Preparation Of Elastin-like Polypeptide And Silk Complex Scaffold Mimicking 3D Microenvironments For Bone And Cartilage Tissue Engineering

The repair of bone and cartilage defects resulting from trauma, inflammation, tumor resection and abnormal skeletal development remains a clinical challenge. Currently, autologous bone graft transplantation is the most successful means for bone defect repair. However, harvesting autologous bone graft requires a secondary operation and may cause severe donor site morbidity. Recent advances in bone tissue engineering provide a promising strategy for bone and cartilage defect repair and regeneration through the seeding of Bone marrow mesenchymal stem cells (BMSCs) or chondrocytes onto selected scaffold. Cell niches in bone and cartilage are 3D microenvironments composed of hydrated, cross linked networks of extra cellular matrix (ECM). The unique structure of nature silk fiber could provide the impressive 3D scaffold. In order to construct the 3D artificial environment, several methods of forming synthetic or semi-synthetic matrices have been developed, such as electrospinning,3D micro/nano-pattern and hydrogel. However, a common problem encountered with many electrospun materials is that the inherently small pores of electrospun scaffolds do not promote adequate cellular infiltration and tissue ingrowth. For the past decade, hydrogels have become especially attractive as matrices for regenerating and repairing a wide variety of tissues and organs. However, one of the main disadvantages of processing hydrogels is the difficulty to shape them in predesigned geometries. The most recent trend is the use of hydrogel systems and cell encapsulation strategies to fabricate gel/cell hybrid constructs. The 3D-Bioplotter and other similar techniques can be seen as the most straightforward hydrogel/cell manufacturing method for designing complex inner and outer architectures, without the need for an additional support structure. However, a quantifiable loss in cell viability was seen, which was caused by a process-induced mechanical damage to cell membrane integrity. In conclusion, most of the current scaffolds are not ideal for clinically applicable bone and cartilage tissue engineering. Aside from good biocompatibility, inter-connective porous structure and proper pore size, and a controlled degradation rate, scaffolds for bone and cartilage tissue engineering should also facilitate MSCs and chondrocytes adhesion, proliferation, differentiation and bone and cartilage matrix formation.A novel design of silk fiber-based scaffold has been developed. Its unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation of MSCs along the osteogenic lineage, have made silk a favorable scaffold material for bone tissue engineering. Meanwhile, the unique structure of nature silk fiber provides an impressive 3D scaffold and allows chondrocytes attachment. The construction of a silk fiber-based scaffold provided a feasible kind of functional biomaterial for the repair of bone and cartilage defects.Elastin-like polypeptide (ELP) has found utility in tissue engineering, not only because they are biocompatible, biodegradable, and non-immunogenic, but also because their amino acid sequence and molecular weight can be precisely controlled at the genetic or synthetic level, affording exquisite control over final protein functionality. In order to mimic bone and cartilage ECM, ELP and silk fiber scaffolds were blended to construct the scaffold.ELPs and Silk microfibers were treated with varied methods, (i) Dehydrothermal treatment (DHT) (ii) The samples were mixed with genipin. (iii) The samples were mixed with proanthocyanidin (PA) (iv) The samples were mixed with PEG and glutaraldehyde (GA). The resulting materials were characterized by scanning electron microscope (SEM) observation, mechanical properties and cell proliferation assay. The results demonstrated that the S-ELP-DHT composite scaffolds for the development of biomaterials constructs with properties comparable to those made with ELP and silk.The S-ELP-DHT scaffold was characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle measurement, release rate of ELP, degradation rate of ELP-silk scaffolds and mechanical properties. The results demonstrated that (i) DHT did not affect the chemical structure of the ELP; (ii) The S-ELP-DHT exhibited bigger pore size and higher porosity (over 85%) than those reported previously, meanwhile, S-ELP-DHT formed a single intact sheet with internal porosity which could loading with the cells; (iii) the ELP incorporation within the silk fibers improved significantly the elastic modulus of S-ELP-DHT; (iv)The S-ELP-DHT degraded slowly, even over 61% of the initial mass left after 60 days immersion into DMEM. This degradation rate was proper rate for bone and cartilage reconstruction.The S-ELP-DHT scaffold was characterized by cell attachment assay, cell proliferation assay and cell differentiation assay. The results demonstrated that (i) the scaffold had the ability to support the cell attachment. MSC seeding efficiency on the S-ELP-DHT scaffold was over 79%, and chondrocytes seeding efficiency on the S-ELP-DHT scaffold was over 74%; (ii) The amount of cells adherent to the S-ELP-DHT scaffold was obviously higher than those on other surfaces, and higher viability and proliferation rates were obtained; (iii) the scaffold had the ability to support the differentiation of MSCs along the osteogenic lineage and production Col II and AGG by chondrocytes.The S-ELP-DHT scaffold was characterized by ectopic bone formation and cartilage formation assay. (i) All of the animals survived the experimental procedure, and no visible inflammatory reactions, infections, or extrusions were observed. (ii) The S-ELP-DHT degraded slowly, which was proper rate for bone and cartilage reconstruction. (iii) The micro-CT images demonstrated that new bone formation in the S-ELP-DHT scaffold/MSCs composite, meanwhile, the histological analysis indicated that the ossification was in the manner of endochondral ossification. (iv) The elastic modulus and the GAG content of the S-ELP-DHT scaffold/chondrocytes (obtained from elastic cartilage) two months post-transplantation were similar to those of the natural fibrocartilage, but different from elastic cartilage and hyaline cartilage.In summary, we report for the first time that the BMSCs and chondrocytes were seeded into Silk-ELP-DHT scaffolds as implant to investigate the bone and cartilage repair. The results revealed that the Silk-ELP-DHT scaffolds had the ability to support the cell attachment, proliferation and the differentiation of MSCs along the osteogenic lineage. Meanwhile, the Silk-ELP-DHT scaffolds encouraged that more mature cartilaginous tissues differentiated into mature chondrocytes, produced more collagen and polysaccharides production of cartilage and maintained chondrocyte phenotype in vivo. The S-ELP-DHT scaffold/MSCs composites two months post-transplantation were similar to the natural bone. Meanwhile, the S-ELP-DHT scaffold/chondrocytes composites two months post-transplantation were similar to the natural fibrocartilage. This study could influence the design of a more suitable microenvironment to encourage bone and cartilage repair in the future.