Fabrication And Structure Characterization Of Tissue Engineering Heart Valve Scaffold Based Collagen/Polyurethane By Electrospinning Method

The technology of tissue engineering (TE) aims to generate new or substitute or malfunctioning and could well become an alternative method to whole organ transplantation. It is the use of combination of cells, engineering and materials methods, and suitable biomechanical and physic-chemical factors to improve or replace biological functions. The Principle of tissue engineering is deriving cells from human body, then culture and seeds it on biocompatible scaffold in vitro. After the new tissue is formed, it will implant to human body to substitute the damage organs. So the scaffold selection is one of the most important factors of tissue engineering success. The method used in this research to fabricate tissue engineering heart valve scaffold is also using this principle. With the selection of thermoplastic polyurethane (TPU) and collagen materials, this research will fabricate tissue engineering heart valve scaffold to mimic biological and structural properties of natural heart valve tissue using electrospinning method.The first part of this research is co-electrospun of TPU and collagen. Co-el ectrospun is frequently used method of tissue engineering scaffold fabrication. A serious of thermoplastic polyurethane (TPU)/collagen blend nanofibrous membranes were prepared with different weight ratios and concentrations via electrospinning. The two biopolymer were all used 1,1,1,3,3,3,-hexafluoro-2-propanol(HFP) as solvent. The electrospun thermoplastic polyurethane-contained collagen nanofibers was characterized using scanning electron microscopy (SEM), XPS spectroscopy, atomic-force microscopy, apparent density and porosity measurement, contact angle measurement, mechanical tensile testing and viability of Pig iliac endothelial cells (PIECs) on blended nanofiber mats. Our date indicate that fiber diameter was influenced by both polymer concentration and blend weight ratio of collagen to TPU. The average diameter of nanofibers gradually decreases with increasing collagen content in the blend. XPS analysis indicates that collagen is found to be present at the surface of blended nanofiber. The results of porosity and contact angle measurement suggest that with the collagen content in the blend system, the porosity and hydrophicity of the nanofiber mats is greatly improved. We have also characterized the molecular interactions in TPU/collagen complex by Fourier transform (FTIR). The result could demonstrate that there were intermolecular bonds between the molecules of TPU and collagen. The ultimate tensile stress and strain were carried out and the dates could also prove the analysis of FTIR. These results suggest tha the blend nanofibers of TPU/collagen are designed to mimic the native extracellular matrix for tissue engineering and develop functional biomaterials.The second part of this research was using coaxial electrsopinning method to evaluate another complex method of TPU and collagen. Effective use of polymer nanofibrous scaffolds for tissue engineering relies not only on the construction of the fibers, which can mimic the physical structure of the native extracellular matrix, but also on the biochemistry characteristics of the materials used. One method of functionalizing nanofiber is realized by employing an advanced coaxial electrospinning technology. Through combination of different materials in the axial or radial direction, novel properties and functionalities for nano-scale devices can be anticipated. The unique core-shell structure offers a number of potential benefits. For example, the core materials should provide certain properties required by the tissue to be repaired, while the shell materials could be tailored to provide or endow additional properties, such as biocompatibility or hydrophilic properties. The major advantage of this core-shell nanostructure is the potential to obtain a combination of properties of different kind of materials. Collagen functionalized thermoplastic polyurethane nanofibers (TPU/collagen) were successfully produced by coaxial electrospinning technique with a goal to develop biomedical scaffold. A series of tests were conducted to characterize the compound nanofiber and its membrane in this study. Surface morphology and interior structure of the ultrafine fibers were characterized by scanning electron microscopy, transmission electron microscopy and atomic force microscopy, whereas the fiber diameter distribution was also measured. Porosities of different kinds of electrospun mats were determined. The surface chemistry and chemical composition of collagen/TPU coaxial nanofibrous membranes were verified by X-ray photoelectron spectroscopy and Fourier transform infrared spectrometry. Mechanical measurements were carried out by applying tensile test loads to samples which were prepared from electrospun ultra fine non-woven fiber mats. The results demonstrated that coaxial-electrospun composite nanofibers has the characters of native extracellular matrix and may be used effectively as an alternative material for tissue engineering and functional biomaterials. But the major disadvantage of coaxial electrospinning was the low production and unstable process parameters.The next part was the collection and analysis of aligned electrospun TPU/collagen complex nanofibers. To design an ideal scaffold for tissue engineering, various factors should be considered, such as pore size and morphology, mechanical properties versus porosity, surface properties and appropriate biodegradability. Tissue engineered constructs must exhibit tissue-like functional properties. Electrospun thermoplastic polyurethane/collagen (ES-TPU/collagen) is elastomeric and allow for control of the fiber diameter, porosity and degradation rate. ES-TPU/collagen scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues. We developed ES-TPU/collagen scaffolds under variable speed conditions and measured fiber alignment by fast Fourier transform (FFT). By using FFT to assign relative alignment values to an electrospun matrix, it is possible systematically evaluate how different processing variables affect the structure and material properties of a scaffold. TPU was suspended at certain concentrations and electrospun from 1,1,1,3,3,3,-hexafluoro-2-propanol (HFP) onto rotating mandrels (200-4000RPM). Scaffold anisotropy developed as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. The circular and axial bi-directional mechanical properties of complex nanofibers were also characterized. With better understanding of the aligned nanofibers, this part research helped the design of heart valve scaffold fabrication.The biocompatibility characterization of electrospun nanofiberous membrane was the next part of this research. Pig iliac endothelial cells(PIECs) cultured on blend nanofibers was compared with that of cells cultured on coverslips (control), pure collagen nanofibers, TPU nanofibers and TPU co-electrospun/coaxial electrospun nanofiber. The conclusions were listed below:(1) Co-electrospun:It was revealed that all the nanofiber mats had good cell viability than coverslips and cell viability had no obvious difference among the blend nanofiber mats at 24 hours, but cell proliferation was very fast and the highest MTT absorbance index could reach 0.7. On days 3, among the different blend nanofibers, the one with the TPU to collagen blend weight ratios of 3:1 had showed better cell viability than the others, at the same time blend ratios of 3:1 had most excellent cell viability on days 5 and 7. Thus, compared with pure collagen and TPU, blend nanofibers could provide better growth condition for cell proliferation. In our studies, the one with TPU to collagen weight ratios of 3:1 might offer the most suitable qualification for cell culture. (2) Coaxial electrospinning: Coaxial electrospun nanofibers was significantly more favorable (P<0.05) for cells proliferation than that of roughly collagen-coated TPU, pure TPU and control. There was no significant difference in the two different core concentrations compound nanofibers, but cell proliferation of core concentration of 6wt% exhibited little better than the concentration of 3wt%. Comparing the coaxial electrospun nanofibers and collagen nanofiers, the proliferation date and statistical test indicated that there is no significant difference among them. (3) The cell proliferation guidance of aligned complex nanofibers was studied and the effect was limited. For cell alignment effects, coaxial electrospinning nanofibers exhibit better performance than co-electrospun fibers. In future research, the growth factor should be added in the blending system to facilitate the cell alignment formation. (4) The mats were placed in desiccator to crosslink using glutaraldehyde (25%water solution) steam for two days. The results showed that glutaraldehyde could not solve collagen dissolving in culture media. So new cross-linking agents should be discovered in future research.The last part of this thesis was using fused deposition modeling (FDM) to fabricate heart valve ring, and using electrospun material to fabricate heart valve leaflet. A novel combination method of electrospinning and Rapid Prototyping fused deposition modeling (FDM) have been proposed for the fabrication of final tissue engineering heart valve (TEHV) scaffold. The fabrication of heart ring scaffolds of a desired porosity and characteristics will require careful 3D CAD modelling and the careful selection of FDM process parameters. The FDM process and build parameters affecting the strength and integrity of scaffolds includes the slice thickness, road width, fill gap, hatch pattern type and raster angles. However, it is imperative that operational procedures, databases and algorithms are developed to select the most suitable process parameters for the given micro-architecture and porosity of the scaffolds. Experimental observations reveal that the porosity of the scaffold built on the FDM machine depends upon four main FDM parameters:slice thickness, road width, raster gap, raster angle. A pulse bioreactor system was set up to evaluate the cell retention ability of electrospun scaffolds. This part study created a electrospinning scaffold with FDM heart valve ring and aimed to investigate its biocompatibility and cell retention ability upon exposure to pulsatile flow. Endothelial cells were seeded onto electrospun scaffold, PGA/PLA and collagen-coated bovine pericardium and exposed to shear stresses of 0.062,0.122 and 0.185 N/m2 for a period of 1 to 3 h. High cell retention rate of> 84% was observed in all 3 experimental groups after 1 h of flow. Mean cell retention rate of 80% was maintained in the electrospun scaffold group throughout the exposure time and with increases in shear stress. The good biocompatibility and cell retention properties of electrospun scaffold demonstrated its potential as a biomaterial for tissue engineering of heart valves.Summary of this PhD research, it was found that electrospun TPU/collagen composite scaffold could mimic the biological and structure properties of natural heart valve tissue. Two kind of compound method has its own advantage and disadvantage. The production output of blend electrospinning was large and process parameter was easy to control. But the collagen was partly distributed on the surface of complex nanofibers. The process parameters of coaxial electrospinning were complicated and difficult to control. But the coating efficiency was high and collagen could distribute at the surface of compound nanofibers. The mechanical property of coaxial electrospinning scaffold was better than co-electrospun scaffold. With the rotation collector, aligned complex nanofiber was collected and its bi-directional mechanical properties were characterized. Its anisotropic properties could mimic natural mechanical properties of heart valve. Combination of electrospinning and FDM and design of pulse bioreactor characterization, the results showed that electrospun scaffold has excellent cell retention ability. This research represented references for fabrication of tissue engineering heart valve scaffold based on natural/polymer nanofibrous composites. It also provided scientific basis and experimental dates for the future tissue engineering artificial organs development and final clinical applications.