Printability Optimization Of Gelatin-alginate Bioinks By Cellulose Nanofiber Modification For Potential Bone-ligament Interface Bioprinting

As a major part of the human musculoskeletal interface system,ligaments insert into the bone through a specialized interface tissue called enthesis.This interface tissue is a transitional area between different biochemical and mechanical properties and is essential for the interaction between soft and hard tissue in musculoskeletal motion.Bone-ligament interface(BLI)permits the gradual increase in stiffness,thereby minimizing stress concentrations and allowing for effective load transfer from ligament to the bone.Unfortunately,the highly specialized biofunctions and structures of this interface mean that it is extremely difficult to recover after musculoskeletal injures.Traditionally,clinical interventions in BLI injures involve autografts or allografts of injured ligaments.However,Long term review studies have shown the formation of mechanically inferior fibrovascular tissue at the interface between ligament and bone with poor long-term outcomes.In the tissue engineering area,attempts to mimic the intricate microstructural features of BLI have also been limited by lacking advanced scaffold fabricating methods as well as mimicking the complex cellular components of this structure.As a group of additive,bottom-up,nature-like technologies,which has made it possible to spatially pattern cells,bioactive factors and biomaterials in 3D.The wide application on biofabricating functional musculoskeletal tissues such as muscle,cartilage and bone has been frequently reported.However,there have been few bioprinting applications of BLI tissues until now.Mainly because of the lack of printability and mechanical strength presented by currently available bioinks,as well as limitation in bone-ligament interface relevant cell source.Thus,improving the printability of bioinks,as well as exploring new bioprinting candidates in terms of BLI-relevant cellular components,are urgently needed.To overcome the current challenges,current work aims to verify the possibility of introducing bioprinting technology into the field of tissue engineering BLI tissues.Initially,a micro-extrusion bioprinting system was developed using gelatin-alginate thermal-responsive bioinks.Cellulose nanofibrils(CNF)were applied to modify the performance of gelatin-alginate based bioinks.Then,both preosteoblast cells and fibrochondrocytes were used in the bioprinting process to assess the biocompatibility and feasibility of the bioprinting system and explore the methods for bioprinting BLI tissues.Section 1.The development and optimization of micro-extrusion based bioprinter.Based on summarizing the basic principle of bioprinting approaches,as well as our present demand in bioprinting BLI tissues,a micro-extrusion based bioprinter(BP-1)was designed and built by our group.Systematic characterization methods were also applied to optimize the printing quality and biocompatibility of the bioprinter.Section 2.Preparation,characterization and printability optimization of the gelation-alginate-CNF bioinks.The bioinks were mixed with CNF to improve printability and rheological properties.The physicochemical properties of the CNF-modified bioinks were characterized.Printability tests were then performed to select the best formula for our targets.The modified bioink presented significant enhancement in rheological properties,mechanical performance as well as bioprintability.Moreover,benefited from the application of CNF,the self-support feature was discovered in the modified bioink samples,which was in favor of bioprinting complex bionic structures.Section 3.Bioprinting and biological assessment of major cellular components of BLI tissue.Both preosteoblast(MC3T3-E1)cells and primary rabbit fibrochondrocytes(rFCs)were used in the bioprinting process to assess the biocompatibility and feasibility of the bioprinting system and explore the methods for bioprinting BLI tissues.To the best of our knowledge,this is the first time that rFCs have been applied in bioprinting approaches.Subsequently,the bioprinted structures were cultured in vitro for 14 d and then underwent histological and immunohistochemical assessments.The samples were bioprinted without compromising cellular viability and biofunction.The existence of collagen I,II and X was confirmed by immunohistochemical stains.In conclusion,a micro-extrusion based bioprinter using paper pulp-based nanocellulose-modified gelatin-alginate bioinks was developed in our study and the ability of biofabricating the major cellular components of BLI tissues were verified.To the best of our knowledge,this is the first time that this has been reported.The bioprinted structures permitted cell viability and physiological function after 14 d of incubation.The current study demonstrates the feasibility of applying bioprinting approaches to the reconstruction of BLI tissues.The use of bioprinting approaches might accelerate the progress of tissue engineering in BLI biofabrication and finally permit the evolution of clinical therapeutic methods.Further research will focus on developing a multichannel bioprinting system with multi-material bioprinting features.Gradient cellular structures will be bioprinted simultaneously.In vivo implantation tests of the bioprinted samples will be performed to verify their potential for vascularization and physiological function.