Research Of Gel/n-HA/PLGA Bone Tissue Engineering Scaffold Based On 3D Printing Technology

In recent times,the tremendous progress has evidenced the advancement of various methods in generating the three-dimensional(3D)porous scaffolds.The current traditional methods include particle leaching,gas foaming,micro-ball sintering,and electrospinning,among others.However,the applicability of these methods is limited due to many shortcomings,such as the residues of the solvent the difficulty in controlling the shape of the orifice,and multiple preparation steps,among others.3D printing technology has emerged as the most promising approach to overcome the aforementioned limitations of all traditional methods and produce the porous scaffolds on demand without using any pore forming agent.We fabricated the composite 3D porous scaffolds based on gelatin,nano-hydroxyapatite,and poly(lactide-co-glycolide)(Gel/n-HA/PLGA)using the 3D printing and freeze-drying hybrid technologies for bone tissue engineering.Further,the degradation and biological properties of these composite scaffolds were systematically elucidated.Firstly,the preparation conditions,as well as the ratio of Gel/n-HA,filling fluid and the PLGA scaffolds were optimized.The hydrophilicity of the PLGA scaffold was improved with the anhydrous ethanol,and the Gel/n-HA mixture was filled in the scaffold.Then Gel/n-HA/PLGA composite scaffold was obtained after freeze drying.The change of physical and chemical properties of PLGA scaffolds in the process of high temperature and humidification were measured.Further,the effect of various factors including filling spacing and angle,the thickness of the mechanical properties and porosity of scaffolds were also investigated.The results indicated that the highly porous PLGA scaffolds were obtained at the optimized conditions(weight of PLGA: 1.6-2.0 g,the room temperature: 30 ℃,the printing temperature: 195 ℃,the nozzle inner diameter: 0.4 mm,the printing pressure: 0.4 MPa,and the printing speed: 10 mm/s).The PLGA scaffolds resulted in the rate of water absorption of 23.31 ± 3.53% after its treatment with anhydrous ethanol for 9 h.The pore with the size of 100 μm-300 μm was obtained at the pre-freezing temperature of-80 ℃,and the Gel/n-HA concentration of 20 mL/4 g.Further,the scaffolds were systematically characterized using various techniques such as fourier transform infrared spectroscopy(FTIR),X-raydiffraction(XRD),differential scanning calorimeter(DSC),and thermogravimetric analysis(TGA)indicating that the only physical process,which can treat the PLGA scaffolds with anhydrous ethanol and high-temperature melting.The mechanical properties,as well as porosity of the composite scaffolds,were affected by the filling space,the thickness,and the filling angle.Next,the degradability in vitro of Gel/n-HA/PLGA composite bone scaffolds was investigated by measuring the rate of water absorption and weight loss,pH value,compression strength,the compression modulus of the scaffolds immersed in the phosphate buffered saline(PBS)for 10 weeks.Moreover,the changes in the process of degradation were observed by capturing the microscopic images.The results indicated that there was no significant change in the pH value of the medium and the scaffolds remained undisturbed,and the water absorption rate was recorded as 54.98 ± 3.03%.However,we observed specific changes in the architecture relevant to the integrity of scaffolds,i.e.,compression strength decreased to about 20 MPa,and the compression modulus decreased from 76.97 ± 7.66 MPa to 49.7 ± 8.39 MPa.In addition,it has shown micro-sized pores on the surface of PLGA,and the porous structure of Gel/n-HA filler appeared collapsed after nine weeks of the study.The degradation process of Gel/n-HA/PLGA composite scaffold was gentle,and it still maintains good mechanical properties during degradation.Finally,the biological performance of Gel/n-HA/PLGA composite scaffold in MC3T3-E1 cells was investigated for osteoblast growth and differentiation.The cytotoxicity,blood compatibility and heat source reaction of composite scaffolds were studied.The cells were co-cultured with the composite scaffold,and the adhesion,orientation,secretory protein content on the scaffold were investigated.Then,the investigations were continued in finding for whether any sort of cytotoxic substances during the 10 week of degradation of composite scaffolds affect the growth of the cells.The effects of degradation products on the behavior of MC3T3-E1 cells were also investigated after three months of compound degradation.Finally,the effects of composite scaffolds on the secretion of osteocalcin and type I collagen in MC3T3-E1 cells were detected.The results have shown that the composite scaffolds exhibited no significant cytotoxic effects on the cells.Moreover,the composite scaffolds had shown excellent blood compatibility and had not caused any signs of heat source reaction.Also,the composite scaffolds had no effect on the protein secretion from MC3T3-E1 cells.It is evident from the confocal laser scanning microscopic(CLSM)images that the MC3T3-E1 cells were arranged and distributed systematically on the composite scaffold after 12 hours.The adhesion rates of common 3D printed scaffolds,humidification scaffolds,and the composite scaffolds were 44.63 ± 3.59%,51.52 ± 8.24% and 72.35 ± 7.15%.Furthermore,the degradation solution of the scaffolds could also promote the proliferation of osteoblasts,which was confirmed by the synthesis of alkaline phosphatase and total protein.Furthermore,the composite scaffolds could also promote the proliferation of osteoblasts,which was confirmed by the synthesis of osteocalcin and type I collagen.In conclusion,Gel/n-HA/PLGA composite scaffolds with excellent mechanical properties and biocompatibility were highly promising in promoting MC3T3-E1 cells adhesion and growth.Moreover,there were biocompatibility issues during the process of degradation,as the resultant products had significantly promoted the proliferation,alkaline phosphatase and total protein synthesis of MC3T3-E1 cells,the composite scaffolds had significantly promoted osteocalcin and type I collagen.synthesis of MC3T3-E1 cells,thus promoting the ossification function.The use of 3D printing technology combined with freeze-drying technology can make highly porous 3D scaffolds without the use of an organic solvent.This innovative hybrid approach for generating the Gel/n-HA/PLGA scaffolds with great benefits will undoubtedly find the potential ways in the treatment of bone defects.