No abstract

A poly (lactide-co-glycolide) (PLGA) scaffold filled with fibrin gel, mesenchymal stem cells (MSCs) and poly(ethylene oxide)-b-poly (L-lysine) (PEO-b-PLL)/pDNA-TGF-β1 complexes was fabricated and applied in vivo for synchronized regeneration of cartilage and subchondral bone. The PEO-b-PLL/pDNA-TGF-β1 complexes could transfect MSCs in vitro to produce TGF-β1 in situ and up regulate the expression of chondrogenesis-related genes in the construct. The expression of heterogeneous TGF-β1 in vivo declined along with the prolongation of implantation time, and lasted for 3 and 6 weeks in the mRNA and protein levels, respectively. The constructs (Experimental group) of PLGA/fibrin gel/MSCs/(PEO-b-PLL/pDNA-TGF-β1 complexes) were implanted into the osteochondral defects of rabbits to restore the functional cartilages, with gene-absent constructs as the Control. After 12 weeks, the Experimental group regenerated the neo-cartilage and subchondral bone with abundant deposition of glycosaminoglycans (GAGs) and type II collagen. The regenerated tissues had good integration with the host tissues too. By contrast, the defects were only partially repaired by the Control constructs. qRT-PCR results demonstrated that expression of the chondrogenesis-marker genes in the Experimental group was significantly higher than that of the Control group, and was very close to that of the normal cartilage tissue.

Combination of gene therapy with tissue engineering can enhance the interplay between cells and matrix, leading to better restoration and regeneration of tissues and organs in vivo. In this study the PLGA/fibrin gel hybrids were employed to load lipofectamine/pDNA-TGF-β1 complexes and mesenchymal stem cells (MSCs) (experimental group), acting as a cartilage-mimetic tissue platform. The gene complexes distributed more evenly in the hybrid scaffolds, whereas they adhered onto the pore walls of the PLGA sponges. The filled fibrin gel rendered gene release in a slower manner, too. Moreover, the fibrin gel entrapped MSCs and contributed to a higher cell loading density in the hybrid constructs. In vivo assay showed that in the defects implanted with the experimental constructs both gene and protein expression levels of TGF-β1 were significantly higher than those of the fibrin-free group at weeks 1, 3, and 6 after surgery. The full articular cartilage defects repaired by the experimental group for 12 w were resurfaced by neo-tissues with a similar thickness, cell arrangement, and color to the normal neighboring cartilage and abundant glycosaminoglycans.

The poly(lactide-co-glycolide) (PLGA, LA/GA 75/25) sponges with different weight average molecular weights (Mw 52, 122, and 177 kDa) were fabricated and were used to build the constructs of PLGA/fibrin gel/mesenchymal stem cells (MSCs)/transforming growth factor-β1 (TGF-β1). The PLGA 177 with the highest Mw (177 kDa) had the fastest degradation rate at the initial stage, whereas the PLGA 122 had the moderate degradation rate and smallest mass loss. After implantation in rabbit knees for 12 weeks, the full-thickness defects (both cartilage and subchondral bone were destroyed with a diameter and depth of 4 mm) repaired by the PLGA 122 group had formed a hyaline cartilage-like tissue with abundant glycosaminoglycans on the top layer and subchondral bone on the bottom layer. The group also achieved the best macroscopic (11.3 ± 0.8) and histological scoring (Wakitani, 0.5 ± 0.6). To unveil the mechanism of the cartilage repair outcome and the PLGA degradation behaviors, the chondrogenesis-related genes, inflammatory cytokines, and matrix metalloproteinase (MMP) activity were analyzed by quantitative reverse transcription-polymerase chain reaction at week 1, 3, and 6 postsurgery. At each time point, the regenerated tissues by the PLGA 122 group had the highest mRNA expression of SOX9 and collagen type II, but the smallest mRNA expression of interleukin-1β and tumor necrosis factor α, and MMP-13 and MMP-3. In summary, as a scaffolding matrix, the PLGA with different Mw shows a huge difference in cartilage regeneration in vivo. The one with a moderate Mw (122 kDa) causes the weakest inflammatory response and results in the best cartilage regeneration.