Biological Basic Research On Synthesis Of New Materials For Skin Tissue Engineering And Its Surface Modification

Continuous self-renewal of normal skin epidermis, dermis and subcutaneoussubsidiary organ damage by external impact, through its own physiologicalregeneration and repair of skin tissue, namely compensatory regeneration. However,for large area burns, soft tissue trauma, congenital nevus and skin necrosis diseasewill lead to full-thickness skin defects, skin normal physiological repair systems facesignificant challenges, has been unable to meet the need for tissue regeneration, skintransplant. Supply patients with autologous the defect covered most traditional skingraft technique is a single-layer skin transplantation, which is currently the clinicalgold standard of treatment. This type of single-layer contains all skin epidermal tissuestructures, but only a portion of the leather structure, often resulting in the formationof scar. Therefore, the development of a full-thickness skin of normal anatomy andfunction of the skin analogues is the solution to donor shortage of skin and scarformation is the most effective way.PLGA is a non-toxic biodegradable high molecular weight organic polymer, itsmetabolism in the body through the first polyester hydrolysis to lactic acid andglycolic acid, and subsequently excreted completely decomposed into carbon dioxideand water, good biocompatibility, has passed U.S. FDA, was formally included aspharmaceutical excipients into the United States Pharmacopoeia, widely used inpharmaceutical, medical engineering materials and modern industrial fields. Is oftenused in tissue engineering and regenerative medicine the electrospinning film madetherefrom, is considered to be a two-dimensional bracket having goodbiocompatibility. PVA hydrogel as the advantages of artificial dressing is rich inwater content, can not only keep the wound moist, absorbent can absorb tissueexudate, at the same time, the interior of a large number of through pores in favor of nutrients and metabolic waste transport, and because the town pain effect, so widelyused in the field of tissue engineering. This study combines the advantages of both,and physical cross-linking of the two composite a double stent to build a skin-likestructure, to make it more suitable for clinical bone skin damage repair needs.Gelatin extracted from animal bone or connective tissue, non-toxic, is widelyused in food, pharmaceutical and tissue engineering. In skin tissue engineeringresearch in the field, the gelatin in addition to having a role in promoting cell adhesionand growth, and can also be used as a substrate matrix extracellular matrix resulting incompetitive effect, prevent the wound site of the protease to digest the extracellularmatrix. Past, gelatin is mainly based on physical mixing applications in tissueengineering, thereby enhancing the biocompatibility of the scaffold. Although thismethod is simple, can improve the biocompatibility of the material to a certain extent,but only through the coating, physical adsorption, or a method of mixing a materialsurface modified poor stability, easy to fall off. Currently, the surface modificationmethod of tissue engineering materials including surface finishing, physicaladsorption, and the surface of the plasma polymerization, photochemical fixed.Compared with the other biological material surface modification method, lightfixed technology has prominent features:①both ideal surface interface performance,but also the nature of the material itself does not affect;②the operation is simple,rapid response, and low cost;③versatility, suitable for almost all biological polymermaterials;④can be any target molecule into derivatives with photoactive groups, andphotolysis conditions are very similar;⑤light fixed a variety of biological moleculeshaving good stability, and its biological activity is almost unaffected; The⑥apply asurface modification of the complex material in a variety of shapes, such as the use ofquartz optical fiber guide ultraviolet, above and according to the need for any part ofthe surface of the material by size or regional processing to obtain medical polymermaterials to meet different clinical requirements;⑦can greatly reduce the purpose ofcoating reagents disorderly medical polymer materials surface cross-linking, the targetmolecules in the surface of the material in a highly ordered state, to avoid the use of other methods, the activity multiplied loss of the physical and biological properties ofthe coating and the surface of the biological material and other surface propertiesgreatly reduced defect.The gelatin is collagen hydrolyzate, and its main by the same composition andmolecular weight distribution of a wide amino acids, and generally has a molecularweight in the tens of thousands of ten thousands. Gelatin on the molecular structurecontains a large number of hydroxyl groups and a small amount of carboxyl groupand an amino group, with a strong hydrophilic. Thus, the amino group in the gelatinmolecule can occur with the azide group of the photoactive condensation reaction toform esters.Accordingly, the present study was to further enhance the biological activity ofthe scaffold material of the artificial skin substrate to investigate the impact of lightfixed to its surface-modified biocompatible biological material for tissue engineering.一、Preparation and characterization of a photoactive gelatin1. The Synthesis and Characterization of Az-G photoactive gelatinThrough the activation of carboxyl group, the condensation reaction, dialysis andfreeze-dried after a series of processes, the photoactive gelatin prepared Az-G, thephotoactive chemical composition was visually observed as a white flocculent solid,soft and sticky.Respectively, using ultraviolet spectroscopy, infrared spectroscopy and nuclearmagnetic resonance of three methods, the characterization of the molecular structureof the photoactive gelatin Synthesis of Az-G: a multifunctional microplate reader,measured within the range of230-800nm gelatin, AZ-G an ultraviolet absorptionspectrum of the photoactive gelatin and4-azido benzoic acid results in the UVabsorption spectra at268nm wavelength,4-azido acid azide group has characteristicpeaks of pure gelatin is not displayed any absorption peak, and Az-G photoactivegelatin characteristic peak appears at270nm; Fourier transform infrared spectroscopycompare the structural differences of the Az-G photoactive gelatin and the gelatinalone, the synthetic compound of the gelatin in the2100nm at a wavelength of a downward peaks, the peaks belonging to the characteristic peaks of the azide does notappear pure gelatin IR spectra of the peaks; NMR results show, the the gelatincompounds obtained by the condensation reaction appears on the position of theδ7.8917,7.7263the characteristic peaks in the benzene ring peak, while the the puregelatin map not the peaks.2. Az-G photoactive gelatin preferred crosslinking reaction conditionsReference Synthesis of Az-G the photoactive gelatin having a photoactive bytheir molecular structure and therefore using ultraviolet spectrometer aqueous gelatinsolution before and after the ultraviolet radiation photoactive full wavelength scan,through the observation of the type of radiation intensity, the type of radiation time ofthe optical activity collagen, thus worked best photoactive gelatin crosslinkingreaction conditions.Results Showing: Ultraviolet absorption spectrum did not change with theextension of radiation time, the irradiation time when the radiation intensity of8×100μJ/cm~2to5T (20s), the UV absorption spectrum changed significantly, i.e. thecharacteristic peaks disappear; radiation time to continue to extend the radiationintensity, the UV absorption spectrum does not change. Irradiation time (20s)5TSynthesis of Az-G photoactive gelatin crosslinking reaction can occur in the radiationintensity of8×100μJ/cm~2. In accordance with this crosslinking reaction conditionsfurther analysis of the UV absorption spectrum before and after the change, and theultraviolet absorption spectrum of the ultraviolet radiation before Az-G photoactivegelatin obvious peak at268.4nm; when the radiation intensity reaches8x100μJ/cm~2, compared with ultraviolet radiation before the radiation time of20s, thephotoactive radiation gelatin UV absorption spectrum at268.4nm at the crestdisappear. Therefore, it is considered that the photoactive gelatin the optimumcrosslinking reaction conditions: radiation intensity is8×100μJ/cm~2radiation timeof20s. 3. The Az-G photoactive gelatin in the fixed effect of the type of materialand the surface of the stentFirst fixed plates table light modification: After drying, ultraviolet radiation,washing and dyeing, Az-G photoactive gelatin is fixed to the inner surface of theculture plate and dyed blue Reference Synthesis of Az-G light active gelatin with lightactivity, and fixed through fixed optical technology in the surface of the culture plate;Subsequently,200mL of PBS buffer added to the light fixed hole of the96-wellplates, were incubated with3,7days in an incubator at37°C, using Coomassie bluedye and found that the culture plate surface is still clearly visible circular blue spotsfixed AZ-G the photoactive gelatin and synthetic AZ-G photoactive Descriptiongelatin was dissolved over time, light fixed with excellent results, will not be affectedby the effects of temperature and solution off, and stable up to7days. In contrast,pure gelatin to be washed off after the ultraviolet radiation, the hole is not formed blueround spots, gelatin alone can not be fixed in the surface of the culture plate.Using the method of fixing the light modification experiments carried out in thetype of polymer film and the surface of the stent. After drying, ultraviolet radiation,washed, stained, Az-G photoactive gelatin either in PLGA and PLA, and PVA filmsurface, or in the the PLGA electrospinning film and PVA hydrogel stent surface, areformed a visible blue circular spot color, and pure gelatin did not leave visible tracesof blue on the polymer surface. Technology by light fixed to achieve photoactivegelatin PLGA, PLA, and PVA films and PLGA electrostatic spinning membrane andPVA hydrogel surface of the stent fixation also demonstrated the synthesis ofoptically active gelatin having optical activity.二、Synthesis of new materials for skin tissue engineering and Cytological studieson its modificated surface1. Preparation of electrospinning PLGA fiber membraneIn this study, prepared by electrospinning PLGA electrospun membrane, theelectrospinning fibers of uniform thickness, diameter in the nanometer range, betweenthe fibers irregular arrangement.2. Preparation of PVA hydrogel by Frozen–thawed methodPVA hydrogel prepared by repeated freezing, the present study was visually observed as a white gelatinous scanning electron microscope PVA hydrogel internalpore structure having a dense, pore sizes, shapes, irregular, the space lattice-shapedstructure is a tissue engineering scaffolds.3. Fabrication and characterization of double artificial scaffolds of PLGA/PVAElectrospinning PLGA film and PVA hydrogel as Artificial dressing defects, thisstudy combines the advantages of both, and the use of physical crosslinking way bothcomposite construct a similar double skin structure artificial bracket.Mechanical properties test results show that, compared with the pure PVAhydrogel scaffolds, composite PLGA electrospun membrane of PLGA/PVA doublestent to the superior mechanical properties of PLGA/PVA double stent to stretchstrength, elastic modulus, up to the point of breaking force energy, tensile strengthstrain, tensile breaking strain gauge tensile maximum load were higher than thesingle-layer PVA hydrogel, and the difference was statistically significant (p <0.05);PLGA/PVA double artificial support compared with the single-layer PVA hydrogeltensile stress at break and tensile breaking load were not statistically significant (P>0.05), but have a similar trend, the PLGA/PVA double stent to The tensile breakingstress and tensile breaking load were higher than the single layer of PVA hydrogel.Determination of PLGA/PVA double artificial bracket and the moisture contentof the PVA hydrogel were81.95±1.880and83.93±3.262, statistical analysisshowed no difference (p>0.05) between. Double dressing and different Time PLGA/PVA PVA hydrogel dehydration rate is not the same, the two dressings in the initialstage of dehydration dehydration rate gradually increases slower dewatering rate is therate of change with time, until constant weight no longer changes; constant weight of135min dehydrated PVA hydrogel of not change thereafter dewatering rate, PLGA/PVA double stent to constant weight is reached only at315min after dehydration; thesame dehydration time after, two dressing dehydration rate is significantly differentPVA hydrogel initially dewatering rate was significantly greater than the PLGA/PVA Duplex dressing (p <0.05), with time, when the the dehydration time reaches 315min, both dehydration rate No statistically significant difference (p>0.05).Description of the the double stent to physically prepared not only has the advantageof rich moisture PVA hydrogel containing PLGA electrospinning layer of the surfacecan stop the rapid loss of moisture, delaying the drying time of the PVA hydrogel,play water retention effect.4. Preparation of patterned PVA hydrogelThe present study was prepared by the mold SYSTEM "patterned" PVAhydrogel, the method to change the structure of the hydrogel surface, rough stripedsurface is conducive to cell attachment and growth,"porous" PVA hydrogel formedon the surface narrow pore structure is conducive to early adhesion of cells.5. The Surface Modification of Biological Basic Research on skin tissueengineering scaffold by Az-G photoactive gelatinThe a study prepared AZ-G photoactive gelatin PLGA film, PVA hydrogelsurface modification, the modified surface can promote the adhesion of L929cells toaccelerate the rate of the adherent cells, and with the time and the photoactive gelatinconcentration increases, cell spreading, growth and proliferation capacity graduallyincreases, without modification of the number of parts of the cell, and the adhesion,growth and proliferation capacity is much lower than the modified portion; thephotoactive gelatin PLGA the electrospinning surface modified did not affectfibroblast adhesion rate, stretch, growth and proliferation of cells had no significantrole in promoting; photoactive gelatin Cytological studies on different tissueengineering scaffold material surface modification that The use concentration of thegelatin of the present study was prepared AZ-G photoactive at0.1mg/mL, the materialsurface can be modified to achieve the purpose of promoting cell adhesion, growthand proliferation.Conclusions: In this study, the photoactive azido group and the gelatin moleculescarboxy occurrence of a condensation reaction, as having photoactive newphotoactive compound-AZ-G is formed gelatin; through the anchoring effect of thesurface of the material and cytologic evaluation proved Az-G light active gelatin stable fixed to the surface of the biological material, will not fall off, and the cellcompatibility, is conducive to cell adhesion, growth and proliferation; This is the firstthe electrospinning film and PVA hydrogel by Acta frozen crosslinked methodcombines the performance of their models and water retention is much better than asimple the PLGA the electrospinning film or PVA hydrogel; mold prepared "porous"PVA hydrogel is conducive to cell adhesion and Growth.