Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-beta-tricalcium phosphate (PLGA-beta-TCP) scaffold (rASCs-COL/PLGA-beta-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-beta-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP; group B), the combination of acellular collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP; group C), and the PLGA-beta-TCP scaffold (group D). 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 of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-beta-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.
"Poly(DL-lactic-co-glycolic acid)/tricalcium phosphate (PLGA/TCP) composite has emerged as a promising bone graft scaffold but its application is limited because it is not an effective hydrophilic material. To tackle this problem, many methods have been introduced , one of which is to crosslink PLGA/TCP with collagen I [5, 6]. Notably, the crosslink of PLGA/TCP and collagen I could accelerate bone repair . "
[Show abstract][Hide abstract] ABSTRACT: Large osseous defect remains a serious clinical problem due to the lack of sufficient blood supply and it has been proposed that this situation can be relieved by accelerating the formation of new vessels in the process of bone defect repair. The aim of this study was to develop a new type of artificial bone by transferring the VEGF gene into marrow stromal cells (MSCs) and seeding them into a porous scaffold.
An adenovirus vector was employed to transfer the VEGF gene into MSCs and expression of the exogenous gene was confirmed by ELISA. Next the transduced cells were seeded into a collagen I modified PLGA/TCP scaffold. The constructed new complex artificial bone was then assessed for biocompatibility in vitro and blood vessel formation and bone formation in vivo.
We found that adenovirus mediated VEGF gene transfer into MSCs sustained VEGF expression in MSCs for 3 weeks. Porous scaffold PLGA/TCP made by rapid prototyping technology exhibited improved biocompatibility resulting from crosslinking with collagen I. Furthermore, the in vivo study showed that large amounts of blood vessels were detected histologically 1 week after artificial bone implantation, and significant bone formation was detected 8 weeks after implantation.
Our findings suggest that gene transfer of VEGF into MSCs combined with PLGA/TCP scaffold enhances bone repair in vivo by promoting vascularization.
Archives of Medical Science 02/2014; 10(1):174-81. DOI:10.5114/aoms.2012.30950 · 2.03 Impact Factor
"Bone formation of adipose-derived stem cells M. Jiang et al. osteogenic differentiation of MSCs (Hao et al., 2008). In accordance with previous studies, higher proliferation rate and increased osteogenesis after combining opASCs with collagen I hydrogel in vitro occurred. "
[Show abstract][Hide abstract] ABSTRACT: To explore the osteogenic potency of adipose-derived stem cells from osteoporotic patients (opASCs).
The opASCs were osteogenically induced in vitro with collagen I hydrogel or in culture plate. Detection of alkaline phosphatase (ALPase) and cell mineralization, and quantitative RT-PCR of collagen I, osteocalcin and bone sialoprotein were undertaken. Proliferation and morphology studies were also performed. After 14 days, opASCs-collagen I hydrogel composite was implanted into nude mice for 4 weeks prior to radiographic and histological analyses.
Staining of ALPase and cell mineralization was strongly positive in opASCs. Fibroblast-like opASCs induced with collagen I hydrogel were evenly distributed and proliferated at a higher rate than in culture plates, showing similar growth curves for both genders. Expression of ALPase activity, cell mineralization and osteogenic-specific genes were higher in opASCs with collagen I hydrogel(male samples exhibited better osteogenicity than female samples) than in culture plates. After implantation for 4 weeks, radiopaque area signifying new bone tissue was observed in opASCs-collagen I hydrogel composite, with no donor gender differences.
opASCs with collagen I hydrogel possesses adequate osteogenic potency and offers new opportunities for osteoporosis-related bone tissue engineering in male and female patients.
Cell Biology International 01/2014; 38(1). DOI:10.1002/cbin.10182 · 1.93 Impact Factor
"In fact, in an ectopic environment no resident osteogenic cells are present and therefore the true potential of the implanted cells can be rigorously assessed. Only very few studies based on such ectopic models managed to demonstrate bone formation in vivo by human (hASC) or rabbit expanded ASC (Hattori et al., 2006; Scherberich et al., 2007; Hao et al., 2008; Jeon et al., 2008; Liu et al., 2008; Müller et al., 2009 "
[Show abstract][Hide abstract] ABSTRACT: The current need for bone grafts in orthopedic and reconstructive surgery cannot be satisfied by autologous tissue transplant due to its limited availability and significant associated morbidity. Tissue engineering approaches could supply sufficient amounts of bone substitutes by exploiting the ability to harvest autologous osteogenic progenitors associated with suitable porous materials. However, the generation of clinically relevant-sized constructs is critically hampered by limited vascularization, with consequent engraftment and survival only of a thin outer shell, upon in vivo implantation. To overcome this limitation, different non-mutually exclusive approaches have recently been developed to promote or accelerate graft vascularization, from angiogenic growth factor gene delivery to surgical pre-vascularization of the construct before implantation. A simple, promising strategy involves the co-culture of vasculogenic cells to form an intrinsic vascular network inside the graft in vitro, which can rapidly anastomose with the host blood vessels in vivo. Recent data have shown that adipose tissue-derived stromal vascular fraction (SVF) may provide an efficient, convenient, and autologous source for both osteogenic and endothelial cells. When SVF progenitors were cultured in appropriate bioreactor systems and ectopically implanted, a functional vascular network connected to the host was formed concomitantly to bone formation. Future studies should aim at demonstrating that this approach effectively supports survival of scaled up cell-based bone grafts at an orthotopic site. The procedure should also be adapted to become compatible with an intra-operative timeline and complemented with the definition of suitable potency markers, to facilitate its development into a simplified, reproducible, and cost-effective clinical treatment.
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