| Objective: Bone defects caused by congenital anomalies, tumors, trauma, and infection are highly common clinical symptoms. Bone defects affect the morphology and function of bone defect positions of patients in varying degrees. Furthermore, bone defects result in serious troubles to the normal lives of patients[1]. At present, several main clinical methods are used to repair bone defects, including autogenous bone graft, allogeneic bone graft, and artificial bone graft. However, all this methods has their limitations [2].The emergence of tissue engineering technology is promising offers promising applications for the repair of bone defects[3]. Essentially, tissue engineering combines bionics ideas with medical science. The technology mainly simulates an internal microenvironment to control the formation of tissues and organs in vitro. Bone scaffolds are one of the key elements of bone tissue engineering, and serving the function of being as the basic framework for seed cell adhesion and carrying growth information. The tissue engineering scaffolds materials for bone regeneration should be highly bioactive, degradable, mechanically strong, osteoinductive, and osteoconductive[4]. Biphasic calcium phosphate ceramics(BCP), which consist of different proportions of hydroxyapatite(HA) and beta-tricalcium phosphate(β-TCP), have been extensively applied as bone tissue engineering scaffold material, drug sustained-release material, and dental implant surface coating material because of their excellent qualities, such as biocompatibility, osteoinductivity, and adjustable degradability[5]. Several conventional methods were previously used to fabricate porous scaffolds, such as phase separation, gas foaming techniques, particulate leaching [6-7] and so on. However, none of these conventional methods can accurately control pore size, pore morphology, overall porosity and the interconnectivity of the scaffolds. Furthermore, the mechanical property of these scaffolds is usually poor. Recently, to better strengthen the mechanical properties and control pore structure, 3D printing technology was used to fabricate porous scaffolds. One of significant advantages of this technique is that the pore structures of the scaffolds can be concisely designed by through layer by layer plotting until the overall structure is completed[8]. Therefore, in this study, we applied 3D printing to prepare BCP(%HA/%β-TCP: 30/70) scaffolds and studied the biocompatibility and physicochemical properties of the scaffolds.Metheds: 1. 3D printing technology was used to fabricate BCP(%HA/%β-TCP: 30/70) scaffolds(cylinders, diameter=10mm, height=3mm). Prepared mound was used to fabricate BCP cylinders, which were the same shape with the scaffolds.2. The surface topography of the scaffolds was evaluated using an atomic force microscope(AFM) and a scanning electron microscope(SEM).3. The relative total porosities(P) of the porous scaffolds were calculated by the gravimetric method.4. The compressive strength and Young’s modulus of the BCP scaffolds(cylinder, d=10mm, h=3mm) were tested using an Instron Universal Testing Machine.5. There’re three groups, BCP scaffolds leach liquor, phenol solution and high glucose DMEM culture medium supplemented with 10% FBS. CCK-8 assays were performed to evaluate the cytotoxicity of the three groups after 1,3, and 5 days of in vitro culture.6. There’re three groups, BCP scaffolds, BCP cylinders and blank control group. CCK-8 assays were performed to evaluate the cell proliferation of the three groups after 1, 3, 5 and 7 days of in vitro culture.7. The morphology of the attached cells on the two types of samples was studied by SEM.8. Data were analyzed using SPSS software package for Windows. The results were reported as means ± standard deviations values(SD) and comparison between different groups was made using one-way analysis of variance(ANOVA). A paired sample t-test was used to compare different groups. A value of P < 0.05 is considered statistically significant.Results: 1. The prepared BCP scaffolds were cylinders(diameter=10mm, height=3mm). BCP cylinders were the same shape with the scaffolds.2. AFM analysis of the BCP scaffolds indicated that the surface was uneven and full of sags and crests. Their surface roughness value(Ra) was 208 nm. SEM micrographs of the BCP scaffolds showed that they had well controlled square macropores and pore size was between 350μm–450μm. In addition the surface morphology of the macropore walls was uneven and there’re numerous micropores(pore size several micrometers).3. The thoretical density(ρt) of the BCP scaffolds was approximately 1.48 g/cm3, whereas their porosity was approximately 52%.4. Compressive strength of BCP scaffolds was roughly 2.77±0.87 MPa,and Young’s modulus was 72.23±9.54 MPa.5. After 1, 3, and 5 days in vitro culture, leaching liquor did not indicate cytotoxicity, whereas 0.1% phenol did.6. After 1 day of cell culture, both blank control group and BCP cylinders demonstrated considerably higher rates of cell proliferation, compared with the BCP scaffolds surface(P<0.05). At the 3-day sampling point, increased number of absorbance observed in the blank group was shown to be statistically significant with respect to BCP scaffolds(P<0.05), whereas no statistical difference could be established between the BCP scaffolds and cylinders. Then, after 5 days of in vitro culture, no statistically significant difference occurred among the three groups. However, after 7 days, the measured cell number on the BCP scaffolds surface was higher at this time point compared with those of the other two groups(P<0.05).7. After 24 h of in vitro culture, a well spread morphology of MG-63 cells adhered successfully to the surfaces of the BCP scaffolds and BCP cylinders.Conclusion: To prepare BCP(%HA/%β-TCP=30/70) scaffolds, 3D printing technology was successfully applied. The resulting materials showed a well-controlled square pore shape, had appropriate porosity and perfect interconnectivity, and featured surface roughness that was favorable for cell adhesion. Moreover, their high specific surface area ensured the steady proliferation of MG-63 cells. The mechanical properties of the scaffolds make them perfect candidate materials for the repair of non-load bearing bone defects in the future. |
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