Construction of injectable tissue engineered nucleus pulposus by using complex of platelet-rich plasma gel scaffold and adipose derived stromal cells in vitro

English Keywords:Tissue engineering nucleus pulposus  Platelet-rich plasma gel  Adipose derived stromal cells  Rabbit

English Abstract:

  【Abstract】 Objective：To explore the feasibility of complex of platelet-rich plasma（PRP） gel scaffold and adipose derived stromal cells（ADSCs） as seed cells to construct injectable tissue engineered nucleus pulposus in vitro.Methed：The third massaging rabbit ADSCs were identified by flow ctytometry in vitro，the ADSCs were seeded into ＰＲＰ gel scaffolds and continued culturing in vitro.At the 2nd，4th and 8th week，the PRP gel-cell complexes were examined macroscopically and histologically.The vitality of cells in the PRP gel scaffold was measured by BrdU immunofluorescence method.The glycosaminoglycans（GAG） in the PRP gel-cell complexes were measured by spectrophotometry.Messenger ribonucleic acids（mRNA） of hypoxia-inducible factor-1α（HIF-1α），Aggrecan，type Ⅱ collagen were determined by real-time PCR.Result：Flow cytometry showed that ADSCs were positive for CD90 and negative for CD45.Macroscopically，the PRP gel-cell complexes presented with smooth surface and well elasticity at each time point.The safranin O staining showed no positive dyeing cells at the 2nd week，weak positive dyeing cells at surrounding region at the 4th weeks，and enhanced obviously at 8th week，which was visible to surrounding cells with red positive dyeing.The results of HE staining and scanning electron micrograp（SEM） demonstrated that the cells were well-distributed in the reticulate scaffold.BrdU immumofluorescence showed that most ADSCs can survive and proliferate in the PRP gel scaffold.Positive cells at the 4th week were much more than those at the 2nd week（P<0.05），and significant differences were also noted between the 4th and 8th week（P<0.05）.The content of glycosaminoglycan at the 4th week was higher than those at the 2nd week（P<0.05），and significant differences were also noted between the 4th and 8th week（P<0.05）.Messenger ribonucleic acids（mRNA） of 3 genes at the 4th week were much more than those at the 2nd week（P<0.05），and significant differences were also noted between the 4th and 8th week（P<0.05）.Conclusion：The PRP gel scaffold allows for rabbit ADSCs differentiated into nucleus pulposus-like cells after compound culture in vitro，the complexes can be used as injectable tissue engineered nucleus pulposus.