Out Of Body Study On Biocompatibility Of Human Umbilical Cord Mesenchymal Stem Cells And Spinal Cord Biological Frame

Objective: Restoring function to the injured spinal cord represents one of the most formidable challenges in regenerative medicine. Transplantation is widely considered to be one of the most promising therapeutic strategies, but it determined by implantation fusion with broken ends of spinal cord and axons regenerate and cross the lesion or reconnect with neurons on the opposite side to any significant extent to recover their neuro function. So spinal cord reconstruction, taking advantage of engineering technology of tissue engineering frame material, spermo-cell etc, is a hot topic in recent years.Our prior research has indicated that the spinal cord tissue of dog by chemistry extracted has three-dimensional network structure and provided a good supporter for human umbilical cord mesenchymal stem cells to grow and differentiate. We utilized the soft spinal cord tissue to make a kind of elder pith,surrounded by natural biological membrane to make a new kind of spinal cord biological frame which is spiral round tube with cerebrospinal fluid pathway. The object of our research is to study the biocompatibility of human umbilical cord mesenchymal and spinal cord biological frame, to provide more evidence for application in experimental or clinical spinal cord transection injury in future.Methods: 1. making elder pith of spinal cord biological fame: Spinal cords were excised about 5cm, cleaned from external debris and treated with chemical detergents to become acellular allografts.The method for isolation of cellular nerve tissue was based on a procedure developed by Sondell et al. Isolated Spinal cords were cleaned from external debris and immersed in distilled water, which was replaced several times during a 3 hours period at room temperature. The tissues were then exposed to 4% Triton X-100 in distilled water, overnight at room temperature, followed by a 12 hours period at room temperature in a solution of 4% sodium deoxycholate in distilled water. The extraction-procedure was then repeated four times. The extracted elder pith of sinal cord biological frame was divided into three parts: most of them, after being dealt with sterility test, were stored in phosphate-buffered saline (PBS, pH 7.2) at 4℃until use; some were made into frozen section for observation under electron microscope; the left were stored after vacuum freeze-drying treatment.2. The culture of HUMSCs: The full-term umbilical cords from healthy children were washed with PBS and umbilical arteries and vein were stripped off. Then remaining parts of umbilical cord were cut into small fragments of 1mm in diameter and cultured in growth medium (HG-DMEM) consisting of 20%FCS, 2mL-glutamine (L- Glu), 100 units/ml peniciline, and 100 ug/ml streptomycin. 5-7 days after primary culture, a great number of cells came out of fragments and became adherent to the flasks. When fibroblast-like cells reached confluence around 10 days, they were detached with 0.2% trypsin and were passaged. The third generation of human umbilical cord mesenchymal stem cells (HUMSCs) was collected in a plastic tube contained culture medium and preserved in -196℃nitrogen canister for further use.3. The natural biological membrane round tube which is spiral with cerebrospinal fluid pathway was partially cut for section and internal surface observation under scanning electron microscope, the remaining part were put on the elder pith of sinal cord biological fame and stored at room temperature for later use.4. Study the biocompatibility of HUMSCs in spinal cord biological frame. The cryopreserved HUMSCs were defrosted. The anabiotic cells were digested with 0.2% trypsin, centrifuged up to a density of 1×10 6/ml, injected into supporting frame, immersed into culture solution for 48,72, 96 hours. The growth of HUMSCs was observed by light, Hitachi scanning electron and transmission microscopy.Results: 1. About 5 days after primary culture, adherent cells came out of fragments, forming a uniform radiating appearance. When the cells reached confluence around 10 days, they produce rich endochylema, axon elongated and became cerioid. They were detached and passaged at a ratio of 1:2-1:3. Cells became adherent and formed a compact, disordered appearance in 30 minutes after being inoculated. After 3 passages, these cells maintained their homogenous appearance. Tested by MSCs, CD29, CD44, CD105 are positive and HLA-DR negative, which is in accordance with the characteristics of mesenchymal stem cell.2. The extracted spinal cord, whose diameter thickened and fissure of the hinder part widened, was oblate, ivory, and soft. The spinal cord transaction extracted with HE coloretur was compact, showed Honeycomb grids, and no cells were found in it. medullary sheath and cells disappeared under scanning and transmission electron microscope (TEM), leaving the layer alone. Nerve fiber matrix and basal lamina formed round, regular lacune. Diameter of The cavity of the neuron under the scanning electron microscope (SEM) supporting frame is the same as that of the neuron. Elder pith of sinal cord biological fame stored in the cryodesiccate state was still oblate, its diameter shrinked, it turned harder, and its tissue in the section became more compact.3.Natural biological membrane round tube is spiral with cerebrospinal fluid pathway. Under SEM, it is composed of several layers, between which, some thick material was visible, and collagen in the internal surface showed honeycomb appearance.4. HUMSCs were injected into the sinal cord biological fame and cultured in vitro. The transplanted cell could survive and grow in the neuroframe at 48, 72, and 96 hours under light and electron microscope. In the frozen section (FS) neuroframe punctiform and asterism-like cells were observed grow in the matrix. Their ecphyma grew and some of the cells became polygon and got connected to each other. Under SEM, cells, attached to the grid wall, were in good state and mivrovilli on its surface were visible, neural ball appeared. Neural ball grew bigger, and formed many ecphymas.Conclusions: The revival HUMSCs from cryopreserved state could survive and grow well in the sinal cord biological fame when cultured in vitro. They divided into neuron cells, produced neuron balls, grew and formed synapse structure. The honeycomb-like structure provided favorable microenvironment for exogenous cell outgrowth, indicating a better compatablity between the sinal cord biological fame and the HUMSCs. Combined with the present finding the spinal cord biological fame transplantated human umbilical cord mesenchymal stem cells could be used as allografts for repair in experimental or spinal cord transection injury.