Experimental Study On Culture Of Bone Marrow Stem Cells Combined With Chitin In Vitro

PrefaceSyringeable hydrogel is a kind of relative novel material for cartilage tissue engineering which has been rising in the International for recent years, which provides a type of new cell - fixing mechanism. There are some superiorities for syringeable materials using in cartilage - defecting repairment, such as good combination and engomphosis between materials and the cell repaired. They are also easy to mold;Cells and growth factors not only can mix with the materials thoroughly and evenly, but also can be operated by arthroscopy or other microin-vassive methods, These operations are safe , effective and low cost. In this experiment we utilized syringeable hydrogel—Chitin as the stem cell vector of bone marrow matrix, and co - cultivated in vitro to provide some experimental foundations for the construction of tissue - engineering cartilage tissue.Materials and Methods1. Experimental animals;10 adult SD rats. The weight of male rat is 120 to 140g, which is provided by the experimental center of China Medical University.2. Main agents and equipmentsChitin (SIGMA company) ,DMEN(GIBCO company) ,Neogenesis cattle serum ( GIBCO company) ? Percoll gradient separating medium ( PHARMACIA company, density;1.073 g/ml) , Insulin - like growth factor (IGF - I ), Transfer growth factor(TGF - β₁,Sigma company) .Vitamin, Dexamethasone,CO2 incubator, Inverted microscopy3. The reparation of chitinPut lOg chitin (SIGMA company) into a mortar, Add 40ml acetone and grind for 10 mins under 10°C;Add 400ml cooled and concentrated HCL slowly into it while grinding and grind thoroughly until it become pasty. Then filtrated by glass cotton. Take the filtrate slowly into a beaker which is filled with 2000ml, 50% ethanol, stirring tensely while adding, And then made the beaker standing. Waiting until the colloidal chitin separated out and removal the fluid above. We only collect the colloidal chitin. Use stilled water to flush the colloidal chitin until ph value is equal to 7, and metered volume to 1000ml by distilled water and cold preservation for preparation.4. The isolation N cultivation and identification of BMSCs Experimental animals are extracted some bone marrow from femur under a-sepsis condition after they are anesthetized by 4% pentobarbital (anticoagulation processed);Multiproportion diluted by DMEM (1:1). Add to the previous prepared test tube with percoll separating medium. Centrifuge for 20min at 1200r/ min, Collect the boundary layer of mono - nucleus cells, Flush with DMEM and repeat for 2 times. Then add the solution into 24 pore - shadow mask which contains 10% new cattle serum / DMEM. Cultivate it routinely on the condition of 37"C N 5%CO2 and saturated humidity, Removal the sticky cells after 48 hours and change the culture solution, Change the solution every other day, Digest it by 0. 25%trypsin,and serial subcultivation. OLYMPUS inverted microscopy can be used to observe the cellular morphous and the growth state. When the stem cells of primary bone marrow proliferate until forming a single layer sticking to the wall, Then we collect the cells. Part of the cells get alkaline phosohotase dyeing, alizarin red dyeing and Mallory dyeing.5. Serial subcultivation % Inducement and IdentificationClassify the fourth generation into three groups, The experimental group is inoculated into the 24 pore - shadow mask by the concentration of 1 x 105, Add 2ml DMEM specific culture solution without serum ( Dexamethasone 4mg/L, TGF - fr lOng/ml.IGF - I 150ng/50ul, Vitamin C 50ng/l) into the three groups separately. And mix it with quantities duplex of chitin uniformly, Then cultivate and induce it. The control group is inoculated into 24 pore - cultivateplate with the same concentration, And cultivate and induce with the same DMEM specific culture solution without serum;The blank control group is inoculated into 24 pore - cultivate plate with the same concentration, But cultivate without any other solutions. Inlay some slides, Cultivate in a 37°C s 5%CO2 incubator, Change the solution every week for two times. Then take out the slide on 7 *\ 14th and 21st day after inducement and observe the slides under the inverted microscope. Detect type II collagen differentiated from cartilage cells which are differentiated from BMSCs and its HE dyeing and Alcian blue dyeing by immunohistochemistry method.6. TEM observation (TEM;transmission electron microscope)Collect the original generation of BMSCs cells and its cells after cultivated and inducement for 14 days. Put these cells into a 1. 5ml centrifuge tube ,Then fixed by 2% glutaral for 24hours. TEM observe the ultra micro - morphous of cells on the ultrathin slide routinely.7. The content analysis of type H collagenCo - cultivation for 7 x 14 and 21 days ,and process these slides by immunohistochemistry dyeing and take slides randomly. 20 slides each group, Detect the immunohistochemistry results under Linzux imaging system. Measure its brightness and count the percentage.8. Statistic processingStatistically analyze the data by SPSS11. 5 soft ware , The variability and significance adopt t test.Result1. Electron microscopy examinationUnder TEM, ruga - like small prominence on the cellular surface is one of the ultramicro - structure characters in cartilage cells. While in the experimental group ,the cartilage cells are round or oval with some typical ruga - like small prominence, There are many RER( rough endocytoplgsmic reticulum) in cellular plasma, regular ranged,, The endoplasmic reticulum cisterna distend slightly, filled with granular materials, And less mitochondrion , Intranucleus euchromo-some increase . While the cartilage cells are small and even less and also become fusiform in the control group.2. Histological detectionHE dyeing: After the 4th generation of BMSCs, the cellular appearance become stub form and triangular form, Nucleus become blue. The cellular mor-phous become short alter co - cultivation, which is square and ranged in a road -paving stone like appearance. By Alcian blue dyeing , extracellular matrix takes on blue, which show that the secretion of extracellular matrix is very productive and the matrix is alkaline.3. Immunohistochemistry detection of type H collagenThe immunohistochemistry of type II collagen secreted by cartilage cell is positive in experimental group, in which the cartilage cell is differentiated from 7th 14th and 21st day BMSCs cultivated. Some brown granules distribute in the cellular plasma, The dyeing of matrix is more significant than that of the control group.4. The content analysis of type II collagen (Table 1)Table 1 the content percentages of type fl collagen after cultivation and inducementGroupGroup for co - cultivation and inducementGroup for monolayer cultivation and inducementBlank control group* * vs * P<0.05DiscussionIt is an active approaching topic in orthopedic filed that repair the articularCultivationtimenCount ofgroup 1232070.380.589.6"2020.629.230.4*20000cartilage - defecting by hyaline cartilage which is constructed by tissue engineering method.The studies on tissue engineering includes three aspects: the isolated cultivation of seed cells % surrogate of extracellular matrix x researches on biological materials and construction of tissue - engineering tissues. Stem cells of bone matrix are easily to get because its resource is unlimited and easy to be isolated and cultivated, It also has strong reproductive capacity in vitro. After many times of serial subcultivation , it also has bone formation ability. Meanwhile, BMSCs have many potencies. According to the different environments ,they get different differential pathways , and form hyaline cartilage or subcartilage bone according to themselves'different anatomy positions. Furthermore, BMSCs is easy to get gene modification and adjustment for the expression of needed cytokines. So , as an important seed cell for cartilage tissue engineering, BMSCs is to be thought highly of by people more and more.Syringeable hydrogel is a relative novel kind of material for cartilage tissue engineering, Its superiorities are that: good combination between materials and the repairing tissues, easily mold, uniformly and sufficiently mixed materials with cells and (or) cytokines. It can be operated by arthroscopy or other micro-invassive methods as well. All these treatments are safe N simple N effective and low cost, And wont leave over obvious scar. At the same time, syringeable materials is suitable for exerting some pressure on cells, the straining of gel produce some force stimulations on cells.Chitin is a natural biological macromolecular saccharan. It possesses good biological compatibility and biological degradation. All of its degraded products are avirulentxnon - stimulating xnon - immunogenicity and non - mutagencity to human bodies. Furthermore, this kind of materials has the function of inhibiting bacteria and mould.In this experiment, BMSCs show good survival abilityxsticky abilityxreproduction ability and redifferentiantion. After induced by cartilage, the cell has typical ruga - like small prominence under TEM, The organelles become developed. All of these display good phenotype of cartilage cell. The cells are round or oval by HE dyeing. Extracellular matrix secretion is very productive underAlcian blue dyeing. Immunohistochemistry detections of type II collagen are positive in experimental group. We can see some brown granules distributed in cellular plasma, especially around cartilage cells. These granules range in trabs form with obvious boundaries to the around tissues, The dyeing of matrix is more obvious than control group. The content analysis of type E collagen in experimental group is also more significant than that of the control group(p <0,05).It has been proved by experiments that Syringeable hydrogel-----chitin canprovide an demanded 3D space for the inducement and differentiation from BM-SCs to cartilage, which is profit to the directed differentiation of BMSCs to cartilage cells and the synthesis of extracellular matrix . Meanwhile, extensive material resources xhigh safety x extensive anti - biotic effects > low cost and syringe-ability , all of these advantages made BMSCs tissue - engineering cartilage become a good natural biological vector.Discussion1. Stem cells of bone marrow matrix have the character to be induced and differentiated into cartilage cells, This character made it as seed cell for the tissue engineering2. Syringeable hydrogel chitin is a ideal natural biological vector for tissue engineering cartilage...