The Research Of Osteogenic Effects And Osteogenesis Mechanism By Freeze-dried Tissue Engineered Bone With Abundance Functional Protein

Background and objectives: Currently the demand of bone repair materials wasenormous in clinical and also growing, but the research was in the bottleneck period. Thethree elements of tissue engineered bone contain a lot of problems to block the development.The first problem was vascularization when tissue engineered bone implanted, studies foundthat most of seed cells were dead because of the nutrients and oxygen lacking; Although theapplication factors can circumvent the vascularized problem, we felt quite helpless whenfaced with the complex environment and factors in vivo. Our group also have done someresearches on vascularization, like others there is a huge of death after implanted the TEB intovivo, but a strange phenomenon was found the osteogenic effect of bone tissue engineeringwas very good, even close to autologous bone. This phenomenon was also consistent withmany other researchers’ results, so could we think that the deadseed cells play an active rolein the subsequent bone repaired? The current studies found that MSCs can be called factorwarehouse because they secrete large amounts of cytokines (growth factors, raising factors,inflammatory cytokines, etc.) and regulate the process of cell recruitment, osteogenesis,angiogenesis and bone remodeling by paracrine or autocrine manners. Cell lysates canstimulate proliferation and differentiation of cells in vitro. Based on the above researches, weconceived that lyophilize and lysate the cells in the TEB, and with the help of different kindsof cytokines alone or combined actions to promote bone repaired in vivo.Methods:Part one: First, we selected the hUC-MSCs as seed cells in our study. Detected thestructure of umbilical cord by a cross-section’s HE stain and distribution of MSCs byimmunofluorescence, and then isolated cells according to the results. In order to fast,effectively, standardly and quality-controlledly harvest the cells, we invented the repeatedtissue culture dish. We compared the differences between repeated tissue culture dish and ordinary dish, and collected the number of floating tissues, the number of primary cells cellsand other data. Then we cultured the tissues repeated, harvested the cells of T1-P3, T3-P3andT5-P3, and identified the cells by pluripotent and the surface molecules by Flow Cytometrythree times. In order to verify the bone repaired ability of repeatedly cultured cells, wedesigned vitro and vivo experiments. In vitro experiments, we detected cell growthmomentum, and tested ALP, RUNX2and OC gene levels by RT-PCR and protein expressionlevels after induced to Osteogenesis. In vivo experiments, we constructed TEB with T1-P3and T5-P3separately, and then implanted them on the surfaces of the left and right iliac of thenude mice to observe the ability of ectopic bone formation. Eight weeks after operation, theMicro-CT, HE and Masson staining was taken to detect repeated cells’ osteogenesis in vivo.Part two: Freeze-dried-TEB (FD-TEB) was constructed and tested in vivo experiments.Frist, constructed TEB by h-DBM and cells harvested in the Chapter II, calculated the cellpercentage on the scaffold. Using light microscopy, scanning electron microscopy, Laserscanning confocal microscope and HE staining to make sure enormous cells on the surface ofh-DBM. The tissue-engineered bone were dealed with deep cryogenic freeze-dried,qualitatived by scanning electron microscopy and then measured the total proteins at differentpoints to chose the optimal time of cultivation. Using Raybiotech protein chip to determinethe amount of21kinds of osteogenic factors and43kinds of raised inflammatory cytokines inthe DBM. FD-TEB were chosed to observe sustained release in vitro. Vivo osteogenic effectof FD-TEB wasexamined by semi-situ femur defected in the nude mice when the FD-TEBwere implanted into the left femur and TEB were implanted into the right side of the femur.3,6,9weeks after operations, choosing Micro-CT to detect the amount of new bone and newbone mineral density, HE and Masson staining to observe the cells and extracellular matrixstructure of the new bone.Part three: We examined the osteogenic effect of the FD-TEB in the large animal models,we chose goat femur defected models. First we separated and identified the goat UC-MSC,the methods were similar to the Chapter III. Specifically, using Flow cytometry to detect theparticular cell surface molecules and pluripotent to detect the stem cells. Constructed the goatTEB, taking light microscope and scanning electron microscopy to judge the success, andthen freeze-dried. Using scanning electron microscopy to determine constructed situation. A2cm defected was maked in the goat femur bone, FD-TEB and DBM were transplanted into the left and right femur defects seperately.2,4weeks after surgery radionuclide imaging wastook to determine blood perfusion of the defects in femurs.2,4,8,16weeks X-ray observationof the progress of the defect repaired,8and16weeks using CT scan to determine the volumeof new bone and CT values, and compared the effect of both sides. Simultaneously HE,Masson staining were performed to observe the new cells and extracellular matrix. Liver,kidney function and blood routine were examinated after operation.Part four: FD-TEB similar as TEB has a problem of high prevalence when transplantedinto body. In order to overcome this problem, we first constructed FD-TEB according to thechapter Ⅳ, made vancomycin alginate beads with a concentration of50mg/ml of vancomycinand16%of alginate solution, and then put it into the mixture solution with fibrinogen andKH2PO3, finally placed it into a thrombin solution (400IU/ml), so there would be a layer offibrin gels on surface of the microspheres to increase the sustained release time. In vitroexperiments we tested the sustained release of vancomycin concentrations of theFG-Vanco-AB, then built antibacterial-FD-TEB by stereo microscope.Results:Part one: The repeated culture dish overcome the problems of tissue floated in theseparative process and tissue’ low utilized rate. When cultured for12days, the experimentalgroup had no floating tissue blocks, while the control group were13(n=3). Separatedsuccess of the tissues is (86±2.7)%over (44±4)%, n=3; p <0.01, with a statisticallysignificant difference. The number of cells after culture for12days were (24.7×104±3.6×104)VS (5.1×104±1.0×104), n=3; p<0.01. Identified repeatedly cultured cells positively expressedCD44, CD73, CD90, CD105, negatively expressed CD34, CD45, can be successfullydifferentiated to osteogenic, adipogenic, chondrogenic. In vitro the doubling time of cellsfrom the three cultured population are32h, and with similar period of adjustment, exponentialgrowth phase, andplateau phase. As quantified by real-time RT-PCR, there was no significantdifference in gene or protein expression among three cell batches, ALP, RUNX2and OC.8weeks after implantation, the BV/TV of T1-P3, T5-P3were (44.2±5.3)%VS (48.5±4.4)%respectively, BMD were (0.41±0.09) g/cm2VS (0.44±0.08) g/cm2.Part two: We found the rates of the cells adhere to the scaffolds were (82.4±3.3)%, usinglight microscopy, scanning electron microscopy, Laser scanning confocal microscopeandHE staining, we all found there were enormous cells on the scaffolds, the cytoplasm were flat, stretch and proliferated with times. In vitro experiments, cell proliferation was into the plateauphase from10day onwards. The weight of the protein after cleavage was11991ug/ml andafter lyophilized it was7544ug/ml, lyophilized protein preserved was (0.65±0.03)%(n=8).In10days, there was a high expression of raising factors, growth factors, inflammatoryfactors, osteogenic factors. Sustained release experiments in vitro also found that the totalprotein detected in21days were38ug/cm3. In vivo experiments the situ bone defects werebasicly repaired after3weeks, while the control group did not completely repaired in the situ,6weeks in the experimental group the ectopic BV was (0.5±0.13) mm3, bone density was:(0.21±0.07)g/cm2; while there was no ectopic bone formation in the control group; After9weeks, the experimental group had a large number of ectopic ossific shadow, while thecontrol group appeared more heterotopic ossification. The ectopic BV is (1.2±0.26) mm3inthe experimental group, while the control is (0.4±0.18) mm3, bone density are:(0.35g±0.11)g/cm2VS (0.27±0.11) g/cm2.Part three: After identification, the isolated cells were goat’s MSCs and can differentiateinto bone, fat, cartilage. Using light microscopy and scanning electron microscopy to observethe FD-TEB and TEB like in the chapter III, a large number of cells and extracellular matrixwere on the scaffold, proved FD-TEB were successfully constructed. After implanted into thebody, the blood pool in left and right were (410±74.5) VS (373±45.8), phase bone scan were(513±62.9) VS (381±64.3) in the2weeks, when after4weeks the blood pool were (559±98.1)VS (378±84.6), bone scan were (1349±103.1) VS (870±90.7), Compared the left and rightside of the defected situ, all have statistically significant difference. After8weeks new bonevolume in the left and right were (1518±159.8) VS (1039±178.2) mm3, CT value were(620±68.8) VS (400±86); after16weeks new bone volume in the left and right were(2532±189.3)VS (2421±111.7) mm3, CT value were (813±104.8) VS (785±118.2). All hadstatistically significant difference between the different groups. White blood cells, aspartateaminotransferase, creatinine, albumin had no meaningful change after operation.Part four: Similar to the previous, scanning electron microscopy confirmed FD-TEBwere successfully constructed, there were a lot of uplift and extracellular matrix proteins onthe TEB after lyophilization. The FG-Vanco-AB produced according to the optimizedprogram when release in vitro, the concentration of vancomycin was greater than bactericidalsensitive break point concentration (5mg/ml) was lasted24days. The FG-Vanco-AB and FD-TEB were constructed together, can be placed in-80℃for long-term preservation.Conclusions: This study arranges a spectral and efficient method to obtain MSCs fromWharton’s jelly in umbilical cord, adherent culture and repeated culture system to maximizethe use of umbilical cord tissue. The cells harvested by repeated cultured system wereidentified not only hUC-MSCs, but also exhibited no significant difference in osteogeniccapability in vitro and vivo. Successfully constructed FD-TEB, and there are a large numberof cytokines presented in the surface of the scaffold when in vitro experiments, and in vivobone defect repaired experiments whether nude mice models or goats models both confirmthat FD-TEB is significantly better than the DBM, so we can conclude that cytokines aftercells lysis promote the bone repaired. Successfully constructed AB-FD-TEB, and find therelease effect is satisfactory in vitro experiments.