Development Of "Green" Thermo-sensitive 3D Printed Bone Scaffolds

Purposes:Large-scale bone defects lack self-healing ability,so bone substitute materials are used to support and promote their osteogenesis.An ideal bone substitute material needs to have sufficient mechanical strength,sufficient porosity,good bioactivity and biocompatibility,and sufficient supply.3D printing can fabricate bone scaffolds that meet the above requirements and can be prepared into specific shapes according to the needs of patients.Among various 3D printing techniques,extrusion-based printing is most commonly adopted due to its relatively simple set-up,low operation cost,and high printing speed.In this study,chitosan(CS)was dissolved by a repeated freezethaw method in alkali/urea solution,to obtain a matrix material with higher mechanical strength and good biocompatibility.Subsequently,hydroxyapatite(HAP)and lyophilized platelet-rich fibrin(L-PRF)powder were combined into the CS solution for co-printing,which endows the scaffold with good osteoconductivity and the ability to release growth factors.The final composite scaffolds were tested to screen out bone scaffolds with excellent performance.Method:1.CS is dissolved in alkali/urea solution by repeated freeze-thaw method,and then different mass fractions of HAP are added into CS solution to prepare CS-HAP bioink.The viscosity of the composite ink was the largest when the mass fraction of HAP was 20% by rheology,and it was used as the ratio of the subsequent experiments.2.L-PRF powders with mass fractions of 0.5%,1%,and 2.5% were added to the screened CS-HAP bioink,and the rheological properties were optimized with acetic acid-gelatin solution.The rheological properties of the optimized PRF-CS-HAP(P-CH)composite ink was tested to evaluate its printability.Subsequently,the P-C-H composite ink was 3D printed at 4?,and crosslinked with deionized water at 37?.Then,the excess water and solvent in the 3D scaffold were removed by freeze-dried.3.The composition and crystal structure of the composite scaffolds were analyzed by Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD),respectively,and the pore size and internal structure of the composite scaffolds were observed by scanning electron microscopy(SEM).4.The mechanical,hydrophilic,degradation,and sustained-release properties of the composite scaffolds were tested.Subsequently,the scaffolds were co-cultured with MC3T3-E1 cells to detect the biocompatibility of the composite scaffolds and the activity of growth factors in PRF.Results:1.C-H,0.5%P-C-H,1%P-C-H,and 2.5%P-C-H composite inks were prepared respectively.The rheological analysis showed that all of them had "shear-thinning" behavior and were thermo-sensitive,which were shown as storage modulus was positively correlated with temperature.2.The 3D scaffolds were successfully printed with four kinds of inks.After SEM scanning,it was observed that the pore diameter of the four scaffolds was about 500-700 ?m,and the lines were continuous and uniform.According to FTIR analysis,the characteristic peaks of CS and HAP were found in the spectrum;through XRD analysis,it was found that the crystal structure of HAP in the scaffold did not change.3.With the addition of L-PRF,the compressive properties of the scaffolds gradually decreased,but they still met the mechanical strength of cancellous bone.All4 groups of scaffolds showed good water absorption capacity,and the water absorption rate exceeded 300%.After the degradation performance test,it was found that the composite scaffolds had a relatively stable degradation rate,but the 2.5%P-CH scaffolds had broken lines after 80 days of degradation.Subsequent p H detection showed that the p H value of the scaffolds after degradation was mostly maintained between 7.5-8.4.4.The sustained release performance of the composite scaffolds was measured for 35 days,and it was found that the release of growth factors could be detected in the composite scaffolds within 35 days,and the release level showed a decreasing trend with the extension of time.After MC3T3-E1 cells were co-cultured with 4groups of scaffolds,it was found that all 4 groups of scaffolds could promote cell proliferation,and the 2.5% P-C-H scaffolds had the best ability to promote cell proliferation,which proved that the composite scaffolds had good biocompatibility and PRF was still active after lyophilization and printing.Conclusion:The 2.5% P-C-H scaffold has good biocompatibility and the ability to promote cell proliferation.Although its mechanical strength is lower than that of other groups,it still meets the strength requirements of cancellous bone.Therefore,the 2.5% P-C-H group is considered to be the most potential composite material for bone scaffolds.