Fabrication Of Mineralized Nanofibers And Electrospun Nanoyarns For Bone Tissue Engineering Applications

Bone defects caused by trauma and bone related diseases are the challenge in clinic, and with the development of the society and aging population in recent yeas, the problem becomes more serious. There are several methods to repair the bone defects in clinic, such as allograft, autograft and xenograft. Autogenous iliac crest bone grafting is regarded as "gold standard". However, the morbidity of iliac crest bone harvesting, including chronic donor site pain, infection, fracture, hematoma, increased operation time and costs, is reported to be as high as30%. Engineered bone graft substitutes have emerged as a new way to solve the problem by the development of tissue engineering. Natural bone tissue encompasses of several hierarchical levels over many length scales. Its microstructure is consisted of osteons with Haversian canals ranging from10to500μm. Its nanostructure shows collagen fiber assemblies of collagen fibrils ranging from a few hundred nanometers to1μm and bone mineral crystals embedded within collagen fibers. The main components of native bone are inorganic hydroxyapatite (HA) and organic collagen type I. It is important to fabricate ideal bone graft substitutes by biomaterials and tissue engineering to mimic the structure, component and function of natural bone tissue.To mimic the natural bone extracellular matrix (ECM), nanofibers were fabricated by electrospinning and mineralized with ten times simulated body fluid (10SBF). The morphology, number and growth sites of the apatites and the structures of the nanofibrous mats were controlled by external mineralized conditions, mineralization time and nanofiber’s component. Scan electron microscopy (SEM) was used to observe the morphology of the mineralized nanofibers. It was found that irregular apatites were deposited on the surface of the nanofibrous mats under static condition. In contrast, sphere-like apatites were observed on/in the nanofibrous mats under shaking and nano-sized sphere-like apatites were observed after adding collagen in the component of nanofibers. With mineralized time extending, the diameter and the number of the apatites increased, and the structure of the nanofibers couldn’t be maintained. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectrometer (EDS) were employed to analyze the sphere-like apatites. Contact angle test and mechanical test were performed to analyze the hydrophilic property and mechanical property of the nanofibrous mats, respectively. It was found that nano-sized sphere-like apatites significantly improved the hydrophilicity of the composite nanofibrous mat and enhanced its mechanical property as well. In vitro study, SEM images showed that human fetal osteoblasts (hFob) proliferated well with normal cell morphology on mineralized composite nanofibers and much better mutual integration. In addition, MTT assay more clearly showed that the apatites on composite nanofibrous mat improved the proliferation of hFob. Furthermore, we found that alkaline phosphatase (ALP) activity and osteocalcin expression were both enhanced on the composite nanofibrous mats. The above results indicated that mineralized composite nanofibrous mats have good biocompatibility and better osteoinductivity.To study the effects of mineralized composite nanofibrous mats [M-P(LLA-CL)/COL] on the proliferation and differentiation behaviors of human bone marrow mesenchymal stem cells (hMSC), hMSC were seeded on M-P(LLA-CL)/COL. The morphology of cells on M-P(LLA-CL)/COL was much better than that on P(LLA-CL) and P(LLA-CL)/COL nanofibers as indicated by SEM images. The integration between the cells and mineralized composite nanofibers was much better. MTT results showed that hMSC proliferated well on M-P(LLA-CL)/COL. The results of ALP activity test, mineralization nodules characterization and immunofluorescence staining showed that mineralized composite nanofibrous mats enhanced the differentiation of hMSC. This phenomenon might be contributed to the bioactive function groups on collagen and sphere-like apatites. The effect of apatites with nano-topological structure on the mineralized nanofibers might be the other probable explanation. The above results indicate that M-P(LLA-CL)/COL nanofibers have good biocompatibility and better osteoinductivity. It has a great potential application in bone tissue engineering.To mimic the microstructure of native bone tissue, nanoyarns were fabricated and incorporated into three-dimensional (3D) porous collagen scaffold. Electrospun nanoyarns maintained the morphology and structure of electrospun nanofibers and its micro-structure makes it easy to manipulate for further usage. Long P(LLA-CL) nanoyarns with extremely high orientation were fabricated by a dynamic liquid support system. The nanoyarns were chopped into short nanoyarns with length around1-2mm without entanglement. The morphology of the nanofibers was maintained as well after the process.3D porous scaffold with nanoyarns were fabricated by freeze-drying method. The distribution of nanoyarns in3D scaffold was homogeneous and no entanglement between the nanoyarns was observed by optical microscopy and SEM. Most of the nanoyarns were consisted of the pore walls of3D porous scaffolds. The water absorbance of3D scaffolds was decreased with the mass increase of nanoyarns. The mechanical property was enhanced after the adding of certain amount nanoyarns. The shrinkage was decreased with the amount increase of nanoyarns in3D porous scaffolds. The result of in vitro study showed that proper amount of nanoyarns in the scaffold could enhance the proliferation of the cells, while too much amount of nanoyarns in scaffold could destroy the integrity of the pore walls and lead to negative effect on cell proliferation. The cells on3D porous scaffolds grew along the pore walls, which were observed in SEM images. The results of ALP activity test and osteocalcin staining showed that the adding of proper amount of nanoyarns in3D porous scaffold enhanced the differentiation of hMSC.3D porous scaffold with nanoyarns has a greatly potential application in bone tissue engineering.Traditional bone grafting requires an open surgical approach which has many disadvantages. While minimally invasive procedure (MIP) in clinical treatment reduces patient’s pain and shortens the recovery. There is a n urgent need for the development of better bone graft substitutes for MIP. A novel injectable system, a biomimetic bone substitute consisted of P(LLA-CL) nanoyarns suspended in type I collagen hydrogel, was developed and it could form a hydrogel completely in2h at human body temperature. The distribution of nanoyarns in the hydrogel was homogeneous and no entanglement between the nanoyarns was observed by optical microscopy and SEM. To investigate the mechanical properties of collagen hydrogel (Col) and collagen hydrogel with nanoyarns [Col/P(LLA-CL)], rheological evaluation was performed.The results indicated that the mechanical property of collagen hydrogel was enhanced by incorporated nanoyarns. The result of injectability test showed that Col/P(LLA-CL) was smoothly injected out of the16gauge needle. Overlapping clustered morphology of hMSCs was observed on Col, but no such obvious phenomenon was observed on Col/P(LLA-CL) after long culture time. MTT results showed that hMSCs grew well on Col/P(LLA-CL). The results of ALP activity test and osteocalcin staining showed that the nanoyarns in Col improved the differentiation of hMSC. The P(LLA-CL) nanoyarns not only maintained the structure of collagen hydrogel matrix, it also played an important role in enhancing stem cell differentiation. The above results suggest that collagen hydrogel incorporated with nanoyarns has a great potential in MIP and bone regeneration.