Repair Of Osteochondral Defects Using Bilayered PLGA Scaffolds Loaded With BMSCs In Rabbits

Part. IFabrication and biocompatibility of bilayered poly(lactic-co-glycolide) porous scaffoldsObjective:To discuss the fabricating feasibility and biocompatibility of bilayered poly(lactic-co-glycolide)(PLGA) scaffolds with different pore sizes and different porosities.Methods:By a "room-temperature" compression molding/particulate leaching method, bilayered PLGA scaffolds with different pore sizes and different porosities were fabricated. After being dissolved in dichloromethane, PLGA was mixed with sodium chloride, acting as porogen, with different sizes (50-450μm), or PLGA was mixed with the same size sodium chloride by different mass ratio. Then these mixtures were filled in the mould and compressed for24hours. After demoulding, the columnar mixtures were formed. Each two of columna mixtures with different sizes or different contents of porogen were glued with dichloromethane, and then were tailored to specific size. The fabrication of bilayered scaffold was completed after the porogen was leached with deionized water. In addition, these belayered scaffolds were seeded with bone marrow mesenchymal stem cells (BMSCs) from rabbit. After being cultured in vitro for one week, the composites of cell-scaffold were oberserved under scanning electronic microscope (SEM).Results:We successfully obtained different bilayered PLGA scaffolds with4mm in diameter and5mm in height (the upper layer:1mm and the lower layer:4mm), including five kinds of different pore sizes and three kinds of different porosities. Under the observation of SEM, we found the good morphology of pores, interconnection in pores and in the well-integrated two layers in the scaffolds. After the scaffolds seeded with BMSCs were cultured in vitro, the cell adhered to the wall of pores well in all the scaffolds and extral cellular matrix deposition was found.Conclusions:By a "room-temperature" compression molding/particulate leaching method, bilayered PLGA scaffolds with different pore sizes and different porosities in the two layers can be fabricated. The bilayered PLGA scaffolds have good biocompatibility. Part. ⅡThe effects of pore size in bilayered poly(lactide-co-glycolide) scaffolds on restoring osteochondral defects in rabbitsObjective:The present study aims at investigating the in vivo effect of pore sizes in bilayered scaffolds on restoring osteochondral defects and the result of bilayered PLGA scaffolds repairing osteochondral defects in rabbits.Methods:By a "room-temperature" compression molding/particulate leaching method, five groups of integrated bilayered poly(lactic-co-glycolide)(PLGA) scaffolds with different pore sizes were fabricated and the size of porogen was50-450μm. After bone marrow mesenchymal stem cells (BMSCs) from New Zealand White rabbit were labeled with Dil, BMSCs1×106cells/μL were evenly seeded drop by drop into the chondral layer of bilayered scaffolds. The composites of cell-scaffold were cultured in vitro for one week and then were observed under SEM. These bilayered scaffolds, which were seeded with or without allogenic BMSCs, were implanted into the osteochondral defects (4mm in diameter and5mm in depth) in the knee joints of rabbits. Six or twelve weeks after implantation, the samples were harvested and sectioned for histological analysis and immunohistochemistry. At6weeks, some samples were cut into halves, frozen-sectioned and stained by DAPI to track BMSCs under the observation of fluorescent microscope. At12weeks, the samples were cut into halves and were examined the relative expression of genes (Collagen type Ⅱ, Collagen type Ⅰ and Aggrecan) by real-time PCR.Results:Five kinds of bilayered PLGA scaffolds with different pore sizes (50-450μm) were obtained. BMSCs grew well in the scaffolds under the observation of SEM. At6weeks after implantation, Dil-labeled BMSCs survived in the defect region under the observation of fluorescent microscope. At12weeks, Cell-seeded scaffold B (100-200μm pore size in the chondral layer and300-450μm in the osseous layer) showed that the neotissue was integrated completely with native tissue without obvious borders. The defects repaired with cell-seeded scaffold B were entirely filled with a smooth cartilage-like tissue similar to the adjacent normal cartilage. The tissues repaired by other implants were not as smooth as those by cell-seeded scaffold B. The mean total score for cell-seeded scaffold B was the lowest, which was significantly different, compared to other implants (P<0.05). Immunohistochemical experiments demon-strated stronger expression of collagen type Ⅱ in the articular surface and robust expression of collagen type I in the subchondral region. In cell-seeded scaffold B group, the relative level of collagen type Ⅱ was significantly higher than those from other experimental groups (P<0.05). Although the mean level of aggrecan was higher in cell-seeded scaffold B, the difference was not statistically significant compared to other four groups (P>0.05). As for the relative level of collagen type Ⅰ, we observed a significant difference between scaffolds B and D (300-450μm pore size in the chondral layer and100-200μm in the osseous layer)(P<0.05), and the difference among other scaffolds was not statistically significant (P>0.05).Conclusions:The integrated bilayered PLGA scaffolds support the simultaneous regeneration of cartilage and subchondral bone. And the group of100-200μm pore size in the chondral layer and300-450μm in the osseous layer is optimal for bilayered PLGA scaffolds to repair the osteochondral defect. Different pore sizes in the bilayered scaffolds have a remarkable effect on repairing the osteochondral defect. Part. ⅢThe effects of porosity of bilayered poly(lactide-co-glycolide) scaffolds on repairing osteochondral defects in rabbitsObjective:To investigate the feasibility of bilayered PLGA scaffolds with different porosities repairing osteochondral defects in rabbits and analyse the in vivo effect of porosity in bilayered scaffolds on repairing osteochondral defects.Methods:The porogen, sodium chloride with200-300μm, was screened and mixed with PLGA by different mass ratio. Three kinds of integrated bilayered PLGA scaffolds with different porosities were fabricated by a "room-temperature" compression molding/particulate leaching method. Mechanical test was made for three kinds of bilayered scaffolds. After bone marrow mesenchymal stem cells (BMSCs) from rabbit were labeled with DiI, BMSCs1×106cells/μL were evenly seeded into the chondral layer of bilayered scaffolds. The composites of cell-scaffold were cultured in vitro for one week and then were observed under SEM. These bilayered scaffolds, which were seeded with or without allogenic BMSCs, were implanted into the osteochondral defects (4mm in diameter and5mm in depth) in the knee joints of rabbits. Six or twelve weeks after implantation, the samples were harvested for gross evaluation and sectioned for histological analysis and immunohistochemistry. At6weeks, some samples were frozen-sectioned and stained by DAPI to track BMSCs under the observation of fluorescent microscope. At12weeks, the samples were cut into halves and were examined the relative expression of genes (Collagen type II, Collagen type I and Aggrecan) by real-time PCR.Results:Three kinds of bilayered PLGA scaffolds with different porosities (77%-92%) were obtained and the pore size of these scaffolds was200-300μm. The mechanical properties of bilayered scaffolds were measured, For scaffold A (chondral layer:92%and osseous layer:77%in porosity), two slopes were observed in the stress-strain curves:E1(3.8±1.1MPa) reflects the modulus of the cartilage layer, and E2(29.1±5.0MPa), of the subchondral layer. For scaffold B (chondral layer:85%and osseous layer:85%in porosity) and C (chondral layer:77%and osseous layer:92%in porosity), only one slope was measured and the compressive modulus was16.1±3.2MPa and0.7±0.2MPa, respectively. Six weeks after implantation, Dil-labeled BMSCs survived in the defect region under the observation of fluorescent microscope. At12weeks, Cell-seeded scaffold A showed that the neotissue was integrated completely with native tissue without obvious borders. The defects repaired with cell-seeded scaffold A were entirely filled with a smooth cartilage-like tissue similar to the adjacent normal cartilage. The tissues repaired by other implants were not as smooth as those by cell-seeded scaffold A. The mean total score for cell-seeded scaffold A was the highest (Maximum-score), which was significantly different, compared to scaffold C(P<0.05). Immunohistochemical experiments demonstrated stronger expression of collagen type Ⅱ in the articular surface and robust expression of collagen type Ⅰ in the subchondral region. In cell-seeded scaffold A, the relative level of collagen type Ⅱ was significantly higher than that from scaffold C (P<0.05). Although the mean level of aggrecan and collagen type Ⅰ was higher in cell-seeded scaffold A, the difference was not statistically significant compared to the cell-seeded scaffold B and C (P>0.05).Conclusions:The integrated bilayered PLGA scaffolds with different porosities support the simultaneous regeneration of cartilage and subchondral bone. And the group of92%porosity in the chondral layer and77%porosity in the osseous layer is optimal for bilayered PLGA scaffolds to repair the osteochondral defect. Different porosities in the bilayered scaffold have an obvious effect on repairing the osteochondral defect.