Construction Of A New Style Injectable Tissue Engineered Nucleus Pulposus In Vitro

Objectives:1. To observe microstructure and ultrastructure of Platelet-rich plasma gel,and to investigate its potential biological characteristics and the possibility of it used as a injectable scaffold material for tissue engineering nucleus pulposus.2. To explore the feasibility of platelet-rich plasma gel as scaffold and adipose derived stromal cells as seed cells construction of injectable tissue engineering nucleus pulposus in vitro.Methods:1. Twenty-five PRP gel samples were obtained by two-step centrifugation method. Before and after centrifugal platelet cells in the whole blood and PRP were determined by Automatic Blood Cell Analyzer. TGF-β1,PDGF-AB in the PRP gel and the whole blood were measured using the enzyme-linked immunosorbent assay method. and then PRP gel samples were observed by macroscopic observation,HE dyeing,transmission(TEM) and scanning electron microscopy (SEM).2. The third generation rabbit adipose derived stromal cells were isolated and cultured in vitro.After identified with flow cytometry,the adipose derived stromal cells were seeded into platelet-rich plasma gel scaffolds,and continued culturing in vitro.At the 2th,4th and 8 week,the cell-scaffold complexes were examined by macroscopy,scanning electron microscopy,histology stained and glycosaminoglycan quantitation measurement;the vitality of cells in the PRP gel scaffold was measured with BrdU immunofluorescence method.Messenger ribonucleic acids(mRNA)of HIF-1α,Aggrecan, typeⅡcollagen were determined by Real-time PCR.Results:1. The platelet concentration of PRP is approximately 458% of whole blood. TGF-β1,PDGF-AB were found in high concentrations in PRP gel samples. Both SEM and TEM showed that PRP gel mainly contains two components: a fibrillar material with striated band similar to fibrin filaments and a cellular component that contains platelet cells. 2. Results of flow cytometry showed that ADSCs were positive for CD90,CD105 and negative for CD45,CD34. Macroscopically,the cell-scaffold complexes were solidied into gel with smooth surfaces and better elasticity at each time points. The results of safranin O staining confirmed that the cells in platelet-rich plasma gel were capable of producing ECM.The content of glycosaminoglycan in cell-scaffold complexes cultrured at 4 weeks was higher than those at 2 weeks(P<0.05).and significant differences were also noted between 4 and 8 weeks(P<0.05). The results of HE staining and SEM demonstrated that the cells were well-distributed in the reticulate scaffold. BrdU immumofluorescence method showed that most ADSCs can survive and proliferate in the platelet-rich plasma gel scaffold at the 8 weeks.The gene expression of HIF-1α,Aggrecan,typeⅡcollagen were upregulated in the cell-scaffold complexes at 2 ,4and 8 weeks(P<0.05).Conclusions: PRP gel has good scaffold material characteristics for tissue engineering. The platelet-rich plasma gel scaffold makes it possible for rabbit ADSCs differentiated into nucleus pulposus-like cells after compound culture,and may be an ideal injectable scaffold material for constructing tissue engineering nucleus pulposus.