Construction And Dynamic Culture Of Tissue Engineered Bone Under Mechanical Environment

Objective:1. To develop a novel bioactive and degradable scaffold with mineralized collagen-polyose based composite by biomimetic synthesis for bone tissue engineering and explore the compatibility of osteoblast culturing on it. offer important experimental data bases for further application in bone tissue engineering and clinic.2. To design and build a novel dynamic strain and circulating-perfusion bioreactor system for 3D tissue engineering bone cultured in vitro and and test its capability. Offer an ideal culture device and technical platform to cultivte tissue engineered bone and to explore the influences of dynamic strain on the behavioral responses of bone cell populations within three-dimensional scaffolds.3. To initially investigate the mechanical response of tissue engineering bone cultured in the novel dynamic strain and circulating-perfusion bioreactor system. Offer important experimental data bases for the study on the mechanism of osteoblastic biological responses to dynamic compressive strain under three-dimensional culturing.Methods:1. Collagen type I and sodium hyaluronate was dissolved in 0.1% acetate solution and stirred at room temperature (The weight ratio of collagen and sodium hyaluronate was 10:1) for 30min. Then,they were reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide by controlling pH at 4.75 for 2 hour.Following, the cross-linking products were rinsed thoroughly in 40% ethanol solution, in O.lmol/L disodium hydrogen phosphate solution (PH 9.1), in lmol/L sodium chloride solution and finally in deionized water for ten times.Using the cross-linking products of collagen type I and sodium hyaluronate as the template,the calcium phosphate was deposited on it to produce a mineralized composite.The porous bredigite scaffolds were prepared by liquid phase separation after the mineralized composite combining with polylactic acid (PLA) and NaCl. The Composite materials and scaffolds were investigated by x-ray diffraction(XRD),scanning electronic microscopy (SEM).Pore size and porosity were observed with light microscopy and scanning electron microscopy(SEM).Measured the 2h water absorption rate of the scaffold materials at 37℃. The mechanical properties of the scaffolds were tested in the Instron Model 5865 mechanical testing machine under saturated humidity and after degraded for 1,10,20 days.2. MC3T3-E1 preosteoblast cells were seeded onto the nHACP/PLA scaffold and cultured in a-MEM (Minimum Essential Medium, Alpha Modified) supplemented with 10% fetal bovine serum,100 mg/L penicillin and 100 U/L streptomycin. Cell Cuonting Kit-8 assay(CCK-8) were performed to analyze the growth and proliferation of MC3T3-E1 osteoblast-like cell on the scaffolds after 1,3,5,7,9 and 11 days of culture. ALP activity assay were performed to analyze the osteogenic differentiation of MC3T3-E1 cells after 1,3,5,7,9 days of culture. Cellular morphology and extracellular matrix secretion of MC3T3-E1 cells on the scaffolds at different culture time were observed by inverted phase contrast microscope,fluorescence microscope and scanning electron microscope.3. Design a novel dynamic strain and circulating-perfusion bioreactor system for 3D tissue engineering bone.At first,The design philosophy of the bioreactor system for bone tissue engineering was specified.The mechanical loading system and the 3D circulating-perfusion system were designed respectively. The mechanical loading system consists of three parts:a rigid frame, An intelligent type piezoelectric ceramic actuator and a PZT driving power. The circulating-perfusion 3D culture system is consist of culture chambers, medium reservoires,a multi-channel two-directional peristaltic pump and liquid transfer lines. A special culture chamber for three-dimensional perfusing and compressive loading was also designed and developed. Built a prototype machine of bioreactor according to the design scheme and tested its functions:①The bioreactor was placed at 37℃in CO2 incubator containing CO2 of 0.05 volume fraction,and placed at super clean bench for 2h loading every day. The sterility of culture devices was tested by extracting medium from it to perform bacterial culture every day for 5 days.②The average flow rates of every liquid transfer lines at different rotate speed were measured.③After loading the scaffold into culture chamber and inputting the parameters of working frequency,displacement value and working time into the computer interface. The accuracy and stability of loading device were tested by calculating the the deviation value of the actual working Data and the preset value.4. MC3T3-E1 preosteoblast cells were seeded onto the nHACP/PLA scaffold and cultured in a-MEM at 37℃.After 4h of static culture, these cell-seeded scaffolds were divided three groups:the static culture group,the perfusion culture group and the perfusion culture and loading group.The static culture groups were cultured in the petri dish.The perfusion culture groups were cultured in the culture chambers of bioreactor and given a circular flow with flow rate of 10 mL/min. The perfusion culture and loading groups were cultured in the same condition of the perfusion culture groups,but they were dynamically loaded in compression using a sine wave at 1 Hz,3500μεin the culture chambers for 2 h every day. Cell Cuonting Kit-8 assay(CCK-8) were performed to analyze the growth and proliferation of MC3T3-E1 osteoblast-like cell in different groups at day 5 and day 10 of culture. ALP activity of celles in different groups were tested after 10 days of culture.The morphology and distribution of cells in the scaffolds in different groups were observed by the cell tissue section examination under microscope and scanning electron microscope. At day 10 of static culture, cell-seeded scaffolds of experiment groups were dynamically loaded in compression using a sine wave at 1 Hz,3500μεfor 3 h,and the non-loaded cell-seeded scaffolds were treated as control groups.The expression levels of mRNA and matrix proteins in different groups were detected by RT-PCR and Western-blot at 12h after loading.Results:1. Both degree and the size of HA crystals in the composite were low,which were simiar to that of nature bone. The SEM micrographs showed that the pore size was about 200μm to 650μm. The scaffolds possessed 82% of porosity.The shape of pore was nearly round shape or irregular square shape. The elastic modulus of the scaffolds tested under saturated humidity at 37℃was 6.834±0.404MPa, similar to that of nature cancellous (spongy) bone. After degraded for 1,10,20 days, The elastic modulus was respectively 5.785±1.887MPa,2.532±0.194MPa and 2.333±0.384MPa. The 2h water absorption rate of scaffold materials at 37℃was 134%±24%.The nHACP/PLA scaffold materials had proper pore size, porosity, water absorption ability and mechanical propert.2. Cell Cuonting Kit-8 assay results showed that the OD values of MC3T3-E1 cells on the scaffolds (nHACP/PLA) raised gradually along with time. The growth curve tend to flat after five days, suggested that MC3T3-E1 cells grew well on the scaffolds and reached the the growth flat period.After 3 days of culture,the activity of ALP in scaffold group was higher than those in the control group(P<0.01), suggested that the nHACP/PLA scaffold materials could increase the activity of ALP in MC3T3-E1 cells. The observation results of MC3T3-E1 cells mixed culture with scaffold materials showed that calcium released from the scaffold materials to form regional increased uptake,and the cells had trend to grow towards the materials. The observation results of tissue section examination and scanning electron microscope showed that cells spread well on the scaffolds, the extracellular matrix secretion can be see in the pore of scaffolds.3. A novel dynamic strain and circulating-perfusion bioreactor system was built in this experiment. The bioreactor system possesses 12 liquid transfer lines,can connect 12 culture chambers. Every liquid transfer lines could provide a circular flow with flow rate ranging from 0.17 mL/min to 20 mL/min. contmlled.There were no germ grown in culture medium detected by means of bacteria cultivation.The dynamic strain loading system could work according the dictates which had been set up in the computer. The actual working frequency and working time of loading system both accorded with the parameter setup in the the computer interface.The deviation value of actual displacement produced by loading system was about 0～1.2‰of preset value.4. The cell-seeded scaffolds were cultured in the dynamic strain and circulating-perfusion bioreactor system or in the petri dish.By day 5 or day 10 of culture, Cell Cuonting Kit-8 assay results showed that the OD values of the perfusion culture and loading group were higher than those of the static culture group and the perfusion culture group,and the OD values of the perfusion culture group were also higher than those of the static culture group(P< 0.05). Results of ALP activity:By day 10 of culture, the ALP activities were significantly different among the the perfusion culture and loading group,perfusion culture group and the static culture group [(8.936±0.589), (7.759±0.466)and(5.673±0.427) U/gprot, P<0.05]. The perfusion culture and loading group or perfusion culture group was higher than the static culture group. The observation results of tissue section examination and scanning electron microscope showed that cells spread and grown well in the pores of scaffolds of every group. The cells showed polygonal shape or shuttle shape,and connected with each other by the pseudopodia from it. The cell density of the perfusion culture and loading group or perfusion culture group was higher than the that of static culture group,and the cells tended to present multilayer growth tendency in these two group. Especially in the perfusion culture and loading group,cells prensented higher density and excreted more extracellular matrix. Cells and extracellular matrix fused into large patches in some pore of scaffold in this group. Cell distribution was uneven on all scaffold in different group,and "cavitation" could be seen in every group. Especially in static culture group,there was only little amount of cells even no cell grow in some pores of the scaffold. Matrix protein gene expression of type I collagen (Col I), osteopontin (OPN) and osteocalcin (OCN) were measured by RT-PCR at 12h after a single bout of 3 h of loading. Loaded samples showed higher mRNA expression levels of Col I, OPN and OCN than that of control group (non-loaded samples), which was similar to the proteins expression levels detected by Western-blot.Conclusion:1. The novel scaffolds were similar to nature cancellous (spongy) bone both on structure and on property, and might be used as one of the optimal scaffold material for bone tissue engineering.2. The bioreactor system could provide compressive strains with different magnitude and frequency components and different perfusion flow to the cells within cell/scaffold constructs which were cultured in it. It could be controlled accurately and has the advantages of easy operation and steady performance. The bioreactor system could keep good air—tightness and sterility,so as to it could offer a suitable condition to the tissue engineering bone to grow in a long peiod. It could be an ideal culture device for tissue engineering bone and used to explore the influences of dynamic strain on the behavioral responses of bone cell populations within three-dimensional scaffolds.3. The bioreactor system has a promoting effect on the proliferation and osteogenic differentiation of MC3T3-E1 osteoblast-like cell on the cell/scaffolds which were cultured on it. The dynamic culture method by coupling mechanical stimulation of fluid shearstress and axial compressive stress might be a good model for improving the quality of tissue engineering bone cultured in vitro,which could be a good esearch platform of cell mechanobiology in three dimensional culture in vitro.