The effect of BMSCs transplantation combined with cryopreservation on trachea allograft epithelium regeneration / 中华胸心血管外科杂志

Objective To investigate the role of BMSCg on enhancing the implant survival and bacheal epithelium regeneration. Methods After transplanted with cryopreserved 2 weeks and 6 weeks allocraft, PHK-26 labeled 3-5 passage BMSCs were injected into the recipient rats via tail vein. Rats in the control groups were injected with the same amount of PBS.We observed the histology of the transplanted trachea including epithelium growth and regeneration, and the PKH-26 fluorescence levels at the para-anastomotic trachea to evaluate the role of BMSC transplantation on the epithelium regeneration. Results Rats from BMSCs injection group survived a long period. Histological observation showed that the tracheal lumen was covered by psudo-striated ciliated columnar epithelium. The cartilage structure was intact. There are no signs of denaturation and necrosis. In the PBS injection group, epithelium regeneration is better in PBS-6-week group than PBS-2-week group. The longest survival time in PBS-6-week group was 32 days, whereas it was 10 days in PBS-2-week group. In BMSCs injection group, rats in BMSC-6-week groups survived longer than 8-week group(12 rats were terminated at 1 week, 4 weeks and 8weeks as planned). There was one rat who survived and were terminated at the designated 8 weeks time point (there were 8regenerated epithelium was similar in the two BMSC transplanted groups. PKH-26 labeled BMSCs migrated to the implant site and showed red fluorescence, with most red fluorescence shown at the anastomotic part. Conclusion BMSCs can migrate to the impaired tissue to repair it. BMSCs may exert their reparation function via enhancing epithelium regeneration.

Repair of sciatic nerve defects with tissue engineered nerves constructed with marrow stromal cells / 中国组织工程研究

BACKGROUND: Schwann calls are seed cells for constructing tissue engineered peripheral nerves. But their in vitro isolation, culture and purification are difficult. Acellular nerve allografts (ANA) have a great effect on repairing peripheral nerve defects, and it can induce marrow stromal cells (MSCs) into Schwann-like calls. Accordingly, MSCs can be used as seed calls theoretically in constructing tissue engineered peripheral nerves, instead of Schwann cells OBJECTIVE: To observe the repairing effect of tissue engineered peripheral nerves constructed with MSCs on sciatic nerve defects and to assess the feasibility of repairing peripheral nerve defects with MSCs as seed calls. DESIGN, TIME AND SE'I-rlNG: A randomized control animal experiment was conducted in the laboratory of Basic Medicine Collage of Dali Collage from July to December in 2008.MATERIALS: A total of 30 SD rats were divided randomly into 3 groups, with 10 in each group. In MSCs+ ANA group, end-to-end anastomosis were performed with 10/0 non traumatic suture to broken ends of rat sciatic nerves and tissue engineered nerves cultured using MSCs combined with ANA; In ANA group, ANA were bridged to broken ends of sciatic nerves; In METHODS: The 10ram sciatic nerve defects in rats were repaired using MSCs-constructed tissue engineered peripheral nerves, whose repairing effects were evaluated at week 12 post transplant through sciatic function index (SFI), gastrocnemius wet weight recovery rate, S-100 immunohistochemical staining and electron microscope observation, etc. MAIN OUTCOME MEASURES: Morphological changes of calls were observed during the culture of compounds; SFI and gastrocnemius wet weight recovery rates were detected after transplantation; New myelinization, axon growth and nerve fiber distdbuUon were observed with toluidine blue staining; Schwann calls growth and nerve fiber regeneration were observed with transmission electron microscope combined with S-100 protein immunohistochemical staining method.RESULTS: The detection results showed that SFI and gastrocnemius wet weight recovery rate were higher in the MSCs+ ANA group than in the ANA group (P < 0.05). Compared the ANA group, the MSCs+ ANA group had more S-100 proteins expressed in its compounds obviously, and the number, the diameter of its myelinated nerve fibers as well as its myelin sheath thickness were all greater than those of the ANA group (P < 0.05). The repairing effect of the MSCs+ ANA group was close to that of the autotransplantation group.CONCLUSION: MSCs-constructed tissue engineered nerves have a better effect on repairing peripheral nerve defects than ANA, and MSCs as seed cells have a high value in peripheral nerve tissue engineering.

Combination of basic fibroblast growth factor and brain-derived neurotrophic growth factor influences the differentiation of adult rat hippocampus neural stem cells into neurons / 中国组织工程研究

BACKGROUND: Differentiation of neural stem calls (NSCs) was mediated by many environmental factors. Several factors can induce NSCs to differentiate into neurons in varying degrees and it is now a focus on the control of NSCs differentiation.OBJECTIVE: To study the effects of combination of basic fibroblast growth factor (bFGF) and brain-derived neurotrophic growth factor (BDNF) on the differentiation of NSCs into neurons.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Neurotomia Laboratory of China Medical University in May 2008.MATERIALS: Three adult male SD rats were provided by Experimental Animal Center of China Medical University.METHODS: Dispositions to the rats were consistent with ethical standards of animals. The rat brain hippocampus was removed sterilely. After trypsin digestion, NSCs were cultured in serum-free medium. Cell suspension was prepared and diluted when the diameter of the fourth passage of clone sphere was 200 μm by mixture of DMEM/F12 containing 2% B27, 20 μg/L of epidermal growth factor (EGF), and 20 μg/L bFGF. Monoclonal calls were passagad. NSCs were divided into blank control, bFGF, BDNF and bFGF+BDNF groups by different growth factors added into the media. Fetal bovine serum of 0.1 volume fraction was added in blank control group. The media in the other three groups were added bFGF, BDNF and bFGF+BDNF respectively for 1 week.The concentration of bFGF was 10 μg/L and the concentration of BDNF was 200 μg/L.MAIN OUTCOME MEASURES: Immunocytochemistry staining was used to identify NSCs as well as to detect the differentiation of NSCs into neurons.RESULTS: The monoclonal calls expressed nestin and the differentiated call expressed neuron specific enolase and glial fibrillary acidic protein. Compared to blank control group, the proportion NSCs into neurons in the bFGF group, BDNF group and bFGF+BDNF group were much higher (t=3.409-7.558, P < 0.05), with the highest in bFGF+BDNF group (t =7.558, P < 0.05).CONCLUSION: Combination of bFGF and BDNF can promote the differentiation of adult hippocampus NSCs into neurons.

In vitro culture and purification of Schwann cells / 中国组织工程研究

Schwann cells are the seed cells of tissue engineedng in the regeneration of peripheral nerve.so whether we can harvest enough Schwann cells of high purity is very important in the tissue engineering.Culture and purification technology of Schwann cells has been improved day by day.The classical cultural methods include tissue-clump cultural method and enzyme digestion cultural method.On the basis of the classical cultural methods,someone uses new technologies to delete fibroblasts,such as addition of cytosine arabinoside,addition of antimitogenic agents.The latest purified methods include magnetic activated-cells separation,co-culture with three-dimensional scaffolds and laminin-coated wells purified method.The aim of the skills above is to provide enough Schwann cells for the tissue engineering repeir of degenerated peripheral nerves.There are many ways of Schwann cells culture and purification.and we have made a primary progress,but it still needs further studies before the entire achievemenL such as long cycle,low activity in cultured calls,and the instable biological characteristics during passage culture,etc.

EXPERIMENTAL STUDY OF THE REPAIRING EFFECT OF ACELLULAR NERVE ALLOGRAFTS ON THE SCIATIC NERVE GAP OF RAT / 解剖学报

Objective To observe the repairing effect of the acellular nerve allografts on the sciatic nerve gap of rat. Methods The acellular nerve allografts,treated by hypotonic-chemical detergent,were put on the 10 mm gap of the sciatic nerve in the rat.The action potential of the regenerated nerves was determined by the electrophysiologic method 13 weeks after operation.The morphology of the regenerated nerves was observed under light microscope and electron microscope,and the results were analyzed statistically. Results No inflammation and rejected reaction were found in the period of 13 weeks after operation in the operated and control groups.There was no significant difference in number of the regenerated nerve fibers,diameter of the axons,and the thickness of the regenerated myelinated nerve between the experimental group and control group.Conclusion The present results indicated that the acellular nerve allografts had good biocompatibility for the host rat in vivo and might as a bridge promote the regeneration of the injured sciatic nerve.;

EXPERIMENTAL STUDY ON THE RECONSTRUCTION OF THE NERVE-MUSCLE STRUCTURE AND FUNCTIONAL RECOVERY OF THE SCIATIC NERVE GAP BY ACELLULAR ALLOGRAFTS IN RAT / 解剖学报

Objective The experiment was designed to investigate the effect of acellular nerve allografts on the functional recovery and reconstruction of the nerve-muscle structure of the sciatic nerve defect in rats. Methods Acellular nerve allograft was transferred into the defected rat sciatic nerve with 10mm long.The wet weight of tibialis anterior was weighed at 12 and 24 weeks postoperatively compared with control group.The conducted velocity of regenerated nerve and the effect of regenerated nerve on tibialis anterior were investigated by electrophysiologic test,and silver staining combined with AChE histochemical methods were used in the experiment separately. Results The wet weight of tibialis anterior and the conducted velocity of regenerated nerve in experimental group were similar to those in control group in 12 and 24 weeks after transplantation.The positive acetylcholinesterase(AChE)histochemical reaction was observed in the tibialis anterior at 12 weeks with deeper staining and located in the middle of tibialis anterior tidily at 24 weeks after operation.The regenerated nerve bundles and nerve terminals were found to grow into the motor end-plate of the tibialis anterior in silver staining combined with AChE staining in experiment group.Electromyogram showed that the regenerated nerve has innervated tibialis anterior already.Conclusion The results indicated that extracted nerve allografts as a bridge can promote the motor functional recovery and reconstruction of the nerve-muscle structure of the defected rat sciatic nerve.