The Application Of Injectable Cell-microsphere-gelatin Complex Combined With Platelet Lysate On Dental Regeneration

During the therapy process in dental clinic, dentists are often faced with problems like the narrow operative field and approach, the complex and variable anatomic morphology of common treatment sites and the limited operation time. These clinically practical difficulties make the use of biodegradable scaffold performed in vitro very limited and are disadvantageous for the popularity of tissue engineering techniques in the field of clinical treatment. Because of certain flow ability, injectable scaffold could transport the carried cells or bioactive substances to a specific site through simple and invasive injection method, making them crystalize in vivo to adapt to irregular lesions. In our previous study, we constructed two kinds of injectable biodegradable scaffolds, that is, nanofibrous microspheres(NF-MS) and gelatin scaffold. We later found that NF-MS could obviously promote the adhesion and proliferation of cells as well as tissue regeneration compared with traditional solid-interior microspheres(SI-MS). As a biomaterial that is characteristic of hydrogel and can lead to in vivo crosslinking curing, gelatin scaffold can encapsulate cells or bioactive substances and keep the aggregation of encapsulated substances like cells at specific sites for a set amount of time. The present study tried to construct the new mode of combined usage of the two biomaterials described above. Taking dental pulp stem cells(DPSCs) as seed cells and platelet lysate(PL) as growth factors, we performed initial basic study on the feasibility of the new mode to apply in the regeneration of dental pulp-dentin complex, which may provide new thoughts and experimental foundation on subsequently related research and clinical application. The main results and conclusion of this study are summarized as follows:1. Under the cultivating mode of DPSCs combined with microspheres, NF-MS could obviously promote the adhesion, proliferation and mineralization of DPSCs in vitro compared with SI-MS. Moreover, the introduction of PL could not only synergetically enlarge the advantages of NF-MS described above, but also make the activity of DPSCs on SI-MS much higher than that on NF-MS without the stimulation of PL at late culture phase, which indicated that PL could make up for the shortcomings of SI-MS to some extent. We injected the cell-microsphere complex with 7 days of in vitro mineralization subcutaneously into nude mice and found that the graft of NF-MS/PL group had more newly formed tissue and higher level of calcium than other groups. Moreover, the formation of dental pulp-dentin complex could also be observed histologically in that group. However, the quantitative indexes between NF-MS group and SI-MS/PL group are not significantly different, and generation of new tissue in SI-MS is comparatively weaker that the above three groups. Take together, these results demonstrate that when DPSCs are cultured with microspheres, NF-MS combined with PL can significantly promote the generation of odontogenic tissue involved in cell-microsphere complex both in vitro and in vivo.2. Under the cultivating mode of DPSCs combined with gelatin scaffold, gelatin could well support the growth of DPSCs. Furthermore, PL that was simultaneously encapsulated into gelatin hydrogel obviously promoted the proliferation of DPSCs during the in vitro culture period of 5 days for the cell-gelatin complex. Moreover, 8 weeks after the implantation of cell-gelatin complex into nude mice, only sporadic calcified tissues without characteristic structure could be observed in both Gelatin group and Gelatin/PL group. Collectively, these results indicated that although gelatin with some nutritive substances like PL could support and promote the activity of DPSCs at given time,the application of gelatin alone could not help the formation of characteristic hard tissues involved in DPSCs, especially for the material property of odontogenic tissues. Therefore, the effect of cell-galetin complex in the growth of DPSCs should be compared with the previously described cell-microsphere complex in subsequent experiments.3. Under the combined culture mode of cell, microsphere and gelatin, we adopted staging recombined way to construct the cell-microsphere-gelatin complex. In that case, DPSCs were first pre-cultured with microspheres in vitro with the stimulation of conditioned medium containing PL, which will adjust the cells to ideal stages. Later on, cell-microsphere complex were encapsulated by gelatin to form cell-microsphere-gelatin complex that could be used for both in vivo and in vitro observation. We found that under the environment of in vitro culture, PL involved in gelatin not only helped to keep the early acquired growth advantages of cells given by NF-MS/PL in the culture period of 5 days, but also further promote the in vitro proliferation ability DPSCs in the cell-microsphere-gelatin culture stage. Furthermore, the implantation experiments performed in nude mice showed that the structure of dental pulp-dentin complex could be observed in both NF-MS/Gelatin group and NF-MS/Gelatin-PL group, whereas in SI-MS/gelatin group and SI-MS/gelatin-PL group there was only osteoid tissue formed. There was no significant difference in calcium content among the groups based on same kind of microsphere. These results demonstrated that under the mode of cell-microsphere-gelatin complex, the effect that PL could promote the formation of odontogenic tissue from DPSCs depends on the prestimulation on cell-microsphere complex. When PL is encapsulated into gelatin, it can only function to keep the proliferation of DPSCs and promote the formation of non-mineralized tissues in vivo. This phenomenon probably results from the fact that gelatin is actually not an effective controlled release system for bioactive substances.In conclusion, the injectable cell-microsphere-gelatin complex constructed in the current study may be well applied in dental tissue engineering, especially for the field of dentin regeneration.