| Collagen is a unique protein family with a distinctive triple-helix structure in the extracellular matrix,and is the most abundant structural and functional protein in the human body.Collagen self-assembles into a fibrous network in vivo,providing a biological scaffold for tissues and organs,and participating in basic physiological activities such as regulation of cell proliferation,migration,and differentiation.Collagen is widely used in the fields of tissue engineering and regenerative medicine due to its excellent properties such as biocompatibility,biodegradability,and bioabsorbability.Currently,collagen is mainly extracted from animal tissues.However,animal-derived collagen has serious issues such as poor water solubility,difficulty in quality control,and risk of disease transmission,which greatly limit its clinical applications.Recombinant collagen,produced by genetic engineering techniques,has significant advantages including good water solubility,stable quality,and no risk of viral transmission,which has attracted increasing attention in the field of medical materials.This thesis aims to prepare a novel of triple-helix recombinant collagen and its functional materials,and systematically study their physicochemical and biological properties.The main contents are as follows:1.Recombinant collagen has been gaining increasing attention as a novel biomaterial in the field of dermatology and bone repair.We have developed a genetically engineered strain of Escherichia coli that efficiently expresses triple-helix recombinant collagen（THRC-1）,enabling the large-scale production of medical-grade THRC-1.We systematically optimized the expression conditions such as the culture medium,inducer,culture temperature,and duration,and successfully achieved the large-scale production of medical-grade triple-helix recombinant collagen（THRC-1）in a 500 L fermenter through high-density fermentation.Furthermore,we established an efficient purification process consisting of nickel affinity chromatography,proteolytic hydrolysis,and ion exchange to obtain high-purity（96%）and high-quality THRC-1.The endotoxin level,residual DNA,and residual E.coli protein content of THRC-1 met the industry standards for medical devices of recombinant collagen.The recombinant collagen possesses a favorable triple-helix structure,high biocompatibility,and biological activity,significantly promoting the proliferation and adhesion of L929 cells.The achievement of large-scale production of medical-grade triple-helix recombinant collagen provides a solid foundation for expanding the clinical applications of recombinant collagen-based biomaterials,with significant economic and social value.2.Collagen self-assembles in the body to form a 3D fiber network,providing structural integrity and mechanical strength to connective tissues.However,currently reported recombinant collagen lacks the ability to self-assemble into fibers,which severely hinders their application in tissue engineering and other fields.Herein,we have for the first time constructed a self-assembling recombinant collagen that mimics the natural collagen structure and fiber morphology,which is rich in tyrosine residues.The designed recombinant collagen comprises of a central（Gly-Xaa-Yaa）n triple helical domain and N-terminal/C-terminal GYY domains.The introduction of GYY does not significantly affect the stability of the triple helical structure of recombinant collagen,and it can induce the self-assembly of recombinant collagen into a nanofiber morphology.Upon exposure to[Ru（bpy）3]Cl2and visible light,the recombinant collagen underwent covalent cross-linking of tyrosine residues,leading to the formation of a hydrogel with remarkable mechanical properties.The recombinant collagen hydrogel demonstrated excellent biocompatibility and bioactivity,significantly promoting the proliferation,adhesion,migration,and differentiation of HFF-1 cells.The novel self-assembling recombinant collagen with multi-level self-assembly structure and biological functions closely mimics the natural collagen protein,and has great potential for applications in tissue engineering and medical materials3.Biomimetic scaffolds with exceptional biocompatibility and biodegradability have extensive application in the field of bone tissue engineering.However,the development of advanced biomimetic scaffolds that can mimic the composition,structure,and function of natural bone tissue remains a significant challenge.Herein,we have for the first time constructed a biomimetic three-dimensional porous scaffold of mineralized recombinant collagen-sodium alginate（MRCSA）with supreme healing efficacy for critical-size cranial defects.In situ biomineralization using recombinant collagen as the unique biotemplate results in the formation of nano-hydroxyapatite with desired low crystallinity and well-ordered mineralized recombinant collagen with fibrous morphology.The MRCSA scaffold finely mimics the organic-inorganic composition and the uniform porous nanostructures of natural bone,which exhibits excellent biocompatibility,adequate biodegradability,superior cellular activity and exceptional osteogenic differentiation capability.Magnetic resonance imaging（MRI）,Micro-computed tomography（micro-CT）and histological characterization of rat models of critical-size cranial defects have consistently demonstrated that the MRCSA scaffold has regenerated an abundance of new bones to refill the defect sites with much higher bone mineral density,bone volume/total volume as well as trabeculae.The novel biomimetic scaffold showcases immense potential applications in orthopedics and plastics.4.The construction of collagen-based scaffolds with uniformly distributed hydroxyapatite remains a significant challenge in the field of orthopedics.Herein,we have for the first time developed a biomimetic collagen composite matrix-hydroxyapatite（CCMH）scaffold with enhanced healing efficacy for critical size bone defects.The collagen composite matrix（CCM）scaffold with an oriented structure was generated by unidirectional freeze-casting.The CCMH scaffold was manufactured by peristaltic mineralization to in situ forms uniformly distributed nano-hydroxyapatite on the CCM scaffold.The CCMH scaffold closely recapitulates the composition and anisotropic channel-like morphology of native bone,which endows it with high biocompatibility,excellent cellular activity,and unparalleled osteogenic differentiation capability.Moreover,the CCMH scaffold displays superior bone regeneration capabilities,as evidenced by various imaging techniques including magnetic resonance imaging（MRI）,micro-computed tomography（micro-CT）and histological analysis of rat critical-size cranial defects.The innovative combination of the unidirectional freezing technique and in situ peristaltic mineralization method provides a novel strategy for the development of three-dimensional porous scaffolds with exceptional biocompatibility and osteoinductivity.These biomimetic scaffolds offer immense potential for the field of bone repair and regeneration,paving the way for advanced therapeutic strategies with remarkable clinical implications.5.High-performance biocompatible batteries are crucial for the development of personalized medical devices such as flexible electronic skin,pacemakers,and neural stimulators.Herein,we have for the first time developed a one-pot collagen-assisted biomineralization strategy to create hierarchical CuO nanostructures.Varying the concentration of recombinant collagen in the reaction system can delicately tune the morphologies of copper oxide mesocrystals.The as-prepared leaf-like CuO mesocrystals exhibited an attractive electrochemical performance,and may have great potential as a promising anode material for lithium-ion batteries.Notably,CuO nanomaterials can significantly promote the adhesion of HFF-1 cells,because of the collagen agent to endow the CuO nanomaterials with high biocompatibility and bioactivity.We provide a new strategy for the development of CuO nanomaterials with well electrochemical performance and biological functions,which have promising applications in implantable health-care electronic.In conclusion,the paper focuses on the preparation and characterization of recombinant collagen and its biomaterials.Initially,the large-scale production of medical-grade triple-helix recombinant collagen was successfully achieved,followed by a comprehensive characterization of its physicochemical properties and biological activity.Subsequently,a covalently self-assembled triple-helix recombinant collagen hydrogel enriched with tyrosine was constructed,marking the first instance of biomimetic replication of natural collagen fiber morphology and hydrogel formation capacity.Furthermore,a biomimetic three-dimensional porous scaffold of mineralized recombinant collagen-sodium alginate（MRCSA）was successfully developed,effectively mimicking the structure,composition,and function of natural bone tissue.Moreover,a biomimetic scaffold of collagen composite matrix-hydroxyapatite（CCMH）was successfully fabricated through the utilization of a pulsatile mineralization method.CCMH scaffold closely recapitulates the composition and anisotropic channel-like morphology of native bone and offering effective treatment for critical-sized cranial bone defects.Lastly,the synthesis of the recombinant collagen-copper oxide nanomaterials with exceptional electrochemical properties and biological functions,has laid a solid groundwork for the production of intelligent and flexible materials.These research achievements provide novel insights and approaches for the application and advancement of recombinant collagen and its biomaterials. |
