The Fabrication Of Dentin-plasma Scaffolds In Vitro Studies

Objective:To explore the possibility of dentin and plasma using as the biological scaffolds. To preliminarily evaluate the biocompatibility of two scaffords. To find the most suitable manufacturing methos for the dentin and plasma scaffolds. Expect to construct a suitable strategy for clinical application of dental pulp biological treatment. To provide new basics and references for biology treatments of dental pulp or periapical diseases.Methods:The human dental pulp tissues were separated and cultured by using tissue block culture method and tissue block enzyme digestion method respectively. The form of DPCs was observed. And the capacity of mineralization was confirmed by alkaline phosphatase, alizarin red and von kossa staining.Respectively, DPCs were seeded on treated dentin and untreated dentin scaffold in vitro. After DPCs were grown on dentin scaffold for1day,1week, or1month, cell morphology was examined by scanning electron microscope (SEM). Extracellular matrix components were analyzed by using energy spectrum analysis (ESA)At the same time, DPCs, which were transfected with green fluorescence protein, were seeded in the plasma scaffold with different proportions in order to find a proper method to make plasma scaffold. The living conditions of DPCs were observed by using laser scanning confocal microscope (LSCM). And the degradation time of plasma scaffold were recorded during culture in vitro.Results:Culture and osteogenesis induction of DPCs:The original generation of DPCs which is cultured by using tissue block culture method needed about1month, while using tissue block enzyme digestion method needed2or3weeks. And then DPCs can be stably passaged, nearly7days before another generation formation. After osteogenic induction, DPCs showed a positive reaction for alkaline phosphatase, alizarin red and von kossa staining.The SEM and ESA showed that treated dentin root canal walls were cleaning, dentinal tubules could be clearly observed.DPCs could adhered firmly on dentin scaffolds, and they extended well. One week later cells secreted lots of calcium rich extracellular matrix. Especially, DPCs processes were observed extending into dentinal tubules which were just like odontoblastic processes.Plasma were collected by different centrifugation methods. Compared to the cell contents of Plasma which were collected by different centrifugation methods.For the first time, using200×g centrifugal force centrifuge10min. For the second time using200×g centrifugal force centrifuge10min two.By this method, platelet contained in the plasma which we collected were1.5times larger then whole blood. And lack of hemameba and erythrocyte.So it can be chosed as a proper plasma collected method.The LSCM observe showed that DPCs extend well in the plasma three-dimensional scaffolds. Adding100μL DMEM into1ml plasma when making plasma scaffold, by this method, the scaffold could maintain a long time in vitro, and DPCs distribution in this scaffold was relatively concentrated. So it could be used as plasma scaffold production proportion.Conclusion:1. Dental pulp cells is rich, containing a few undifferentiated mesenchymal cells, and is expected to become one option of seed cells for pulp regeneration.2. DPCs growth well both on the dentin scaffolds and in the plasma scaffolds in vitro. And then the degradation time of plasma scaffolds was proper for dental pulp regeneration. As a result, dentin and plasma scaffolds could be used as biology scaffolds for dentin pulp regeneration.