| As the most important organs of human, bone plays the irreplaceable role in life activities, but the bone defect is a common and frequently-occurring disease clinically. Bone defect or functional disorder of patients in our country each year can reach more than millions of people. Congenital developmental disorders, trauma, infection, tumor, senile osteoporosis caused by fracture all can lead to bone defect. The bone defect repair methods mainly include autograft, allograft bone transplantation, prosthesis, the artificial bone replacement materials such as metal, polymer materials and so on. But the methods above all have their own defects, such as limited source, leading to traumatic, rejection, limited biocompatibility, lack of corrosion resistance and mechanical properties so on. Further more, some supercritical bone defect was limited by the ability to repair itself, intervention and appropriate external implant material were needed to achieve the purpose of healing supercritical bone defect just like skull defect. So repairing huge and complex bone defect is still a challenging problems in bone reconstruction field, there is no clear effective treatment for it.Tissue engineering become one of the important and potential alternative treatment approache in tissue regeneration. In the typical tissue engineering approach, healthy cells which are from the patient’s own tissue are seeded in and co-cultured with 3D scaffolds prepared from natural or synthetic polymers, then after expanding in cell culture medium, the construction is implanted into the defected area for new tissue regeneration.Bone marrow mesenchymal stem cells (BMSCs) are easily isolated, expanded in culture and have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. In vitro, without osteogenic supplements, BMSCs cultured on CaP related materials could differentiated into osteoblasts. And BMSCs with ceramic scaffolds could be used in vivo for bone formation in ectopic sites or critical sized defects. BMSCs have unique immunoregulatory properties besides their regenerative properties.Another key component for bone repair and regeneration in tissue engineering is the scaffold that serves as a template for cell adhesion and interactions and the formation of bone-extracellular bone matrix to provide structural support to the newly formed tissue. In general 3 D scaffolds for the regeneration should have appropriate criteria to serve this function, including biocompatibility, the appropriate biodegradation rate (during the period of contactinf with the cells and tissues in the body will not soon degradation), mechanical properties similar to those of the bone defaced site and porous microstructure and so on. In order to meet the requir of oxygen and nutrients in the organization cells and wastes discharge in a timely manner, and the formation of blood vessels for organizational growth, large open, interconnected pore structure is also required in the support scaffold. In addition, higher porosity to the three dimensional porous scaffolds can provide cells with larger contact area, and then increase cells adherent quantity, Cai and others reported that best porosity of scaffolds is 90% or more. Hydrogel scaffolds are applicable to be used as cell scaffold or slow release carrier because of the similar bionic structure to extracellular matrix, the three-dimensional network structure, and cell migration and growth promoting properties. Nowdays different methods, such as electrospinning, freeze-drying, cryogelation and gas foaming, etc, have been reported for preparing porous scaffold to be used in tissue-engineering field. Cryogelation is a method that producing gels in moderately frozen solutions of monomers, macromers or polymeric precursors. It was reported that the cryogels prepaired by cryogelation contain interconnected macropores which allowed diffusion of solutes as well as mass transport of nano and microparticles microorganisms and cells.Both naturally derived and synthetic polymers have been used to prepare scaffolds that provide templates for new tissue regeneration. Collagen is present everywhere in the original tissues and gelatin is a denaturated form of collagen. Gelatin is modified collagenprotein which is very cheap, extensive sources with a lot of functional groups and compared with collagen, gelatin has better water solubility and low antigenicity. Gelatin methacrylate (GelMA) hydrogel is widely used for various biomedical applications due to its good biological and physical characteristics of adjustable. GelMA is obtained by modified natural polymer gelatin. Some basic properties of GelMA hydrogel is similar to natural extracellular matrix (ECM), such as the existence of amino acid structure unit which response to cell adhesion, matrix metalloproteinases reaction, allowing cell adhesion and stretching on GelMA scaffold and proliferation. In order to possess specific properties, hybrid hydrogels are constructed from blends of different components. Although GelMA is a cell-responsive material with wide-spectrum of tunable properties, tailored design of hybrid materials has been a useful strategy to improve some characteristics of GelMA for certain applications. For example the addition of gold and other inorganic particles has been used to improve the mechanical strengths, conductivity, response to thermal or magnetic stimuli, and biological functionality of GelMA hydrogels. There is paper also reported that GelMA-hydroxyapatite hybrids was proposed as coatings for titanium (Ti) surfaces to improve the integration between bone implants and titanium. The titanium surfaces were hydroxylated by alkali treatment first and then covalently coated with a GelMA film in this application. Recent research has proved that the GelMA based hydrogels were widely used in the field of tissue engineering, including bone, cartilage, heart and vascular tissue engineering, etc. In addition, in cell research, cell signal transduction, drug and gene delivery, etc, GelMA is also used widely.Bone tissue is composed of minerals and proteins. The minerals are mostly apatites such as hydroxyapatite which is the main inorganic component of natural bone. It is reported that it has excellent biocompatibility with hard tissues and to be osteoconductive and has positive effects on osteoblastic cell differentiation despite its low degradation rate and mechanical strength. Inorganic hydroxyapatite and organic polymer compound can not only simulate the compositions of natural bone, can also greatly increase the mechanical properties of scaffold materials. In spite of this, hydroxyapatite particle size, shape, surface chemical properties and other properties will affect the biological effect of hydroxyapatite on cells.The aim of this study was to develop biodegradable hydroxyapatite-GelMA based cryogels with interconnected macropores to be used as scaffolds in bone tissue regeneration. GelMA and hydroxyapatite-GelMA cryogels containing different concentration of micro-hydroxyapatite particles were synthesized and their chemical structure, swelling ratio, pore morphology, mechanical propertywere characterized. In vitro the BMSCs viability and the cell differentiation were performed to show the biocompatibility of the cryogels and the potential of the cryogels for future tissue-engineering applications.Part Ⅰ:The identification and osteogenic effect investigation of rat bone marrow mesenchymal stem cellsObjectives:To identify the bone marrow mesenchymal stem cells (BMSCs) obtained from the lower limb in rats to ensure its purity and then investigate the osteogenic ability by culturing the BMSCs in an osteogenic medium.Methods:Two 4-week-old male Sprague Dawley (SD) rats were used for obtaining bone marrow-derived mesenchymal stem cells (BMSCs) from the lower limbs in rats by the whole bone marrow adherent culture method. And then the BMSCs were subcultured for several times for the purification, and then (1) to observe the morphology of the cells under a microscope;(2) the cell surface markers of BMSCs incluing CD44, CD45, CD34 and CD90 were identified throuth flow cytometry instrument by using the 4th generation of BMSCs; (3) the BMSCs were cultured in osteogenesis induction medium, ALP staining and Alizarin red staining were used to identify the osteogenetic abilites of the BMSCs.Results:(1) Under the microscope, the morphology of the BMSCs were lile spindle, spindle and polygon at first, after cultured for some time, most of the cells had a spindle shape, and had large cell sizes with a large ovoid nuclei, presented clone-like growth, cells were arranged in swirl when cell fusion rate above 80%. (2) The flow cytometry detection of BMSCs:the characteristic surface antibody positive rate were CD44 (87.78%), CD90 (98.14%) separately, and the uncharacteristic positive rate of antibodies:CD 34 (0.14%), CD45 (0.06%). (3) ALP staining results showed that the BMSCs cultured in the osteogenic medium were dyed bluish violet, proved the result was positive. (4) After cultured in osteogenic medium for four weeks, the BMSCs were stained in red all over the view by alizarin red staining, demonstrating the generation of mineralized nodules.Conclusions:(1) The source of bone marrow mesenchymal stem cells (BMSCs) is very rich, the cells are easily isolated, easy to be cultivated without special culture conditions and ethical limitation, which making BMSCs favorable for applications; (2) The BMSCs from the lower limbs of rats were isolated successfully and then adherented, the phenotype and morphology characteristics of the cells were similar to the BMSCs as reported. (3) The BMSCs was of high purity, can be used in the subsequent experiment in vitro and animal experiment research in the tissue engineering field; (4) The BMSCs obtained from rat lower limbs were proved to have good osteogenic differentiation ability in osteogenic medium.Part Ⅱ:The biological features of micron grade hydroxyapatite and its osteogenic induction effect in vitroObjectives:The micron grade hydroxyapatite (mHAP) were selected to be used in this study. The morphology of the mHAP particles were been characterized and the mHAP particles were co-cultured with MC3T3-E1 Preosteoblast Subclones for exploring its biocompatibility with cells and osteogenic induction effect in vitro.Methods:In this study, the micron grade hydroxyapatite (mHAP) were selected to be used. First of all, the morphology and the surface charge of the mHAP particles were characterized by scanning electron microscopy (SEM) and melvin Zeta potentiometer respectively.And then the cell biocompatibility and osteogenic induction effect of the mHAP particles with this kind of morphology were examinatied in vitro:(1) Different concentrations (Oug/ml,250 ug/ml,500 ug/ml, 1000 ug/ml) of mHAP particles were co-cultured with MC3T3-E1 cells and then the proliferation activity of cells were detected by using CCK-8 assay at different time point. (2) Based on the results of the proliferation activity, the optimal concentration of mHAP was chosen to co-cultured with MC3T3 El cells, and the biocompatibility of mHAP was detached by live/dead dyeing method for cells. (3) The mHAP marked with the F1TC fluorescence were preparied in advance according to the literature, and were used to observe the adhesion effect between mHAP and cells. (4) Alkaline phosphatase staining and quantitative detection of alkaline phosphatase were performed respectively to study the osteogenic induction effect of the mHAP particles, cell ALP activity was investigated using BCIP/NBT and pNPP (Beyotime, CHINA) assays respectively.Results:(1) Under the microscope, the particles exhibited microsphere shape with relatively smooth surface and the larger diameter microspheres were consisted with lots of smaller diameter microspheres. (2) In order to address the cell adhesion with mHAP, as-prepared FITC-mHAP was co-cultured with MC3T3-E1 cells for 24h. Green fluorescence located on the cell surface demonstrated the mutual adhesion and interaction between mHAP and cells. (3) The sizes of the small microspheres were about 5-20μm after being sonicated. The zeta potentials of the mHAP in PBS (PH=7.2) were measured to be-39.84±1.37 mV (n=6). (4) The CCK-8 assay results showed that all of the three different concentration of mHAP could implove cell viability at different levels (p<0.01), among which the 500 μml-1 mHAP performed the best cell viability promotion effect. A higher cell viability promotion effect of mHAP was obtained after being co-cultured with cells for 48hrs compared with 24hrs. (5) Live/dead assay showed that cells co-cultured with the mHAP exhibited high viability, and the mHAP on the cell surface induced cells aggregation to form multicellular spheroids. (6) Cells cultured in different medium (a-MEM medium with or without osteo-inductive reagents) were analyzed using ALP staining. After 3 days, the higher ALP activity was detected in the cells co-cultuerd with mHAP group compared with the control group no matter cultured in a-MEM with or without osteo-conductive reagents. The further quantitative detection of ALP secretion at day 7 showed that cells co-cultuerd with mHAP had higher ALP activity compared with the control cells cultured in a-MEM medium with or without osteo-conductive reagents.Conclusions:(1) The hydroxyapatite used in this experiment have morphology of smooth spherical, mostly close to 100 um in diameter, the surface charge is of negatively charged. (2) The appropriate low concentration of mHAP microspheres (500 ug/ml) have good biocompatibility, can promote the adhesion and proliferation of MC3T3-E1 cells. (3) The ALP secretion can be up-regulated by appropriate low concentration of mHAP microspheres (500 ug/ml), demonstrating the good osteo-inductive effect of the mHAP microspheres.Part Ⅲ:The preparation of micron hydroxyapatite-gelatin methacrylate cryogel and the osteogenesis effect in vitroObjectives:First of all, gelatin-methyl methacrylate graft copolymer (GelMA) was prepared by gelatin (Gel) and methyl methacrylate (MMA) as raw material, and then the inorganic micron grade hydroxyapatite (mHAP) with different quality/volume concentration was added into GelMA compound for cryogelation. not only for the purpose of simulating the compositions of natural bone, but also greatly increasing the mechanical properties of the scaffolds. And the cryogels based on mHAP-GelMA were characterized and the biocompatibility in vitro and osteogenesis characteristics were studied in order to provide the basis for tissue repair.Methods:(1) Gelatin-methyl methacrylate graft copolymer (GelMA) was prepared by gelatin (Gel) and methyl methacrylate (MMA). (2) On the basis of GelMA with the concentration of 0.33%, mHAP with different quality/volume fraction (0%,1%,2.5%,5%,10%) were compound to the GelMA, appropriate concentration of ammonium persulfate (aps) and the four methyl diethylamine (TEMED) were adding into the mix as crosslinking agent and catalyst, cryogels were fabricated through cryogelation method at -20℃. (3) The swelling performance of cryogels containing different concentrations of mHAP preparied above was tested according to the classic methods. (4) The cryogels with different concentrations of mHAP which were prepared under different temperatures, were compressed by the bose electroforce, and then the elastic modulus can be obtained according to the stress and strain parameters. (5) BMSCs cells were seeded on the cryogels, cell adhesion rates were tested by using the CC-K 8 assay. (6) The dry cryogels with different concentrations (0%,2.5%,10%) of mHAP and pure hydroxyapatite were ground into powder and investigated by fourier transformed infrared (FTIR) spectroscopy to confirme the material composition. (7) The morphology of cryogels containing different concentrations (0%,2.5%,10%) of mHAP, as well as the cells grown in the cryogels were observed by scanning electron microscopy. (8) BMSCs grown in different quality/volume fraction mHAP (0%,2.5%,10%) cryogel for 1 day.3 day,5 days respectively, the cell activity was tested by live/dead staining kit. (9) BMSCs grown in different quality/volume fraction mHAP cryogel (0%,2.5%,10%) for 3 days,1 week and 3weeks respectively, after that the total RNA was extracted from BMSCs, and the RT-PCR products were used for real time quantitative PCR for osteogenesis related gene such as RUNX2, OCN and IBSP gene expression analyzed. (10) And same as the above step, BMSCs grown in different quality/volume fraction mHAP cryogel (0%,2.5%,10%) for 3 weeks were used for the immunofluorescence to detact the OCN protein expression.Results:(1) Different cryogels were successed prepared by the following method:on the basis of GelMA with the concentration of 0.33%, mHAP with different quality/volume fraction (0%,1%,2.5%,5%,10%) were compound to the GelMA, appropriate concentration of ammonium persulfate (APS) and the four methyl diethylamine (TEMED) were adding into the mix as crosslinking agent and catalyst, which were put and maintain in-20℃. All the cryogels had good elasticity, the cryogel without mHAP is transparent, while cryogels containing mHAP present white color. (2) Swelling rate detection:the cryogel without mHAP has the fastest water absorption rate within the first 5 min and at last has the maximum equilibrium swelling ratio (Qc= 15.38), by contrast, the water absorption rate and the equilibrium swelling ratio of the cryogel compound with mHAP were decreased(9.05,8.67,5.89, 8.67, respectively) with the increasing of mHAP dosage (1%,2.5%,5% and 10% respectively of mHAP). Of which the 1% group and 2.5% group have little difference on the equilibrium swelling ratio and there is little difference between the 5% group and 10% group; (3) The cryogels containing mHAP (2.5%.10%) have a higer elastic modulus(2.5%:53.47±8.43 kPa and 43.72±7.64 kPa,10%:70.14±10.07 kPa and 54.7±12.2 kPa) compared with cryogels without mHAP (36.02±3.67 kPa and 7.32±1.09 kPa) which were prepared under the same temperature, it means that the mechanical properties increased gradually with the increase of the mHAP in cryogels. While there was rarely difference between the cryogels with the same composition but cryogelation at different temperatures(-20℃ and-80℃). (4) The cells adhesion rates were in complete contrast to the results of the cryogels elastic modulus. Under the same preparation temperature, the cells adhesion rates were decreased gradually with the increase of the mHAP in cryogels, cryogel without mHAP had a higher adhesion rate than 2.5% group (P< 0.01) and 10% group (P< 0.01). By contrast, the temperature had a smaller effect on cell adhesion rate of the cryogels. (5) Fourier infrared spectrum detection showed that the cryogel with mHAP (quality/volume fraction of 2.5%,10%) exhibitied the characteristic absorption peak of pure GelMA and pure HAP in its infrared absorption curve, demonstraing the composite of the two materials. (6) The morphology of different quality/volume fraction mHAP (0%,2.5%, 10%) cryogel were observed by scanning electron microscopy (SEM), images showed that:the cryogel scaffold of each group were present an inter-permeation honeycomb macroporous structure, the aperture of the pure GelMA cryogel was around 20-200 um. with smooth surface. The cryogel contain 2.5% mHAP had similar aperture with the pure GelMA cryogel, while the surface got rough and thickens because of the adding of mHAP. When the adding mHAP increased to 10%, the pore diameter reduced obviously, and more rough surface and thickening wall could be observed with numerous massive protuberant on it, and it looked like having a trend of collapse. (7) The live/dead result of BMSCs in different quality/volume fraction mHAP (0%,2.5%,10%) cryogel showed that, cells on each group material (0%,2.5%,10% cryogel) all had good vitality, red death celsl could rarely be seen. At day 1, cells on the three groups material presented circular cell morphology which were not spreading, and the 10% group’s cells number was less than the other two groups (cryogel with 0% mHAP and 2.5% mHAP). At day 3 and day 5, cell morphology changed to spindle on each group, and the number of cells increased, while 10% mHAP group cells was still less that the other two groups. (8) BMSCs in different quality/volume fraction mHAP (0%,2.5%,10%) cryogels observed by scanning electron microscopy (SEM) and results showed that:overall, each group cryogel had a large number of cell growing on/in it, cells showed spindle shape, more cells could be seen both on the cryogel surface and inside the pore wall of the 0% and 2.5% mHAP groups compared with 10% mHAP group. (9) The osteogenesis related gene expression of BMSCs grown in different quality/volume fraction mHAP(0%, 2.5%,10%) cryogel at different time points 3 days,1 week,2 weeks, result as the following. At day 3, RUNX2, OCN and IBSP gene expression of BMSCs growinig on all the cryogels were all low, and had little difference between each group. While the expression of RUNX2, OCN and IBSP were increased with the culture time prolonged to 1 week and 2 weeks respectively, at the same time, for OCN and IBSP genes, the higher mHAP concentration the cryogel containg the higher gene expression were observed. RUNX2:at day 3,0%> 2.5%>10%***/## at 1 week, 2.5%***> 10%***/##> 0%, at 2weeks,2.5%***> 10%***> 0; OCN:at day 3, no difference between each group, at 1week,10%**/#> 2.5%**> 0%, at 2 weeks, 10%***/##> 2.5%****> 0%; IBSP:at day 3,10% group gene expression was higher than the other two groups (P<0.01), at 1week and 2 weeks,10%***> 2.5%***> 0%, (*means compared with 0% group,#means compared with 2.5% group). (10) The osteogenesis related protein OCN expression of BMSCs growing in different cryogel for 3 weeks:OCN protein expression was incresed with mHAP dose incresed in cryogels, the most amounts of OCN protein were found in 10% mHAP group,and then the 2.5% group, at last the 0% group. This result demonstraing that adding hydroxyapatite could enhance differentiation of mesenchymal cells to osteoblasts.Conclusions:(1) On the basis of GelMA, large porosity cryogels with different performance were succeed through adding different quality/volume fraction of mHAP (0%,1%,2.5%,5%,10%). (2) The swelling rates of cryogels containing different concentrations of mHAP were different, the swelling rates were decreased with the mHAP concentration increased. (3) The cryogels containing different concentrations of mHAP have different mechanical properties, the elastic modulus were increased with the mHAP concentration increased, while the temperature effect on the mechanical performance of the cryogel were decresed with the mHAP concentration increased. (4) The mHAP particles dispersed evenly in the cryogels. as the adding mHAP concentration increasing, the cryogel surface become rough instead of smooth, the scaffold wall thickening, aperture decreased and dense. (5) Compared with pure GelMA cryogel, adding mHAP into cryogel could inhibit cell’s adhesion and proliferation, but on the contrary could promote differentiation of mesenchymal cells to osteoblasts as the osteogenesis related gene and protein RUNX2, OCN and IBSP expression were obviously increased.Part Ⅳ:Micron hydroxyapatite-gelatin methacrylate cryogel 3D scaffold for parietal defects repair in ratsObjective:To implant the hydroxyapatite-gelatin methacrylate cryogel 3D scaffold in the parietal defects in rats, and investigate the osteogenic effect of the scaffolds.Methods:(1) We fabricated the GelMA cryogel containing different concentration of mHAP as shown in Part III. (2) The above 3D scaffolds were planted into a rat parietal defect.12 weeks later, the parietal gross specimen was observed. (3) All the samples were scanned with micro-CT to get the area data of regenerated bone in all the samples. All the scanning section images of the samples were reconstructed into a three-dimensional image for detecting. (4) HE staining and Masson staining will be done for bone repair investigation in each group.Results:(1) The gross images of samples in each group were compared: samples from all groups have flat surface, with no residual defects in the original defect place. (2) As the micro-CT results showed, the GelMA cryogel containing different concentration (0%,2.5%,10%) of mHAP all has certain effect on repairing skull defect, in which the 2.5% group showed the biggest new bone area (17.272±0.807mm2****), and then the 10% group (13.036±0.948mm2**) pure GelMA cryogel without mHAP showed the worst repairing effect (10.658±1.910mm2). The result indicated that the cryogel containing 2.5% mHAP has the best ability of repairing skull defect.(* means compared with the model group).Conclusions:(1) The rat skull critical bone defect model was succed and showed that after 12 weeks the defact cannot be repaired itself without any implant materials, repair performance was poor; (2) The GelMA cryogel containing different concentration (0%,2.5%,10%) of mHAP all has certain effect on repairing skull defect, in which the 2.5% group showed the biggest new bone area. |
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