| The human cartilages are composed of chondrocyte and extracellular matrix(ECM),the form of chondrocytes are hypertrophy and the quantity are less;the ECM of cartilage are compised of type II collagen and proteoglycan. Articular cartilages are all hyaline with little fibers. Trauma and arthritis are the main cause of cartilage injury,the ommilayer injury ofcartilage can be recovered by marrow,but because of without stimulation mechanism,the new tissues are merely fibrocartilages,they can not be coincide with hyaline cartilage in menchanics;the purely damage of articular cartilage can not stimulate chondrocyte to regenerate because of without blood circulation,thus,the plerosis of articular catilage can not depend on the proliferation of local chondrocyte.Ever since,people tried their best to find a way to reconstruct articular cartilage.Following the maturity of cultivation technology on chondrocyte and the development of medical organic materials, technology of tissue engineering come to show its superiovity in articular cartilage plerosis.The core technology of tissue engineering is utilize the suitable seed-cells compound with organic materials to build a cartilage-like tissue in vitro or in vivo,this tissue must be similar with hyaline cartilage in forms, structure and behavior.The cartilage tissue engineering involved in two roof of substance:l)suitable seed-cells;2)degradable scaffold with organic activity.Autologous and allolous chondrocyte are suitable for tissue engineering,but to obtain them is a difficult procedure,and their source are poor,and because they are mature terminal cells,they will lose their chondrocyte phenotype in proliferation ,so they can not satisfied with demand of mass cells in tissue engineering.Autologous MSC can be obtained easily,their source in human are plenty , own active proliferationand potential to difference into chondroblast.Now MSC become the chief seed-cell in cartilage tissue engineer, but the technology to stimulate MSC differentiate into chondroblast is not very successful,,the standard technology is culture the cell-ball in centrifuge tube,the reconstructured cartilage-like tissue is not achieve the mechanics of hyaline cartilage.The materials investigation of cartilage tissue engineering gained correspondent development in two type materials,one is the nature matrix,such as collagen, chitosan, cellucose,et al;the other is artificial synthesis material,such as PLA, PGA,et al.All these materials have deficiency in source, moulding ability and cells recognition.These experiment focus on the chondroblast induction in MSC, the culture technology in monolayer system and triaxial system,in order to explore the ideal condition of chondroblast induction of MSC,the molecul mechanism of maintenance chondroblast phnotype and the interaction between cells and porous materials.This investigation include two parts: l)The primary culture, chondroblast induction and evaluation of MSC. 2)The chondroblast cultured with compound porous materials PDLLA and PDLLA/chitosan.The aim are:l)To examine the proliferation ability and potential chondroblast differentiation ;2)To find an ideal condition stimulated BMSC differentiate into chondroblast;3)To examine the chondroblast proliferation in porous scaffolds and to explore the interaction between cells and materials;4)To examine the release of cytokine in vitro.Part one Isolating, culturing, chondroblast induction and evaluation of human MSC in vitroObjective:To examine the property and proliferation ability of primary culturing MSC ,mvestgate the effect of lightly centrifugation and chemical definition medium to induce MSC differentiate into chondroblast.Methods:A 5ml bone marrow was extracted from the iliac of humanvolunteers.By Percoll fluid and density gradient centrifugation,the MSC was obtained;after the cells filled the bottom of vessel,subcultured them,when they subculture in third generation ,redigested them,500 R/min centrifugate,alter the... |
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