The Construct Of Tissue-Engineering Adipose With Progenitors Of Human Adipose Mesenchymal Stem Cells

The materials used most generally were solicon rubber and fat particle injection auto-transplantation in tissue defect at present. The effection of fat particle injection auto-transplantation is the best among them, it is easy gain from suction adipose apatients, but its suck rate is high , so if we can overcome the shortcomings, we will gain the viable material used in tissue defection. The development of tissue engineering brings the new hope to it.Tissue engineering is a new science of co-development and combination of modern cell biology, biomaterial science and engineering, the aim of tissue engineering is to investigate tissue and organ substitutes. In cartilage tissue engineering, a series of techniques are being developed for culturing and reconstructing adipose tissues both in vitro and in vivo whose structure and function are as same as fat particle injection auto-transplantation . The development of adipose tissue engineering has positive contribution to the repair of the defect of cartilage, and it helps to generate new materials used in tissue defection.Tissue engineering techniques include seed cells, the coculture of scaffold and cells, implantation of cell and scaffold compound. It is the prerequisite to harvest seed cells maintaining stable phenotype, high proliferous activity and with broad extensive source to construct engineering adipose in vitro, and a major challenge to find appropriate scaffold confronting tissue engineering researchers. In this work, we studied the methods of using PLGA as scaffold of engineered adipose, the complexs were implanted into the superiousteum tissue of the banks of the rabbits as implanted materials .The specimens were dissected and examined macroscopically and histologically at different time.Objective: 1.To investigate the biological characteristics of mesenchymal stem cells from human adipose tissue.2.To study an ideal cryopreservation method of Human Bone Mesenchymal stem cells in vitro.3.The study was aimed to evaluate if the modifical in situ cryopreservation could affect the biological function of Human Bone Mesenchymal stem cells in vitro.4.To study the ability of adhesion proliferation and differentiation of Human Adipose mesenchymal stern cells, which were cultured in PLGA , thus to provide a basis for their in vivo lantation. 5.To study the ability of adhesion proliferation and differentiation of Human Adipose mesenchymal stern cells, which were cultured in PLGA , thus to provide a basis for their in vivo lantation. Methods: 1. Adipose stem cells were highly purified and culture expanded The morphology, phenotype and biological properties of the cultured adipose stem cells were observed by flow cytometer. 2. ADSCs of human were collected and cultured,and were conserved 3 monthes in cryoprotective agents by two step method. After 3 monthes,TBR, the expression of ADSCs surface antigen and the potency of various kinds fifferentiation were performed to evaluate change of ADSCs biology characters. At the same time, ADSCs growth carves was described. 3. Mesenchymal stem cells from human adipose were isolated by standard method and characterized with their morphology.cell-surface antigen profile and differentiation repertoire in vitro. The culture-expanded Mesenchymal stem cells were cryopresered in situ with culture medium (containing 10%DMSO and 30% selected FCS in -70℃. Fellowing recovery of cryopreservation,differentiation to adipocytes in vitro and cell cycle analysis were performed to investigate whether the cryopreservation would change the differentiation potential of Human Adipose Mesenchymal stem cells.4.PLA—PGA mix with 1: 1 ratio, and PLGA was gained by insert moldling with organic solvents. In vitro, Human Adipose Mesenchymal stem cells were adhered to PLGA. The attachment and the proliferation of Human Adipose Mesenchymal stem cells in PLGA were analysed by scanning electro microscope, and the morphological change of Human Adipose Mesenchymal stem cells was investigate before and after induction. 5.PLA-PGA mix with 1: 1 ratio, and PLGA was gained by insert moldling with organic solvents. In vitro, Human Adipose Mesenchymal stem cells were adhered to PLGA. The attachment and the proliferation of Human Adipose Mesenchymal stem cells in PLGA were analysed by scanning electro microscope, and the morphological change of Human Adipose Mesenchymal stem cells was investigate before and after induction. Results: 1.Adipose stem cells possessed some unique phenotypes. Adipose stem cell were positive of CD44 CD106 and negative of CD49d CD34 CD45å’ŒHLA-DR. Cell cycle studies showed there were percentage of stem cells which G₀/G₁ 79.1% S 19.7and G₂/M 1.3% respectively. After 7days of adipocyte inducing, about 85%of the cells displayed accumulation of lipid vacuoles, as detected by Red Oil O. After 14 days of osteogenic inducing,ALP stains were positive. 2. Compared to ante- freezing, ADSCs after freezing had no significant effects on procferation and differention abilities to adipocytes and osteoblast. 3.The results showed that after recovery of cryopreservation. There was no changesdetected as compared with the culture expanded Human Adipose Mesenchymal stem in both differentiation potency and growth pottern at 12 weeks. 4.Human Adipose Mesenchymal stem cells can attach, proliferate and secrete extracellular matrix in porous PLGA(pore size: 100-400 pm), whose porosity is 85 % , and Human Adipose Mesenchymal stem cells can differentiate adipoblast like circular under the induction of adipose medium. After 14days, the particulate of PLGA has been overlayed by and extracellular matrix.5.Human Adipose Mesenchymal stem cells can attach, proliferate and secrete extracellular matrix in porous PLGA(pore size: 100-400 pm), whose porosity is 85% , and Human Adipose Mesenchymal stem cells can differentiate adipoblast like circular under the induction of adipose medium. After 14days, the particulate of PLGA has been overlayed by and extracellular matrix. Conclusion: 1.Adipose stem cell from adult human can be induced to differentiate into adipocytes and osteoblast.2. It can more completely biology characters in cryoprotective agents (5%DMS0 -30%fbs-DMEM) by two step method, which is the ideal way to conserve ADSC_s on the condition of extremely low temperature. 3.This optimized short term in situ cryopreservation at -70℃could retain biological characterizes of Human Adipose Mesenchymal stem cells for at least 3 months,and this method may be useful for cryopreservation of Human Adipose Mesenchymal stem cells. 4.Human Adipose mesenchymal stem cells can be adhere to PLGA and proliferate, indicating PLGA can be used as Human Adipose mesenchymal stem cells in Adipose tissue engineer. 5.Human Adipose mesenchymal stem cells can be adhere to PLGA and proliferate, indicating PLGA can be used as Human Adipose mesenchymal stem cells in Adipose tissue engineer.