Experimental Study On FN/CDH Biomimetic Scaffold Promoting MSC Osteogenic Differentiation In Vitro Based Coaxial Electrospinning Technology

There is a large number of patients requiring bone grafts to rebuild bone tissue through regeneration annually due to structural changes and unrecoverable in bone tissue trauma or congenital causes. Currently a variety of methods have been used in bone tissue repair and reconstruction, main way is the usage of autogenous bone graft ?from the patient? allografts ?from a donor? and a variety of biological materials. Autogenous bone graft has long been considered as a golden standard for the treatment of bone defects, although there are successful cases, but this technique is limited by material availability. Allogenic bone graft technique may cause side effects like pathogen invasion or immune rejection. Therefore looking for alternative bone repair material is necessary for the development of bone tissue repair and reconstruction.Successful implementation of engineered tissue bone injury repair consists of three main factors:the seed cells with potential of osteogenic differentiations, it has growth factors which can induced the seed cells to the direction of osteogenic differentiation and a three-dimensional scaffold to support the growth of seed cells. Therefore this study aims to use the coaxial electrospinning technique to build a nanofibers composite scaffold which can slow release osteogenesis induced factor. To provide an ideal seed cells proliferation in vitro, adhesion, differentiation microenvironment and to promote tissue engineered bone injury repair.This research provides new ideas for the study of tissue engineered damaged bone repair.Method:1. Fabrication of different biomimetic nanofibrous with rFN/CDH₁₁ protein concentration. Based on the preliminary work of research group, lab associates Dr. Zhang Yuan synthetic rFN/CDH₁₁ the fusion genes of protein, using pET 22b as a carrier, using Rosetta-gamiTM i ?DE3? as competent cells, by expressing and purifying to get rFN/ CDHi i protein. By using the coaxial electrospinning technique, construct type I collagen containing polyvinyl alcohol ?polyvinyl alcohol, PVA?, polylactic acid-glycolic acid copolymer ?poly ?lactic-co-glycolic acid?, PLGA?,and various concentrations nano bionic bone scaffold with recombinant fibronectin/cadherin (recombinant fibronectin/cadherin chimera, rFN/CDH₁₁) ?Oug/ml,1.0ug/ml,10.0ug/ml,50.0ug/ml,100.0 ug/ml?.2. The characterization of evaluation for different biomimetic nanofibrous with rFN/CDH protein concentration. By using the methods like fourier transform infrared spectroscopy ?FTIR?,X-ray photoelectron spectroscopy ?XPS?, scanning electron microscopy ?SEM?, transmission electron microscopy ?TEM? and protein adsorption, which can understand the composition of the chemical elements, surface structure of onlookers, the onlookers structure of nanofibers and the release efficiency of rFN/CDH₁₁ protein; contact angle analysis ?Contact Angle? and biomechanical analysis method for the determination of hydrophilic/hydrophobic and tensile strength of nano bone scaffold. By evaluating bio-physical and chemical properties of nano bone scaffolds and determine whether compatible with the organization.3. Research in vitro function with different rFN/CDH₁₁ protein concentration of biomimetic nanofibrousBy using human mesenchymal stem cells ?hMSCs? as seed cells and through scanning electron microscopy to observe hMSCs in bone scaffold surface proliferation, adhesion, case shape; toxic effects of MTT assay of cell proliferation and scaffold for cells; quantitative real-time PCR ?QRT-PCR? assay ALP gene, OCN gene, RUNX2 gene expression.Results:1. Fabrication of different biomimetic nanofibrous with rFN/CDH₁₁ protein concentration.According to the most suitable conditions for coaxial electrospinning parameters ?V= 16-18Kv, outside the v= v= 0.4-0.7ml/h, d= 15cm?, rFN/CDH₁₁ protein concentration of nano bionic bone scaffold fabricated several sheets successfully.2. The characterization of evaluation for different biomimetic nanofibrous with rFN/CDH₁₁ protein concentration.?1? SEM observed the bone scaffold surface morphology with uniformity, Nanofibers continuity is better, no beaded fibers, the diameter distribution uniformly. The software Image J measurements showed bone scaffold fiber diameter and added to the rFN/CDHn protein is inversely proportional. ?2? TEM observed the microstructure of the single coaxial nanofibers, have the same axis of the shell-core structure. ?3? XPS data shows 133.0 eV, 164.0eV,190.0 eV and 401.0eV peak spectrum, namely sulfur, phosphorus, nitrogen indicating that type I collagen and rFN/CDH₁₁ protein has been spun into the coaxial fibers. ?4? FTIR results showed a coaxial spinning process does not produce a new chemical group, coaxial fiber has the advantages of each material properties. ?5? Porosimetry showed bone scaffold porosity is between 67-76%, which is proportional to the size of rFN/CDH₁₁ protein concentration. The larger the contact angle measurements described ?6?Contact Angle test specification in the bone scaffold which containing rFN/CDH₁₁ protein concentration, shows that the greater the concentration of protein the better hydrophilic. ?7?Mechanical test results shows that the tensile strength of bone scaffold and elastic modulus is proportional to the bracket the rFN/CDH₁₁ protein concentration ?8?Protein release testing shows that protein early "explosive release" and subsequent slow release effect, achieve the controlled release of proteins. Characterized by the above evaluation,50 ug/ml and 100 ug/ml concentration of rFN CDH₁₁ bone scaffold protein has good physical and chemical properties.3. Research in vitro function with different rFN/CDH₁₁ protein concentration of biomimetic nanofibrous?1? SEM observed the hMSCs vaccination on the scaffold then cultured after 3 days?7 days of cell morphology:, growth state of the cell the 3 days group is preferably a protein concentration of 50ug/ml, embedded grow into the surface of the stent.viscous osteoblasts could be secreted substances secretion of extracellular matrix.7 days group, No significant differences between the protein concentration in the group.?2? The cell adhesion determined by MTT method to detect stents:hMSCs were seeded 0.5h, 1h,2h, the three time points,50ug/ml protein concentrations the OD value of protein concentration in the group were biggest and significantly higher than the negative control group ?p<0.05?. ?3? Determined by MTT method to detect bone scaffold effect on promoting cell proliferation:hMSCs were seeded 2,4,6,8 days within four time points, OD value of protein concentration in the group were biggest and significantly higher than the negative control group ?p< 0.05?. ?4? Real-time quantitative PCR test to induce osteogenic marker genes showed:hMSCs were seeded 1,2,3 weeks, RQ value multiples 50ug/ml protein concentration group and the negative control group RQ value multiples are biggest and significantly higher than the negative control group ?p<0.05?; In the first week, RUNX2 gene expression to highest level, ALPL gene followed; In the second week, OCN and gene expression ALPL elevated, RUNX2 gene expression relative decline; In the third week,the RUNX2 gene expression increased again. Through the above experimental study of cytology,50ug/ml protein concentration rFN/CDH₁₁ bone scaffold proteins promote hMSCs have the best functions of adhesion, proliferation and differentiation.Conclusion:1. Our previous work found rFN/CDH₁₁ fusion protein has good promotion for MSCs cell adhesion, proliferation and ability to differentiate into bone cells direction, combined with the unique coaxial electrospinning shell-core structure, better to achieve a sustained release rFN/CDH protein and slow sustained act on seed cells.2. Coaxial electrospinning construct containing rFN/CDH₁₁ protein nanofiber scaffold is a unique bone shell-core structure,400-800-nm fiber diameter distribution form in disordered arrangement, microscopic structure similar to the spatial structure of extracellular matrix.3. Constructed coaxial electrospinning method comprising rFN/CDH₁₁ bone nanofiber scaffold protein has good hydrophilicity, tensile strength, chemical composition and excellent biodegradability, can compatible with a cell as bionic tissue engineering scaffolds.4. Different rFN/CDHn coaxial electrostatic spinning method to construct protein concentration of nanofibers bone scaffold are all strengthening hMSCs in vitro proliferation and adhesion, HMSCs after the osteogenetic differentiation, osteogenesis markers genes such as ALPL, OCN gene, RUNX2 gene expression level raised. Calcium extracellular matrix mineralization ability enhanced.5.50ug/ml rFN/CDH₁₁ protein concentration coaxial electrospinning nanofibers bone scaffold with excellent bone induction and conduction properties, can be used as an alternative material for future applications of new bone tissue engineering and bone damage repair, but whether plays a good osteogenesis role in vivo, still need further study.