percentage of the defect filled with osteochondral tissue (%) in the osteochondral defects at 8 weeks. Scale bars indicate 1 mm.

Figure 5

Tissue repair in non-weight-bearing trochlear groove defects. (A) Representative micro-CT reconstructions treated with either scaffold-only or scaffold adsorbed with BMP-2. The best, average, and worst repaired samples are presented based on BV/TV. The scale bar indicates 2 mm. (B) BV/TV, trabecular thickness (Tb.Th [mm]), trabecular number (Tb.N [1/mm]), and trabecular separation (Tb.Sp [mm]) in the bone defects after 6 months. (C) RGB (Alcian Blue, Fast Green, and Picrosirius Red) staining and macroscopic cross-sectional view of osteochondral defects treated with either scaffold-only or scaffold adsorbed with BMP-2. The best, average, and worst repaired samples are presented. White squares indicate 6 ∗ 6 mm osteochondral defects. Black arrows indicate the structure with only bone marrow. The scale bar indicates 5 mm. (D) The percentage of tissue volume calculated in the bone defects (BD). (E) The percentage of tissue volume calculated in the cartilage defects (CD).

Figure 6

Bone repair in the weight-bearing femoral condyle defects deteriorated with the adsorption of BMP-2 onto the scaffold. (A) Representative micro-CT reconstructions treated with either scaffold-only or scaffold adsorbed with BMP-2. The best, average, and worst repaired samples are presented based on BV/TV. The scale bar indicates 2 mm. (B) BV/TV, trabecular thickness (Tb.Th [mm]), trabecular number (Tb.N [1/mm]), and trabecular separation (Tb.Sp [mm]) in the bone defects after 6 months. Tissue repair in the femoral condyle defects. (C) RGB (Alcian Blue, Fast Green, and Picrosirius Red) staining and macroscopic images of osteochondral defects treated with either scaffold-only or scaffold adsorbed with BMP-2. The best, average, and worst repaired samples are presented. White squares indicate 6 ∗ 6 mm osteochondral defects. The scale bar indicates 5 mm. (D) The percentage of tissue volume calculated in the bone defects (BD). * p < 0.05 in fibrous tissue, # p < 0.05 in osteochondral (cartilage-like and bone-like) tissue. (E) The percentage of tissue volume calculated in the cartilage defects (CD).