Restoration of osteochondral defects by implanting bilayered poly(lactide- co-glycolide) porous scaffolds in rabbit joints for 12 and 24 weeks

Abstract

Background: With the ageing of the population and the increase of sports injuries, the number of joint injuries has increased greatly. Tissue engineering or tissue regeneration is an important method to repair articular cartilage defects. While it has recently been paid much attention to use bilayered porous scaffolds to repair both cartilage and subchondral bone, it is interesting to examine to what extent a bilayer scaffold composed of the same kind of the biodegradable polymer poly(lactide-co-glycolide) (PLGA) can restore an osteochondral defect. Herein, we fabricated bilayered PLGA scaffolds and used a rabbit model to examine the efficacy of implanting the porous scaffolds with or without bone marrow mesenchymal stem cells (BMSCs). The present manuscript reports the regenerative potential up to 24 weeks.

Methods: The osteochondral defect, 4 mm in diameter and 5 mm in depth, was created in the medial condyle of each knee in 23 rabbits. The bilayered PLGA scaffolds with a pore size of 100-200 μm in the chondral layer and a pore size of 300-450 μm in the osseous layer, seeded with or without BMSCs in the chondral layer, were then transplanted into the osteochondral defect of each knee. The osteochondral defect created in the same manner was untreated to act as the control. At 12 and 24 weeks postoperatively, condyles were harvested and analyzed using histology, immunohistochemistry, real-time polymerase chain reaction, and biomechanical testing to evaluate the efficacy of osteochondral repair.

Results: No joint erosion, inflammation, swelling, or deformity was observed, and all animals maintained a full range of motion. Compared with the untreated blank group, the groups implanting the bilayered scaffolds with or without cells exhibited much better resurfacing, similar to the surrounding normal tissue. The histological scores of neotissues repaired by the scaffold with cells were closer to that of normal tissue. Although the biomechanical properties of neotissues were not as good as the normal tissue, no significant difference was found between the gene levels of neotissues repaired by the scaffold with or without cells and that of the normal tissue. The repair of the osteochondral defect tends to be stable 12 weeks after implantation.

Conclusions: Our bilayered PLGA porous scaffold supports long-term osteochondral repair via in vivo tissue engineering or regeneration, and its effect can be further facilitated under the scaffold seeded with allogenic BMSCs.

The translational potential of this article: The bilayered PLGA porous scaffold can facilitate the repair of osteochondral defects and has potential for application in osteochondral tissue engineering.

Keywords: Bilayered scaffold; Osteochondral defect; Poly(lactide-co-glycolide) (PLGA); Stem cell; Tissue engineering.

Figure 1

(A) Schematic of the osteochondral defect of the medial condyle in the knee joint, as indicated by the black circle. (B) Diagram of a bilayered PLGA scaffold. (C) Pore structure of the bilayered PLGA scaffold observed via SEM. The blue line indicates the boundary of the two layers. (D) An SEM image of BMSCs on the internal surfaces of the chondral layer of the porous scaffold after being cultured for one week. (E) Magnification of the rectangle of (D). The yellow arrows indicate cells adhered to the pore wall of the scaffold. BMSCs = bone marrow mesenchymal stem cells; PLGA = poly(lactide-co-glycolide); SEM = scanning electron microscopy.

Figure 2

Fluorescence micrographs of BMSCs on the chondral layer of porous scaffolds after 7 days of culture in the basal medium. Using the Live/Dead assay kit, it was found that the majority of the adhered BMSCs were viable, as indicated by green fluorescence, with only a few dead cells as indicated by red fluorescence. BMSCs = bone marrow mesenchymal stem cells.

Figure 3

(A–D) Photographs of osteochondral defects created in rabbit knee joints. (A) A defect was created in the medial femoral condyle by applying a surgical drill bit; (B) the defect was 4 mm in diameter and 5 mm in thickness in the medial condyles of the knee joint; (C and D) the medial condyle defect was implanted with the bilayered PLGA scaffold seeded with BMSCs. (E) The global and cross-sectional views of reparative tissues 12 weeks after implantation. (F) The global and cross-sectional views of reparative tissues 24 weeks after implantation. The red circles in (E) indicate the original defect region. The yellow arrows in (F) indicate the interfaces between the neotissues and native osteochondral tissues. At 12 weeks, the defects in the medial condyles were repaired with a bilayered PLGA scaffold with or without cells. The defects were covered with an irregular tissue. The obvious vacancy defect could be seen in the blank control group at 12 and 24 weeks after operations. At 24 weeks, the neotissue in the condyle-implanted cell-seeded scaffold exhibited better resurfacing than that in the condyle-implanted scaffold, as indicated by the red dotted rings in the defect sites.

Figure 4

Histological scores for reparative tissues. The histogram shows that the scores of tissues repaired by scaffolds with or without cells were lower than those of tissues in the blank group, and the scores of tissues repaired by scaffolds with cells were both lower than those of tissues repaired by scaffolds at 12 weeks and 24 weeks after implantation (“*”: p < 0.05). And the scores of tissues repaired by scaffolds with or without cells were higher than the score of the normal tissue. The difference between the group of scaffold with cells and the normal tissue was not significant (p > 0.05). The scores between 12 and 24 weeks for each group did not exhibit a significant difference (p > 0.05).

Figure 5

(A) The histological images of repaired tissues 12 weeks after implantation; (B) the histological images of repaired tissues 24 weeks after implantation (H&E, safranin O/fast green, and toluidine blue staining). The length of the white vertical line is 1 mm, which is used to measure the thickness of the cartilage. The white asterisks indicate the remnant scaffold materials. H&E = haematoxylin and eosin.

Figure 6

Immunohistochemical images of tissues 24 weeks after implantation. For collagen type II, the expression in the chondral region of tissues repaired and of the normal tissue was positive. The images in the first row come from magnification of the blue rectangles in groups of scaffold, scaffold with cells, and normal, respectively. For collagen type I, the expression in the subchondral region of tissues repaired and of the normal tissue was positive.

Figure 7

(A) The relative levels of collagen type II and type I were assessed with real-time PCR in each group. For collagen type I, the relative level in the scaffold group at 24 weeks after implantation was higher than that in other groups. For collagen type II, the relative level in the scaffold with cells group at 24 weeks after implantation was higher than that in other groups, close to that of the normal group. GAPDH expression was used for normalization. All values are expressed as mean ± SD. (B) Nucleotide primers used for real-time PCR. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; PCR = polymerase chain reaction; SD = standard deviation.

Figure 8

(A) Typical stress–strain curve of repaired tissues and normal osteochondral tissues. (B) The compressive moduli in the groups of scaffolds with and without cells and normal osteochondral tissues. n = 5; “*”: p < 0.05.