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Preparation of small intestinal submucosa/ poly(ε-caprolactone-co-lactide) blended nanofiber sheets for tissue engineering and medical applications

초록/요약

In the proliferation of cells, the extracellular matrix (ECM) functions as environmental information transducer, cell morphological inducer and vital factor forming physical properties of tissues. Fabrication of ECM-mimicking scaffold using ECM-based materials brings potential advantages of natural ECM for tissue regeneration by reconstruction of cell and tissue surrounding environment. Small intestine submucosa (SIS) is a member ECM material. SIS consists of over 90% type I, II, III collagen, and various kinds of bioactive factors, benefits cell proliferation, cell migration, angiogenesis, mononuclear infiltration and cellular differentiation. The goal of this study is to use SIS as the standard material to develop a nanofiber structure which is not only received the bioactive characteristic of SIS but also restored ECM structure by electrospinning technique. Practically, SIS possesses good biocompatibility but lacking of electrospinning ability and mechanical properties. Thence, a biocompatible poly(ε-caprolactone-co-lactide) (PCLA) copolymer was added for fabrication composite fibrous membranes, in which, incorporated both nature SIS for cellular attachment and bioactive surface and synthetic polymers for the mainstay of electrospun fibers. In this study, the blended solution of SIS/PCLA with various concentrations were electrospinning to generate the fibrous structure of ECM. Structural characterization of SIS fibrous sheets was observed by scanning electron microscope. Biodegradability and biocompatibility of SIS nanofiber sheets were evaluated by weight loss (in the present of collagenase) and MTT cell proliferation assay. The finding showed that SIS/PCLA blended solutions are able to electrospun to gain 60nm to 700nm fibers, correlated to the amount of SIS in the electrospinning solution. Embedded of drugs into SIS/PCLA nanofibers showed different affected on the fiber size between experimental groups. SIS/PCLA sheets expressed good biocompatible characteristic. In conclusion, we hypothesize from these results that SIS nanofiber sheets can be used as a superior scaffold for tissue engineering applications. For advanced application in the regeneration of tissue/organs, SIS nanofiber sheet was established as a drug delivery system which carried Dexamethasone (DEX) and Silver sulfadiazine (SSD). Using high-performance liquid chromatography (HPLC) and UV-Vis photo-spectroscopy, we investigated release characteristic of these embedded hydrophilic, and hydrophobic drugs in SIS nanofiber. An in vivo biocompatible and inflammation response examinations of the SIS and drug carried SIS nanofiber sheets have carried on showed the potential of SIS/PCLA in the application as a drug carrier. 

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목차

Chapter I: General introduction and aims of this work. 1
1. General introduction. 2
1.1. Introduction to tissue engineering, traditional approaches and micro and nano technologies in tissue engineering. 2
1.2. The demand for micro and nanotechnologies in tissue engineering approaches. 3
2. Aims of this work. 5
Chapter II: Fabrication and characterization of small intestinal submucosa delivered sheets blended with poly(ε-caprolactone-co-lactide) copolymer as a biocompatible scaffold for tissue regeneration. 7
1. Introduction 8
2. Experimental section. 10
2.1. Synthesis of poly(ε-caprolactone-co-lactide) (PCLA) copolymer. 10
2.2. Preparation of small intestinal submucosa powder. 11
2.3. Electrospinning of the SIS/PCLA fibrous sheets. 12
2.4. Characterization of the fiber formation, fiber morphology and orientation of the SIS/PCLA electrospun sheets 12
2.5. Characterization of the fiber pore size and porosity of the SIS/PCLA electrospun sheets. 13
2.6. Characterization of the fiber tensile strength and elongation at break of the SIS/PCLA nanofibrous sheets. 13
2.7. Characterization of the surface wettability of the SIS/PCLA electrospun sheets using water drop angle test. 14
2.8. Characterization of the biodegradation of the SIS/PCLA electrospun sheets in the present of collagenase. 14
3. Results 15
3.1. Physical and optical characterization of the electrospun SIS/PCLA sheets. 15
3.2. Characterization of the degradation of SIS/PCLA fibrous sheets in the collagenase buffer at in vitro condition 20
4. Conclusion 24
Chapter III: In situ incorporation of drugs into electrospun membranes and study of inflammatory response of the host cell towards the drug loaded small intestinal submucosa blended with poly(ε-caprolactone-co-lactide) nanofiber sheets. 25
1. Introduction. 26
2. Experimental section: 27
2.1. Preparation of electrospun SIS/PCLA (5:1) nanofiber sheet and in situ corporation of drugs in the membranes. 27
2.2. Characterization of drugs embedded SIS/PCLA electrospun sheets. 27
2.3. In vivo study of fiber degradation of the SIS/PCLA nanofiber sheets. 28
2.4. Study of drug release assay of the electrospun membranes in vitro and in vivo.29
2.5. Histological analysis. 30
3. Results. 32
3.1. In situ incorporation of drugs into the electrospun sheets. 32
3.2. Surface wettability and fiber degradation of the drug loading SIS/PCLA nanofiber sheets. 35
3.3. Drug release characteristics of DEX and SSD from SIS/PCLA electrospun nanofiber sheets. 36
3.4. Histological analysis of the drug loaded SIS/PCLA 5:1 nanofiber sheets 41
4. Conclusion. 43
References 44

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