| Background and Objectives:With the progress of science and development of our society, environmental factors have been severely change, which results in sharp increase of incidence of massive trauma, severe infectious disease-including tuberculosis-and bone tumors. In most cases of the abovementioned diseases especially tumor, surgeries should be considered and a large amount of tissues will be cut which will lead to massive tissue defect, especially bone defect. In Orthopaedic surgery, treatment of massive bone defect has always been the key point. Bone grafting turns out to be the only way to treat this series of diseases. The sources are from autologous bone, allograft bone, xenograft bone and artificial bone. Autologous bone has always been considered as gold standard of bone grafting material due to its good osteoinductivity and osteoconductivity, and the effect of therapy is good, but it has some shortcomings such as limited sources and range of repair. What is more, the injury of supply area might cause chronic pain and infection. Allograft and xenograft bones do not have the shortcomings of limited sources and injury of supply area, so that it can significantly increase range of repair, but they might cause post-transplantation rejection which affects the long-term effect. Artificial bone is a newly developed bionic bone. With the development of tissue engineering, artificial bone is gradually replacing other grafting bones and starts to be implemented in clinical application. Hydroxyapatie is one of the artificial scaffolds which has similar ingredients as natural bone, and it begins to be widely accepted in Orthopaedic clinical use, and the efficacy is affirmative. On the other hand, pore size and porosity are considered to be play an important role in affecting cell proliferation and other biological activities. In order to observe this, we immersed pre-osteoblasts suspension into porous HA scaffolds and cultured dynamically in vitro to observe the difference in cell proliferation and other biological activities between scaffolds with different pore sizes and porosity. At last, in vivo experiment is implemented to observe the repairing effect of bone regeneration in rabbits。Methods:In the first part, porous HA scaffolds with different pore sizes, different porosity and good interconnectivity were made by extrusion deposition technique and microwave sintering. Second, dry weight of each scaffolds-which are divided into two groups, as known as group A with designed pore size of150μm and group B with designed pore size of300μm-is weighed, then they are submerged in distilled water under vacuum, and the full saturated weight is weighed, permeability(p) is calculated by certain formula, the mean of the six is calculated. Third, the phase composition of sintered scaffolds and original HA powder is tested to determine if there is any phase transition. Last, scaffolds are scaned under SEM for surface morphology.In the second part, MC3T3-E1was obtained from American Type Culture Collection(ATCC) and cultured in DMED-HG medium at37℃in5%CO2. Then cell suspension was dropped axially through the whole column scaffolds with average diameter of12mm and height of5mm(grouped as those in the first part) to make sure initial seeding, then scaffolds were transferred to perfusion bioreactor for5days with average flow of3.4ml/min at37℃in5%CO2。When perfusion culture was finished, cell proliferation between different groups of scaffolds was determined by MTT assay, and cell metabolic activities were determined by glucose consumption. Last, cell adhesion and proliferation is observed directly by SEM scan.In the third part,30New Zealand rabbits were randomly classified into6groups. Bone defect of1.0cm was made in each rabbits, and4different series of HA scaffolds with length and width of5mm and height of10mm were implanted into bone defect of4groups respectively (each group has6rabbits):A, nonporous HA blocks; B, scaffolds with designed pore size of150μm; C, scaffolds with designed pore size of300μm; D, scaffolds with designed pore size of500μm. Group E and F(each group has3rabbits) were designed as positive and negative control respectively.3rabbits from Group A to D were executed at the time of4weeks an8weeks postoperatively respectively, and gross specimen assessment, CT with3D construction and Micro-CT scan were made to observe bone defect repair.Results:Extrusion deposition method and microwave sintering can fabricate pre-designed HA scaffolds with complicated internal structure, the pore size of two groups are (149.0±6.8)μm and (294.3±8.5)μm respectively and porosities are (36.7±0.1)%and (52.2±0.2)%respectively. Phase composition of scaffolds and material show the same XRD spectrum as HA, the full width at half becomes narrower。SEM scan showed the ordinary internal structure of scaffolds, the pore are rectangular with good interconnectivity. Surface morphology showed irregular polygon of HA particles with micropores between them.A large amount of osteoblasts were proliferated on the scaffolds after perfusion culture, MTT assay showed OD/mass ratio of1.31±0.26in group A and1.51±0.43in group B, between which the t-test showed no significant difference(p>0.05). Glucose consumption was (162.38±33.09)μmol/L in group A and (217.97±27.91) μmol/L in group B, between which the t-test showed significant difference(p<0.05). A large amount of osteoblasts were adhered and proliferated on the scaffolds under SEM scan, and they grew gradually from corner to center of the pores. Pseudopodiums were seen in osteoblasts and a lot of extracellular matrix was excreted.Massive specimen observation of animal experiment showed distinct gap between scaffolds and bone after4weeks, which became vague after8weeks in3groups other than Group A. Under CT scan and reconstruction, a little callus was found only in group D after4weeks, and closure of intermedullary space was found in group A, more callus was found in other3groups (most in Group D and least in Group B) after8weeks. Under Micro-CT scan and reconstruction, a few osteoid materials were found in pores of3groups after4weeks, which increased significantly after8weeks.Conclusion: Extrusion deposition method and microwave sintering was a successfully effective way to fabricate complicated porous HA scaffolds with different pore sizes and porosities and good interconnectivities, which will not cause phase transition.Perfusion culture was an effective way to make osteoblasts adhere to and proliferate on the scaffolds. With the increase in pore size and porosity, the activity of osteoblasts increased.Porous HA scaffold had osteoinductivity in vivo, which increased with the increase in pore size and porosity. |
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