The Construction And Determination Of The Sustained-released Microspheres Combing With The Partition Type Tissue-engineered Tube Scaffold Of The Spinal Cord

Objective We prepared the partition type tube scaffold(PtTS)of the spinal cord and chitosan microspheres(CMSs).CMSs could be used to encapsulate platelet-derived growth factor(PDGF).Muse cells were induced and differentiated into neural precursor cells(Muse-NPCs).When PDGF-CMSs were fixed to PtTS and a certain concentration of Muse-NPCs implanted into PtTS,they formed a new artificial tissue engineering spinal cord,which could be used to provide a good preparation method of materials for the future treatment of repairing spinal cord injury in vivo.Methods We prepared PtTS by freeze drying method;by emulsion solvent evaporation technique,we prepared chitosan microspheres encapsulating BSA and PDGF;we used scanning electron microscope to detect the particle size of CMSs;gravimetric method were used to detected the swelling index of CMSs;the BCA assay was used to detected encapsulation efficiency of CMSs;the ELISA kit was used to detect the sustained-release curve of CMSs;density gradient centrifugation and differential adhesion method were used to isolate human bone marrow stromal cells(hBMSCs)from the bone marrow of healthy adult;when hBMSCs were amplificated in vitro,Muse cells were separated from them,neural induction medium could induce Muse cells into Muse-NPCs;PtTS,CMSs were separately co-culutured with Muse-NPCs,CCK-8 assay tested the biological effects of materials on Muse-NPCs;Transwell experiment and immunofluorescence staining studies observed the proliferation,migration and differentiation effects of PDGF on Muse-NPCs;Muse-NPCs were seeded into PtTS,we evaluated the effect of PDGF on the attachmnet and growth of Muse-NPCSs through morphological observation.Results We had prepared PtTS which had a high rate of formation and had better internal porosity which was suitable for the adhesion of CMSs.CMSs showed spherical shape and had reasonable particle size distribution which were suitable for encapsulating BSA,PDGF and other biological macromolecules.We detected swelling index of CMSs and the CMSs incorporating 3mg,6mg and 12 mg BSA.We found that the swelling index of four groups were rising within 1~12h,the swelling index of four groups almost reached equilibrium after 12 h.The encapsulation efficiency of BSA-CMSs was 72.33% ± 1.28%.The CMSs prepared in the presence of 10 mg BSA had cumulatively released about 17% of the total encapsulated BSA in 28 days.After separating Muse cells from hBMSCs,neural induction medium induced Muse cells to differentiate into Muse-NPCs,Muse-NPCs grew in the manner of spherical suspension and had characteristics of neural stem cells in vitro.PtTS and CMSs respectively co-cultured with Muse cells,they had good biocompatibility,PtTS and CMSs had no toxicity on Muse-NPCs.Neural induction medium containing PDGF were able to promote proliferation and migration of Muse-NPCs and promoted their differentiation into neurons.PDGF combined with PtTS to form a composite scaffold could promote the attachment and survival of Muse-NPCs.Conclusions We had successfully prepared PtTS which had a high rate of formation and better internal porosity.We also prepared CMSs with reasonable particle size distribution,they were suitable for encapsulating BSA,PDGF and other biological macromolecules.CMSs were fixed into PtTS,the combination of them were good.PDGF could effectively promote the proliferation and migration of Muse-NPCs and promoted their differentiation into neurons.Muse-NPCs were seeded into PtTS,PDGF could promote the the attachment and growth of Muse-NPCs in the compossite scaffold,they lay the foundation of using new tissue-engineered tube scaffold of the spinal cord for future repairing spinal cord injury in vivo.