The Study On Preparation Of Bone Collagen Gel And Construction Of The Tissue-engineered Bone By Combinating The Gel With HMSCs-microspheres

Objective:The development of bone tissue engineering gives a good way to the treatment of bone defeats caused by trauma,tumor or other bone diseases.It has made great progress in each aspects of the bone tissue engineering during the past decade,and some of the bone tissue engineering products has begun to use in clinic.Now,the study of the bone tissue engineering are mainly focusing on the seeding cells,the tissue construction and the tissue cultivation in vitro before the implantation.In this study,we propose to amplificate human mesenchymal stem cells(hMSCs) with the macropole degraded gelatin microsphere(Cultispher G) in rotary cell culture system(RCCS) and observe the influence on the proliferation and characteristic of the cells;to extract the porcine bone collagen with the enhanced pepsin digestion method and construct tissue engineering bone(TEB) by combinating the collgen gel with the hMSCs-microspheres;to observe the result of ossification after the bone inductive cultivation.Method.1.Ⅰ-collagen was extracted from porcine bone with the enhanced pepsin digestion method established by ourselves.Then,we prepared the collagen gel-solution and freezed drying them into collagen membranes.The cytotoxicity was observed by co-cultivating the hMSCs with the collagen membranes;Histocompatibility and degradation were observed after implanted them into rabbits;and the affects of seeding cells on theβ-TCP scaffolds were also observed when they were coated with collagen;2.HMSCs were isolated in vitro and divided into two groups after they were amplificated to the second generations.In the experimental group,the mixture,1×10~7 cells mixed with 0.25g pretreated CultiSpher G,Dulbecco's Modified Eagle Medium(DMEM) and fetal bull serum(FBS),was put into a high aspect ratio vessel(HARV) with the volume of 50ml and cultivated on RCCS. In the control group,cells were continued to be static cultivated.Specimens were stained with methyl viole(MV) and MTT at different time points,or sliced to observe the cell adhesion and growth on the microsphere under inverted microscope.We sampled at 1,2,3 weeks and observed them by SEM.MTT detection;cell counting were made every day in 2 weeks to observe the growth of the cells in two groups;the changes of cellular characteristics in the RCCS were assessed by detecting the cell cycle and CD34,CD29;3. We constructed the compounds with hMSCs-microspheres and collagen gel as experimental group,the compounds with hMSCs and collagen gel as control group;and observed the changes of the appearance and texture of the tissues after cultivation;4.To construct the experimental group as above and construct the control group by traditional method with DBM as scaffolds;and observe the shapes and distribution of the cells in the tissues and determine the activity of ALP,contents of DNA at different time points during the bone inductive cultivation.Result.1.The collagen extracted by enhanced pepsin digestion method has no cytotoxicity;2.The experiment of implantation in rabbits revealed:there was no obvious toxic reaction.The collagen membranes began to degradate in the 1st week,and be completely absorbed in the 2nd week;3.The seeding efficiency was increased from 46.95% to 60.60%after theβ-TCP scaffolds were coated with collagen,and the cells on the scaffolds,coated with collagen,restored and proliferated more quickly than the scaffolds undisposed by collagen(P＜0.05);4.In the RCCS,most of the cells adhered to the surface of the microspheres after seeding 24h,and the cells proliferated quickly and excreted a lot of matrix,which could bind many microspheres together to become the bolus;MV and MTT staining showed that the cells increased quickly with the cultivation.The HE staining showed a lot of cells was on the surface and some cells in the pores of the microshperes;5. MTT detection and cell counting showed that the exponential phase of the cells in RCCS were 6 days comparing with the 3 days of static cultivation.And the number of the cell at the peak point was 10.75 times of seeding time,while the times of the static cultivation was just 3.17;6.The cell circles determination of the two groups had no difference,most of the cells were in G1 period.In the surface markers tests,two cultivated methods all kept the stem cells' characterics,CD34~-,CD29~+;7.The neutral collagen gel-solution solidificated after 10mins in 37℃,and the cells on the surfaces of the microspheres restored quickly and started to migrated into the gel after 2 hours;8.The complex was very soft and fragile at the beginning of the construction and the mechanical strength and toughness were improved after cultivation;9.The volume of the compounds contracted after the cultivation.Two weeks later,the diameters of the compounds in the experimental group was 85.33%of the beginning,while the control group was 27.33%;10.The contents of the DNA and ALP all increased significantly during the bone inductive cultivation,and the experimental group was faster than the control group.Conclusion:1.The bone collagen extracted with the enhanced pepsin digestion method has the good biocompatibility and solidifcation,which is helpful to the seeding cells for early planting and restoring,so it can be used into the tissue fabrication;2. CultiSpher G microsphere is suitable for hMSCs to adherence and growth.The method to cultivate the hMSCs by using CultiSpher G in RCCS is a efficient way to considerably amplificate the hMSCs,which is capable to supply a large number of stabilized cells for the construction of TEB and the hMSCs-microsphere complexes got from that can be directly used into TEB construction;3.Adding the CultiSpher G microspheres into bone collagen before the solidification can notably improve it's mechanical strength and keep the tissue shape;4.The TEB fabricated by the collagen gel solidification method has a good mechanical strength and ossification after the bone inductive cultivation;5.The method is a optimal way to construct the TEB and has some follwing advantages:to avoid pepsin digestion of seeding cells,to enhance the seeding rate,to operate easily and to activate seeding cells to recover their physical function fastly.