Preparation And Performance Of Coaxial Tissue Engineered Bone Scaffolds Loaded With Multiple Antituberculosis Drugs

The main treatment for severe bone tuberculosis is combination of surgical bone grafting and systemic chemotherapy.The dominant lesions are excised by surgery,followed by medication to the lesion area and reconstruction of bone defects with bone grafting.However,the intraoperative drug treatment only plays a role in assisting sterilization in a short time,but Mycobacterium tuberculosis cannot be completely eliminated,so long-term oral or injection of a large amount of antituberculosis drugs is still required for chemotherapy on the lesions.This will not only cause toxic and side effects to the patient's organs and nervous system,but also lead to low drug concentration at the lesion and poor therapeutic effect due to obstruction of blood circulation,drug metabolism and connective tissue around the lesion,making bone tuberculosis potentially recurrent.Thus,maintaining the drug concentration at the lesion is critical to achieve efficient treatment and rapid repair.Herein,a 3D printing technique combined with INH/SM/RFP multiple antituberculosis original drugs and drug-loaded microspheres was used to construct a degradable drug-loaded tissue engineered bone scaffolds with a multi-gradient sustained-release structure.It can coordinate the suitable mechanical properties with stable drug release,prolong the sustained release time,and provide a good local environment for the cure of tuberculous bone defects.The performance and dose-effect relationship between the composite materials are the key factors for the drug-loaded scaffolds to improve the drug release stability and promote new bone regeneration.According to the unique physicochemical properties of silk fibroin(SF),the specific properties of SF-based composites were optimized by physical methods,such as ultrasound and freeze-drying.Effectively increased content of?-sheets is reported to improve the mechanical strength of composites by 13.91% and29.28%.According to the different functions of the silk structure in the coaxial stent,the composite materials SF/PVA/HA/?-TCP with high drug loading was selected as the inner core substrate,HA/PVA/?-TCP as the outer coating material,and a suitable quantitative range was established to make the material possess adaptive mechanics and degradation properties.The process parameters and preparation process of the microspheres are quantitatively controlled to improve the formation,drug loading and drug release performance.Drug-loaded microspheres were prepared using SF as the matrix material and INH as the model drug based on W/O method.BP neural network-genetic algorithm was used to optimize the process parameters.The response surface method(RSM)comparative analysis indicates the optimized parameters as follows: oil-water ratio of10:1,stirring temperature of 45°C,and stirring speed of 400 rpm.The drug-loaded microspheres with uniform particle size,good integrity,high drug loading and stable drug release were obtained.Furthermore,the ultrasound-repeated freezing-thawing-ultrasound optimization of the SF solution effectively induces transformation of the SF structure,achieving effective adjustment of the degradation rate of the microspheres,and stabilizing the drug sustained-release.According to the difference of drug release behavior between hydrophilic and hydrophobic drug-loaded microspheres,a concentration dependent drug diffusion-dissolution mechanism model was established.The release mechanism of hydrophilic and hydrophobic drugs was explored,and the relationship between the release rate,carrier material and property of the drug were described.The drug distribution in microspheres was modeled by ANSYS.The results showed that hydrophilic drugs are closer to the surface of the microspheres,and the drug release is mainly dominated by diffusion.The hydrophobic drugs are more likely to accumulate in the center,and the drug release is dominated by the dissolution of the drug inside the microspheres.The correlation coefficients of the release fitting results of the three drugs are all above 0.98,indicating a good adaptability of the model for hydrophilic and hydrophobic drugs.It can better guide the design of the microsphere structure and optimization of drug release performance.Coaxial structural design and shape optimization of drug-loading stent.Based on the rheological characteristics of the composite materials in the inner and outer layer of coaxial nozzle,the relationship model between feed rate of cylinder,change of nozzle wall,and silk discharge rate of the nozzle was established.The diameter of the inner and outer nozzles were accurate to 300 ?m and 600 ?m,and the printed silk structure has a high coaxiality.The impact of the inter-relationship between the three dominant process parameters(extrusion speed,filling speed,and layer height)on the quality of the formed scaffolds was comprehensively analyzed,which accurately and effectively optimized and controlled the formation process.3D printing was used to construct a coaxial stent model with controllable microstructure,which provides a basis for diversity of drug delivery from the stent and gradient of drug sustained-release.The influence of drug loading method and drug distribution on the drug sustained-release was explored.the drug-loaded microspheres are used as the first-level sustained-release carrier,the inner core composite material encapsuling the three original drugs and the drug-loaded microspheres as the second-level layer,and the outer encapsule layer is used as the third-level layer.3D printing was used to construct a coaxial drug-loaded tissue engineered bone scaffold with triple drugs,through blending of original drugs and microspheres,possessing high coaxiality of the silk structure,and high overall accuracy of the scaffold.The results of comparative analysis indicate that the three original drugs 94%/6% microsphere coaxial scaffold is selected as the best drug-loaded scaffold.The mechanical strength of the coaxial scaffold is improved by53.09% compared with the uniaxial scaffold.At 12 weeks,the cumulative release rates of INH,SM,and RFP in the 94% original drug/6% drug microsphere coaxial stent were53.46%?85.57%?31.38%,respectively,and the effective drug concentrations were still higher than the minimum.Furthermore,according to the study of release behaviors of hydrophilic and hydrophobic drugs,INH is released via diffusion-controlled mechanism,the first released drug for early-stage treatment.SM and RFP are released via scaffold degradation,erosion and diffusion,realizing long-term stable release for middle and late-stage treatment.The drug-loaded scaffold achieves not only high drug loading and stable drug release,but also function of gradient sustained-release of different drugs in the treatment stage,which provides a theoretical and experimental basis for the research and application of implantable drug release system based on drug loaded tissue-engineered bone scaffold.