Experiment On Tissue Engineered Bone Fabricated By Simulated Microgravity Bioreactor

Background:Bone defects induced by various kinds of causes are commonly seen in clinic. At present, there is still a lack of widely resourced bone repair material that is either cheap or with fine osteogenetic abilities. Tissue engineering technology could prepare bone repair materials with fine osteogenetic abilities in vitro, but there is still a series of technical issues including scaled amplification of seed cells in vitro, high-performance and aequalis construction with seed cells and scaffold materials, centralized supply of tissue engineered bone of large size, optimized cultural time and modality of in vitro culture of tissue engineered bone, etc.Rotating wall vessel bioreactor (RWVB) has advantages of suitable fluid shear stress, good effect of material transfer, high cultural density and etc. In certain conditions, RWVB rotated around the horizontal axis can simulate microgravity environment that will make the culture of cells and tissue get rid of the influence of gravity and achieve 3-D culture which is similar to in vivo living environment, probably benefiting the proliferation of cells and the maturity of tissue more in vitro.Objective:The aim of the study was to apply the simulated microgravity environment of RWVB into the preparation procedure of tissue engineered products, carry out the research on scaled amplification of hMSCs in vitro, dynamic construction and maturity of culture in vitro of tissue engineered bone, observe the influence of simulated microgravity environment of RWVB on the growth of seed cells as well as the construction and maturity of tissue engineered bone, so as to provide foundation for exploring the scaled preparation of tissue engineered products of high quality.Methods:1. The original generation of hMSCs were isolated from bone marrow of the iliac crest of voluntary normal adults by density gradient centrifugation and adherent culture. Culture-expanded were identified with superficial marks and multi-directional differentiation.2. The whole ranged autoserum culture was compared with fetal calf serum culture to detect the influence of autoserum on the proliferation and osteoinductive differentiation of adaptable hMSCs of cattle serum.3. The hMSCs were cultured in the simulated microgravity environment which made by a new RWVB, ie, rotary cell culture system (RCCS), to combine the Cytodex-3 microcarrier. The cell biology of hMSCs was observed, including the morphological observing, cell counting, MTT and CCK-8 to survey the proliferation, detecting the LDH activities as well as the density of glucose and lactic acid in the culture medium, and detecting the influence of this said cultural method on the osteoinductive differentiation of hMSCs.4. After first generation, hMSCs were cultured continually three passages with the method of simulated microgravity and microcarrier, and the cell biology of hMSCs were observed, including the morphological observation, proliferation, osteoinductive differentiation, cell senescence, even including the cellular oncogenicity.5. The tissue engineered bone was constructed with the hMSCs and DBM scaffold materials by using three methods including dynamic method in simulated microgravity culture environment of RCCS, conventional standing method and biphasic seeding technique of fibrin gel. The efficiency of tissue construction and the density of seed cells were compared with these three methods.6. The tissue engineered bone of different kinds were cultured continually in the simulated microgravity environment of RCCS and conventional standing environment. The degree of tissue matured were observed in aspects of cellular proliferation and osteoinductive differentiation.7. The tissue engineered bone constructed by different methods and culture time were implanted into nude mice subcutaneous recipients, where the bone formation was observed by X-ray photograph,histochemistry and immunohistochemistry.Results:1. Four days of hMSCs of original generation, the fibroblast-like cell colony was observed, and hMSCs have fulfilled the bottom of the monolayer cultural bottle in 9-12 days, while the passage cells having done in 4-7 days; the second generation of hMSCs showed CD105 rather than CD45 could induce differentiation of cartilage; 10 days after the induction of osteogenesis, ALP have stained masculine while the immunohistochemistry of osteocalcin and type-I collagen showed positive, which have formed mineralized nodus after 10-12 days.2. There is no significant difference between the amplification of hMSCs and the osteoinductive differentiation of cattle serum group and those of ordinally used autoserum group.3. In the RCCS simulated microgravity culture environment, hMSCs could grow up with adherence to surface of Cytodex-3 microcarrier, and the number of cells will achieve the peak value on the 9th day. The results of cell counting, MTT and CCK-8 methods showed that the log phase growth of plate-cultured hMSCs was the 2nd to the 5th day and that the number of cells achieved the peak value on the 6th day increased to 3.78 times; the log phase growth of hMSCs cultivated by microgravity method was the 2nd to the 8th day, and the number of cells achieved the peak value on the ninth day amplifying to 11.9 times, the cell density of unit culture medium has increased 15.8 times than that in the plate group while the analosis degree of glucose, the activity of LDH and density of lactic acid are significantly lower that those in plate group.4. The ALP activities have all achieved the peak value and there was no significant deviation between hMSCs of microgravity group and that of plate group inductively cultured with bone formation orientation for 12 days. Both group got the positive of ALP obtain, I-type collagen and osteocalcin immunohistochemistry, and formed calcium nodus during in vitro culture. The amplification speed of hMSCs has decreased after the bone formation induction.5. The serial passage of MSCs cultivated by RCCS microcarrier method could be carried out through direct addition of new microcarrier, the passage cells could remain amplification activities, the hMSCs appearedβ-galactosidae positive staining after 4 continuous RCCS generations, being obtained from 4 continuous generations (RCCS culture for 57 days, in vitro culture 72 days in total), the caryotype of hMSCs was normal, the clone formation experiment of soft agar showed negative and the tumorgenesis experiment of nude mouse showed also negative.6. The effectiveness of dynamic RCCS seeding in 1×106 cell/block group and higher density group was higher that that of standing innoculation, but both the innoculation effective of this method and the cell density of tissue block are lower than the innoculation effectiveness of FG biphasic seeding technique in the same density groups.7. The tissue block obtained by RCCS dynamic seeding have achieved higher peak value of cell density than that obtained by conventional standing culture, and the tissue block contained more base material element through the biological observation. biphasic seeding group with 1×106 cell/block density have obtained tissue engineered bone which could achieve the maximum cell density after 12 days of dynamic culture in RCCS.8. X-ray photographing, histology and immunohistochemistry showed that in the subcutaneously bone formation of nude mice: the tissue engineered bone, constructed by biphasic seeding method and cultured 12 days in RCCS in vitro possessed better osteogenetic abilities than that containing the same number of seed cell or that cultured by standing culture with the same period, abovementioned 3 groups of material have all possessed better in vivo osteogenetic effect than solitary DBM group.Conclusion:1. The cells of second generation obtained from dissociating red marrow by density gradient centrifugation and adherent culture have matched the hMSCs features in aspects of appearance, cell surface marker and multi-directional differentiation ability while in possession of higher purity and considerable quantity. Autoserum has no adverse effect on the amplification of adaptable hMSCs of cattle serum and the osteoinductive differentiation.2. The adoption of culturing hMSCs with microcarrier method in the RCCS simulated microgravity environment can achieve 3-D cell culture, increase the number of cells in unit culture medium and the availability of medium, encourage the quick cellular proliferation (mainly by extending the log phase growth) and achieve serial passage without enzymatic digestion RCCS simulated microgravity environment has no significant contribution to the directional induction of bone formation of hMSCs, the speed of proliferation after osteoinductive culture has been slowed down. The hMSCs cultured in vitro with 4 successive generations (RCCS culture for 57 days, in vitro culture 72 days in total), using the microcarrier method in RCCS simulated microgravity environment, have begun to appear cell senescence but still no oncogenicity. So this method is suitable for scaled amplification of seed cells in vitro.3. FG biphasic seeding method can significantly improve the seeding efficiency and the density of seed cells in the tissue engineered bone, compared to standing seeding method and RCCS simulated microgravity dynamic seeding method, and achieve quick construction of tissue engineered bone.4. RCCS simulated microgravity environment can promote the material transfer of the seed cells on microcarrier and in the scaffold, advance the density of seed cells in the tissue blocks, facilitate the seed cells to secrete extracellular matrix, and possess higher bone formation abilities than that constructed by conventional standing culture or instant construction.