Construction Of Biobased Anisotropic Scaffold And Its Biocompatibility Study

In recent years,global petrochemical resources have been progressivelydepleted,while climate change and environmental pollution have intensified alarmingly,posing a serious threat to the sustainable development of the global ecological environment and economy.The development of biomass conversion technology and the utilization of bio-based materials are of great significance to solving the deteriorating environmental problems,as they are considered an indispensable part of the global green economy development.Moreover,it has been emphasized as one of the critical tasks of China’s carbon neutrality and carbon peak promotion project,holding the potential to contribute to a breakthrough for China’s sustainable development.Cellulose,one of the most widely distributed and abundant bio-based materials in nature,has found diverse applications in different areas of daily life,such as food,clothing,housing,and transportation.In recent years,biomedical engineering application has emerged as a highly active field of research and development for cellulose-based materials,especially bacterial cellulose（BC）,owing to its exceptional water-retaining property,mechanical strength,and biocompatibility,making it a favorable choice for tissue engineering scaffolds However,the relatively lower structural and functional fidelity of BC-based biomaterials still cannot fully satisfy the requirements of tissue engineering.Therefore,this study is based on the urgent demand for research and development of renewable bio-based materials,which in line with the imperative of the dual carbon policy.To address the dual requirements of the BC-based biomaterials application in tissue engineering regarding structural and functionality needs,this work proposes strategies aimed at developing bio-based anisotropic biomaterials with high added value for potential bone tissue engineering applications.The research mainly includes the following three aspects:Firstly,the BC in the form of a membrane was obtained through static fermentation using Acetobacter xylinum AS 1.1812 after optimization of the fermentation conditions.An increase in bacterial cellulose production yield can be achieved by changing the composition of the Hestrin-Schramm（HS）medium.The optimized fermentation medium consists of 20 g/L glucose,5 g/L yeast powder,5 g/L peptone,2.7 g/L disodium hydrogen phosphate,0.3g/L Mg SO₄·7H₂O,1.15g/L citric acid,and 1.5%ethanol,with p H=5.Under these optimized conditions,the yield of BC was 836.67±8.3 mg/L,showing an increase of 20.4%compared to the initial conditions.The FT-IR spectroscopy,its derivative spectrum analysis,and the XRD spectrum identification indicate that the fermentation product of Acetobacter xylinum has typical diffraction peaks and crystal lattice structure of cellulose,which can be concluded as bacterial cellulose with a crystallinity of 88.01%.Secondly,2,2,6,6-Tetramethylpiperidinyloxy（TEMPO）-mediated oxidation was used to modify BC.The resultant oxidized bacterial cellulose（TOBC）was applied to construct BC-based scaffolds with anisotropic structures.The average width and length of the TOBC were 26.76 nm and 407.74 nm,respectively.The surface carboxyl content increased from 0.27±0.02 mmol/g to0.75±0.04 mmol/g,and the crystallinity degree increased to 91.39%.The anisotropic structure was constructed using a simple and green wet pressing and limited hydration swelling method.The high orientation of the microstructure of the constructed scaffold was characterized by using optical microscopy,scanning electron microscopy,two-dimensional wide-angle X-ray diffraction,etc.The anisotropic structure was further verified through behaviors such as swelling anisotropy and mechanical anisotropy.The biosafety of TOBC was evaluated by detecting the pyrogen and anaphylactogen in TOBC,i.e.,endotoxin and fungal（1-3）-β-D-glucan content.Results reveal that these levels can meet the relevant criteria in the national pharmacopeia,suggesting the biosafety of the TOBC.In the cell compatibility evaluation of TOBC,direct contact cell culture experiments were conducted using mouse osteosarcoma cell K7M2.The results showed that TOBC exhibited good cell adhesion and biocompatibility and had a significant promoting effect on cell growth.The anisotropic structure inside the scaffold can promote the oriented growth of cells.Thirdly,to further improve the mechanical properties of the scaffolds,we proposed the in-situ mineralization of TOBC hydrogel by using enzymatically induced calcium carbonate precipitation.Through single factor and response surface analysis,we found that the optimal molar ratio of the substrate during in-situ mineralization was M_urea:M_{Ca Cl2}=1:1.The mineralization degree significantly increased under the conditions of TEMPO-mediated oxidation at 37℃for 12hours and swelling time of 12 hours.The orthogonal experimental design was used to optimize the mechanical properties of the scaffold further,and the results showed that the axial Young’s modulus of the scaffold was controllable within the range of 29.38-1378.26 k Pa.The mouse mesenchymal stem cells（BMSCs）were used to study the directional osteogenic differentiation induction potential of the scaffolds.The results showed that the mineralized TOBC scaffold has good biocompatibility and non-toxicity and allows stem cells to grow and proliferate within the scaffold.The levels of osteogenic markers indicate that scaffolds with a higher modulus（209.5±26.3 k Pa）can better promote cell adhesion,proliferation,and osteogenic differentiation.Scaffolds with a lower modulus（31.6±4.3 k Pa）are unsuitable for osteogenic differentiation induction due to their lower osteogenic markers.In summary,this work proposed strategies to construct BC-based tissue engineering scaffolds for potential high-value applications in the biomedical field.This work could provide new ideas for the research and development of non-grain bio-based materials under the dual carbon policy,serving to alleviate and respond to the increasingly short supply of resources and deterioration of the ecological environment worldwide.