Preparation Of ICA/?-TCP/PCL Tissue Engineering Scaffold By 3D Printing And Its Performance Study

In traditional medicine,Icariin(ICA)has been commonly used to prevent osteoporosis and bone trauma.In view of these facts,we used a drug-loaded bone tissue engineering scaffold based on poly-?-caprolactone(PCL)and tricalcium phosphate(?-TCP).Further,the composite material was added with ICA,subjected to low-temperature extrusion-assisted three-dimensional(3D)printing approach and for osteogenic effects of these scaffolds were explored.It was expected to overcome the limitations of bone trauma and repair in the field of bone tissue engineering.Firstly,the preparation and characterization of ?-TCP/PCL(TP)scaffolds were investigated.The TP scaffold was prepared using a biological 3D printer as a blank scaffold for subsequent studies.The whole factor experiment was designed to analyze the experimental results by Minitab software to determine the optimal preparation parameters.Then,the physicochemical,as well as cytocompatibility attributes of the blank scaffold,were investigated.The preliminary results showed that there existed any significant levels of residual organic solvent dioxane,demonstrating that the structures met the requirements of biosafety;FTIR and XRD characterizations were explored to illustrate the material properties of the scaffold after 3D printing and freeze-drying process.The results confirmed that the stent forming effect was excellent and the repeatability was high.The SEM results showed that the surface of the stent prepared by 3D printing was rough and the internal pores penetrated,which facilitated cell adhesion and nutrient transport in the later stage;the compressive strength test was(1.78±0.02)MPa.It meets the requirements of tissue engineering scaffold strength;contact angle test results showed that the contact angle of the scaffold was less than 90°,which was conducive to cell adhesion and growth;the results of cell compatibility studies showed that the TP scaffold environment is suitable for cell adhesion and growth,and the cells secreted phosphoric acid on the scaffold.The enzyme activity increased with the culture time,and the TP composite scaffolds have shown excellent cytocompatibility.Secondly,the preparation and characterization of ICA/?-TCP/PCL(ITP)scaffolds were investigated.By adding different concentrations of ICA to the printing matrix,the optimal preparation parameters were used to prepare scaffolds with different drug loadings,and the cytotoxicity of different drug loading ITP scaffolds was investigated to determine the optimal ICA of the ITP scaffold.The physicochemical properties,the cytocompatibility and biotoxicity of ITP scaffold materials,and the long-term release behavior of ICA and the pH change of the release medium in the simulated body fluid environment were examined.The results showed that the cytotoxicity results showed that the three levels of ICA scaffolds were not toxic to BMSCs(Bone mesenchymal stem,BMSCs)cells,while the scaffolds containing 0.32% have shown the promising effects on BMSCs cells.The ICA loading in the scaffolds was determined to be 0.32% by mass;The 16-week release test showed that the ICA in the composite scaffold was slowly released;the pH of the release medium(from pH 7.40 to 6.91)showed that the material such as PCL in the scaffold was slowly degraded;The results showed that the cells could naturally stretch and grow well on the scaffold,and they could proliferate and differentiate.The cell protein adsorption and secretion on the scaffold showed that the scaffold could enrich the surrounding proteins and promote the secretion of related proteins,which could facilitate the cells on the scaffold.The results of biocompatibility studies showed that the hemolysis rate of ITP scaffold extract was much lower than 5% and has shown excellent blood compatibility.The results of acute systemic toxicity test showed that the intraperitoneal injection of scaffold extract has no significant changes in the activities of animals within 3 days.Rats had normal body weight growth and no significant adverse traits;liver pathological observations showed that the material has no significant toxicity to mouse liver;sensitization test results.Collectively,it showed that the ITP scaffold material has shown no signs of allergic reactions in mice.Finally,the biological properties of ITP scaffolds were investigated.a)The biodegradability of TP in the simulated body fluid was investigated.In the degradation process,the physical and chemical properties of scaffolds,such as surface morphology,hydrophilicity and mechanical properties,as well as the toxicity of degradation products of different groups of scaffolds to BMSCs cells and the effect of degradation products on ALP expression of BMSCs cells were investigated.b)The osteogenic properties in vitro of ITP scaffolds were also examined.The expressions of ALP,OCN,Col-I,Runx2 and other related osteogenic specific factors were investigated to evaluate the osteogenic properties of ITP scaffolds.The results showed that a)the degradation of TP scaffolds has no signs of generation of resultant toxic substances,and the physical and chemical properties of the scaffolds were excellent during degradation.BMSCs cell growth curve showed that b)the cells entered logarithmic growth phase after 4 days of culture and were in growth plateau after 8 days.Giemsa staining of BMSCs cells in logarithmic growth phase showed that the nuclei were purple and healthy.The cytoplasm was pink and the cytoplasm was plump,indicating excellent activity;the calcium nodule staining results showed that the extract of ITP scaffold material had a good ability to promote calcium deposition and calcium production;Moreover,osteogenic specific factors(ALP activity expression,OCN,Col-I,Runx2)test results showed that ICA in the scaffolds could up-regulate the expression of osteogenic differentiation-related genes and significantly promote BMSCs differentiation into osteoblasts.In conclusion,we could successfully prepare an ITP scaffold material with good physical properties,biocompatibility and osteogenic properties by 3D printing technology.Moreover,the loaded drug in the scaffold could successfully maintain its activity and release slowly and steadily for a long time.TP scaffolds were highly compatible resulted in no toxic substances in the degradation process as the degradation products successfully promoted cell growth and proliferation.ITP scaffolds extract could up-regulate the expression of osteogenic differentiation-related genes.The ITP scaffold prepared by this method is expected to be used in the treatment of bone defects.