Effect of TGF-β3 on rabbit nucleus pulposus(NP) cells cultured in three-dimensional polylactic-co-glycolic acid scaffold in vitro / 中国生化药物杂志

Objective To evaluate the effect of TGF-β3 on rabbit nucleus pulposus( NP) cells cultured in three-dimensional polylactic-co-glycolic acid (PLGA)scaffold in vitro.Methods PLGA scaffolds were fabricated by particulate leaching method and soaked in rabbit NP cells suspension(1 × 106/scaffold).PLGA-seeded NP cells were devided into 4 groups: 100 ng/mL TGF-β3/PLGA,500 ng/mL TGF-β3/PLGA,1 μg/mL TGF-β3/PLGA, PLGA control group.Cell proliferation activity was measured using MTT assay.The glycosaminoglycan ( GAG ) analysis were performed by 1, 9-dimethylmethylene blue(DMMB) assay.mRNA expression was measured by quantitive PCR at each time point.Histological observation was performed to elucidate the morphological changes of NP cells in PLGA effected by TGF-β3.Results Higher cellular proliferation activity, GAG production,Collagen type II, Aggrean expression were observed in TGF-β3 /PLGA-seeded NP cells compared with PLGA control group on day-7,day-14,day-21(P<0.05). Higher dose of TGF-β3 exhibited intense cellular proliferation activity and peri-cellular matrix by increasing trend(P<0.05).Histological observation showed TGF-β3/PLGA developed more significant disc cells cluster than PLGA groups on day-21.Conclusion The 3D porous PLGA scaffold-seeded cells using TGF-β3 can promotes cell proliferation, and prompt extracellular matrix(ECM) production.It is a potential biotherapy for the treatment of disc degeneration.

Osteogenic Differentiation of Human Adipose-derived Stem Cells within PLGA(Poly(D,L-lactic-co-glycolic acid)) Scaffold in the Nude Mouse

PURPOSE: The object of this study was to evaluate the development of continuous osteogenic differentiation and bone formation after the subcutaneous implantation of the tissue-engineered bone, in vitro. METHODS: Human adipose-derived stem cells were obtained by proteolytic digestion of liposuction aspirates. Adipose-derived stem cells were seeded in PLGA scaffolds after being labeled with PKH26 and cultured in osteogenic differentiation media for 1 month. The PLGA scaffolds with osteogenic stimulated adipose-derived stem cells were implanted in subcutaneous layer of four nude mice. Osteogenesis was assessed by RT-PCR for mRNA of osteopontin and bone sialoprotein(BSP), and immunohistochemistry for osteocalcin, and von Kossa staining for calcification of extracellular matrix at 1 and 2 months. RESULTS: Implanted PLGA scaffold with adipose-derived stem cells were well vascularized, and PLGA scaffolds degraded and were substituted by host tissues. The mRNA of osteopontin and BSP was detected by RT-PCR in both osteogenic stimulation group and also osteocalcin was detected by immunohistochemistry at osteogenic stimulation 1 and 2 months, but no calcified extracellular deposit in von Kossa stain was found in all groups. CONCLUSION: In vivo, it could also maintain the characteristics of osteogenic differentiation that adipose- derived stem cells within PLGA scaffold after stimulation of osteogenic differentiation in vitro, but there were not normal bone formation in subcutaneous area. Another important factor to consider is in vivo, heterologous environment would have negative effect on bone formation as.[p1]

Chondrogenic Differentiation of Mesenchymal Stem Cells and Its Clinical Applications

Tissue engineering has the potential to provide cartilaginous constructs capable of restoring the normal function of native articular cartilage following joint injury or degradation. One approach to functional tissue engineering of cartilage involves the in vitro cultivation of tissue constructs by using: (i) chondrogenic cells that can be selected, expanded, and transfected to overexpress the genes of interest, (ii) scaffolds that provide a defined three-dimensional structure for tissue development and biodegrade at a controlled rate. Understanding the functional potential of the cells and the signaling mechanisms underlying their differentiation should lead to innovative protocols for clinical orthopaedic interventions. A large number of growth factors and hormones have been implicated in the regulation of chondrocyte biology, relatively little is known about the intracellular signaling pathways involved. We have tried to define the roles of specific TGF-betadependent signaling pathways involved in the regulation of chondrogenesis from human mesenchymal stem cells. Chondrogenesis induced by TGF-beta in alginate bead system was confirmed by examining cartilage specific type II collagen expression and aggrecan, whereas type I collagen expression was not affected by TGF-beta. Type II collagen mRNA expression was expressed strongly during chondrogenesis and MEK inhibition (U0126) resulted in complete down-regulation of type II collagen. In contrast, aggrecan expression was detected in same level by treatment of U0126. These results strongly suggest that the ERK signaling cascade is involved in TGF-beta induced-chondrogenesis signaling pathways and a role of its pathway is necessary over a longer period to promote type II collagen expression. However, their end product properties in vivo have not been well known. In this study, an articular cartilage from chondrogenic MSCs with PLGA scaffolds (75:25 and 65:35) were made and analyzed its biochemical, histological and mechanical properties in vitro and in vivo. And also, we evaluated the cartilage formation in vivo through the injection of cell-thermosensitive gel complex, a newly developed injectable material. At 12 weeks after PLGA scaffolds containing chondrogenic MSCs transplantation, the separated rabbit distal femur showed a good gross articular cartilage appearance in the transplanted site. In indentation test, compare to the native articular cartilage, the engineered cartilage from two types of (75:25 and 65:35) achieved up to 30-60% in mechanical stiffness. And also, a new model for cartilage formation in bladder, at 14 weeks after injection, we could find out mass formation in the submucosal area grossly. H&E staining, alcian blue staining and other special staining confirmed the chondrogenic differentiation in the mass. These cell therapy technologies can provide the possibility of clinical applications for vesicoureteral reflux and reflux esophagitis, and urinary incontinence as well as articular cartilage regeneration.

Comparison of Corneal Substitutes Using PLGA, Collagen, Collagen with Amniotic Membrane Homologous Cornea

PURPOSE: To evaluate the biocompatibility of the corneal substitutes using PLGA scaffold, type I collagen film, type I collagen film combined with amniotic membrane (AM) and lyophilized homologous cornea in severely damaged ocular surface disease. METHODS: Rabbits were distributed into four experimental groups: (1) Type 1 collagen film: group A, (2) Type I collagen film combined with AM: group B, (3) lyophilized homologous cornea: group C, and (4) PLGA scaffold: group D. Corneal substitute-like discs in 7 mm diameter were inserted into the intralamellar stromal pockets of severely damaged corneas, and then corneal windows in 3 mm diameter were made on the upper lamellar cornea in groups A, B, and C. In 2 months of follow up, clinical evaluation including corneal neovascularization, opacity and transparency of the graft materials was performed, and the inflammatory reaction and fibroplasia were evaluated histologically. RESULTS: Corneal windows were completely reepithelized by postoperative day 3 to 9, and more rapid reepithelization was shown in groups B and C, Corneal neovascularization, opacity, and inflammation decreased more in groups B and C. No inflammation and transparent graft material was shown in group C. Histological studies showed many corneal stromal fibroblasts in and around graft materials in groups B and C. CONCLUSIONS: The type I collagen film combined with AM and the lyophilized homologous cornea were more stable in the severely damaged cornea. These results could be useful for the development of corneal substitutes.