| Traumatic brain injury (TBI) is a brain disease caused by external attack on the head. With high mortality and high morbidity, TBI brings heavy burden to the society and family, therefore, it is urgent to find a positive and effective treatment. Stem cell replacement therapy provides a new strategy and hope for the treatment of TBI.Human umbilical cord mesenchymal stem cells (hUC-MSCs),which derived from human umbilical cord tissue, has the characteristics of no ethical arguments, widely source, multi differentiation potential, low immunogenicity, thus, they are the good cell resources in tissue engineering. However, the low survival rate and insufficient quantity of transplanted stem cells still restrict the application of stem cell transplantation. In order to improve the biological activity and therapeutic effect of stem cells, it is essential to create a micro environment suitable for the growth and survival of the transplanted stem cells.The alginate (SA) hydrogel tissue engineering scaffold has a three-dimensional network structure. Similar to the extracellular matrix (ECM), SA provides three-dimensional support for cell growth. Besides, SA provides a channel for nutrient and exchange, as well as the excretion of metabolites . Hyaluronan (HA), one of the main components of the natural extracellular matrix, can bind to the specific receptor on the surface of stem cells, and promote proliferation, adhesion, signal transduction, and differentiation of stem cells. Therefore, in this study, we prepared SA/HA tissue engineering scaffold and explored the therapeutic effects of transplantation of human mesenchymal stem cells loaded on scaffolds for TBI.ObjectivesIn this study, we intend to prepare SA/HA tissue engineering scaffold in vitro, and research their physicochemical properties; study the biological activity of hUC-MSCs loaded on SA/HA hydrogel scaffolds,in order to create a more suitable environment for hUC-MSCs; and then explore the neurological repair effects of hUC-MSCs combined with SA/HA hydrogel scaffolds on TBI rats and its possible mechanism, so as to provide new strategies for TBI treatment by stem cell transplantation.Methods1. Evaluation of biocompatibility of SA/HA hydrogel scaffold and hUC-MSCs in vitroFour different ratio (1%-0.8,1%-0.5,0.5%-0.5,0.5%-0.2) of alginate hydrogels were prepared by internal gel method. The morphology were observed by SEM; The gelation time were measured by tube-tube tilt method. The moisture content and degradation performance were determined by freeze-drying and weighing method.The elastic modulus were measured by rheometer. The expression of hUC-MSCs were detected by flow cytometry. The three-dimensional distribution of hUC-MSCs in hydrogels were observed by laser confocal microscopy. Live-dead staining was used to detect the survival of stem cells in different groups. CCK- 8 assay were performed to detect the proliferation of hUC-MSCs in different groups.2. The effect of SA/HA hydrogel scaffold combined with hUC-MSCs on neuronal repair in TBI rats in vivoTraumatic brain injury model was established by Feeney's weight-drop. The rats were randomly divided into NaCl group, scaffold group, hUC-MSCs group and scaffold + hUC-MSCs group. CV was used to observe the brain defect in rats. The mNSS score was used to evaluate the recovery of motor function in rats. Morris water maze method was used to detect the learning and memory ability of rats.Immunohistochemistry was used to detect the survival and migration of hUC-MSCs.The expressions of ?-? Tubulin and Ki67 were detected by immunofluorescence.The mRNA and proteins e expression of neuronal differentiation associated markers and neurotrophic factors were detected by Western Blot and qRT-PCR.Results1. The alginate hydrogel prepared by internal gel method is translucent, and the shape could be molded according to different mold. The results showed that the hydrogel had a three-dimensional porous network structure. The gel time,water content and degradation rate increased with the decrease of SA concentration and f value, and the elastic modulus decreased with the decrease of SA concentration and f value. Compared with SA group, the viability of S4H1 and S2H1 groups increased(p<0.05), and the cell proliferation ability enhanced (p<0.05).2. The TBI model was successfully constructed by the impact method. Compared with the control group, motor function was significantly improved (p<0.05) in the hUC-MSCs group and scaffold+hUC-MSCs group as evaluated by the mNSS; the learning and memory ability of the rats were able improved(p<0.05); the loss of stem cells in the lesion were reduced (p<0.05);?-? Tubulin+ and Ki67+ cells in the treatment group than that in the control were increased (p<0.05); The expression of NSE, NEUN, MAP2, BDNF and Bcl2 protein in the injured brain tissue were increased (p<0.05). The expression of NEUN, MAP2, BDNF, NGF and NT3 was upregulated (p<0.05), and the above effects in the combined group was better(p<0.05).Conclusion1. The plastic injectable alginate hydrogel could be prepare by internal gel method,and the physical, chemical and rheological properties can be regulated by adjusting the ratio of sodium alginate and f value.2. Alginate gel scaffold could provide three-dimensional support for the growth of hUC-MSCs,HA could increase cell viability and improve cell proliferation,SA/HA hydrogel provided a good environment for the survival of hUC-MSCs.3. Compared with hUC-MSCs alone, transplantation of hUC-MSCs loaded on SA / HA hydrogel could significantly improve the motor function , learning and memory ability of TBI rats, reduce the loss of stem cells, increase the expression of neurotrophic factor and enhance the regeneration of neural cells , thereby improve the therapeutic efficacy. |
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