| The reconstruction of bone defects and nonunions after trauma, tumor resection has long been a problem for clinicians. The most effective and widely used method up to now is autologous cancellous bone grafting. Whereas several unavoidable drawbacks such as limited graft availability, donor site morbidity as well as increased operation time put researchers to find other alternatives. Recently, the cell-based bone tissue engineering has been thought to be a new therapy. In this study, we isolated a new stem cell population from the subcutaneous adipose tissue of Japanese White rabbit. Firstly the multipotency of rabbit adipose-derived stem cells(rADSCs) was explored and then the in vitro osteogenic differentiaton was fully evaluated. After which, the rADSCs was suspended in collagen I gel and combined with a porous PLGA-β-TCP scaffold to fabricate a new biomimetic bone tissue engineering composite: rADSCs-COL/PLGA-β-TCP. The in vitro osteogenic differentiation of rADSCs-COL/PLGA-β-TCP was firstly assayed. Then the osteogenesis of this new biomimetic composite was thoroughly determined at ectopic site and bone defect site.1. In vitro osteogenic differentiation of rADSCs and related researchThe rADSCs were isolated from the subcutaneous adipose tissue of suprascapular region from Japanese White rabbit by collagen I digestion. The primary-cultured rADSCs were firstly identified by STRO-1 immunocytochemistry. Then the multipotency of rADSCs toward osteogenic, chondrogenic and adipogenic lineages were detected under different conditioned media. During the osteogenic differentiation, the specific markers during osteogenesis was examined including type I collagen, alkaline phosphatase and extracellular matrix mineralization. Results indicated that the rADSCs were STRO-1 positive and have multipotency toward osteogenic, chondrogenic and adipogenic lineages. During osteogenic differentiation, the expression of collagen I, alkaline phosphatase and calcium deposit of rADSCs were all positive. In summary, rADSCs have favorable multipotency, especially toward osteogenic lineages. Therefore, the rADSCs can be used as a potential alternative to various kinds of seed cells in bone tissue engineering.2. In vitro and in vivo osteogenic differentiation of a novel bone tissue engineering construct by using collagen I gel to suspend rADSCs in a porous PLGA-β-TCP scaffoldFirst of all, collagen I gel was used to suspend the passage 3 rADSCs in the PLGA-β-TCP scaffold to fabricate a new biomimetic bone tissue engineering composite: rADSCs-COL/PLGA-β-TCP. Meanwhile, the composites by single combination of rADSCs and PLGA-β-TCP (rADSCs/PLGA-β-TCP), combination of acellular collagen I gel and PLGA-β-TCP (COL/PLGA-β-TCP) and PLGA-β-TCP scaffold were also prepared as control groups. Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks'in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in rADSCs-COL/PLGA-β-TCP composite was significantly higher than rADSCs/PLGA-β-TCP (p < 0.05, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in rADSCs-COL/PLGA-β-TCP composite whereas no apparent calcification was observed in rADSCs/PLGA-β-TCP, COL/PLGA-β-TCP and PLGA-β-TCP (n = 4). In rADSCs-COL/PLGA-β-TCP composite, woven bone with a trabecular structure was formed while in rADSCs/PLGA-β-TCP composite, only osteiod tissue was observed. Bone forming area in rADSCs-COL/PLGA-β-TCP composite was significantly higher than rADSCs/PLGA-β-TCP composite (p < 0.05, n = 4). No bone formation was observed in COL/PLGA-β-TCP or PLGA-β-TCP (n = 4). In conclusion, by using collagen I gel to suspend rADSCs in porous PLGA-β-TCP scaffold, osteogenic differentiation of rADSCs can be improved and homogeneous bone tissue can be successfully formed in vivo. Therefore, the successful construction of rADSCs-COL/PLGA-β-TCP composite cast a new light on the various methods for fabrication of bone tissue engineering composites.3. Repair of segmental radial defect in rabbit with a new biomimetic construct by using collagen I gel to suspend autologous adipose-derived stem cells into PLGA-β-TCP scaffold (rADSCs-COL/PLGA-β-TCP)To further evaluate the osteogenesis of rADSCs-COL/PLGA-β-TCP composite, in this study, we explore the possibility of repairing a 1.5cm segemental radial defect in rabbit by using rADSCs-COL/PLGA-β-TCP composite with autologous rADSCs. Firsly, the 1.5cm segemental radial defects were created bilaterally. Then the rADSCs-COL/PLGA-β-TCP composite, rADSCs/PLGA-β-TCP composite, COL/PLGA-β-TCP composite and PLGA-β-TCP were implanted with the untreated radial defect as blank control. 30 rabbits were used in this experiment. 12w, 24w, 36w after implantation, 10 animals were sacrificed and the constructs of different groups were harvested. Radiographic examination with Lane-Sandhu X ray semi-quantitative analysis was firstly carried out. 36w after implantion, microCT scanning was also performed for the constructs. After which, the constructs were processed as paraffin sections for HE staining and modified Masson Trichrome staining. In addition, histomorphometric analyses of new bone formation and scaffold degradation in different groups were implemented at the same time. Radiographic analyses indicated that the subjects treated with rADSCs-COL/PLGA-β-TCP composites exhibited the best therapeutic effect compared with rADSCs/PLGA-β-TCP composites, COL/PLGA-β-TCP composites and PLGA-β-TCP scaffold (p < 0.05, n = 4). 36w after implantation, bone integrity was realized and bone remoulding was almost fininshed in rADSCs-COL/PLGA-β-TCP groups, whereas no radiographic evidence for new bone formation was observed in rADSCs/PLGA-β-TCP groups, COL/PLGA-β-TCP groups and PLGA-β-TCP groups. MicroCT scaning also confirm this point. Similar to radiographic evidence, histological analyses demonstrated that in rADSCs-COL/PLGA-β-TCP groups, new bone tissue was evenly distributed in the pores of scaffolds after 12w implantation, bone integrity was reconstructed but bone marrow was not recanalized after 24w implantation, bone integrity was completed and bone marrow was recanalized after 36w implantation (n=4), whereas in rADSCs/PLGA-β-TCP groups, no apparent bone formation was observed (n=4). Meanwhile, no bone formation was observed in COL/PLGA-β-TCP groups and PLGA-β-TCP groups during the whole implantation period (n=12). Histomorphometric analyses indicatd that the new bone forming area as well as scaffold degradation rate in rADSCs-COL/PLGA-β-TCP groups were all significantly higher than the other three groups at different time points (p < 0.05, n = 4). In conclusion, the rADSCs-COL/PLGA-β-TCP composite exhibits a favorable osteogenesis, biocompatibility and biodegradability during the repair of autologous segemental radial defect model and therefore provides strong support for its possible application in clinical area.
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