3D Macroporous Electrospinning Nanofibrous Scaffolds Of Nature Polymers Made By Ethanol Bath For Tissue Engineering & Clinical Case Report

Tissue engineering is mainly aimed at promoting the recovery and regeneration of tissues to remedy the limitation of self-healing. The physicochemical properties of scaffolds that function as tools for launching cells and cytokines can affect the cell behavior. The ideal scaffolds should imitate the extracellular matrix (ECM) of tissues to be recovered so that they could simulate the cell living environment and accelerate the cell proliferation and differentiation.The scaffolds can simulate the ECM mainly by two ways. One is the imitation of the morphology and structure. Most of the mesenchymal tissue contents abundant collagen fibers, which built the main scaffold of their ECM structure. Thus nanofibers could be ideal materials to make scaffolds for tissue engineering. The arrangement of collagen fibers is different in different tissues. It had been claimed that fiber arrangement of the scaffold could change the morphology and migration of cells, which finally affected the proliferation and differentiation. Nowadays there are three main methods to make nanofibers, including electrospinning, phase separation and self-assembly. The electrospinning uses the high voltage static to attract the polymers in the solution to form small liquid flow; the fibers will be made when the solvent is volatilized. Compared with the other two methods, the eletrospinning is efficient and flexible, there are abundant materials could be made into nanofibers by this method, and the structure of fibers and scaffolds is adjustable.For example, aligned fibrous scaffolds can be made by changing the receiver. So the electrospinning was widely used to make scaffolds for tissue engineering recently.The other way is the imitation of the element. Nanofibers could be made by electrospinning both from the natural and synthetic polymers. The natural polymers have better bioactivity than the synthetic polymers. It contains ECM element polymers and non-ECM element polymers. Gelatin as the production of the hydrolyzed the collagen is a kind of ECM element natural polymer. Studies proved that gelatin has favorable bioactivity with weak mechanical properties. Silk fibrin (SF) is the extract of silk or cobweb, which belongs to the non-ECM element natural polymer. Its bioactivity and mechanical properties are between ECM element natural polymers and synthetic polymers. Gelatin and SF electrospinning nanofibers were applied effectively in all kinds of tissue engineering.However, the conventional eletrospinning has some limitations. No matter the scaffold was aligned or not, the fibers were usually densely packed with small pores, which handled cell infiltration. In addition, the scaffold had paper like 2D structure that limited its application in large tissue defect. There were many methods had been explored to improve the pore size and the thickness of scaffolds. As-spun fibers made from natural polymers, like gelatin and SF, are unstable. So the crosslink treatment is needed to improve their stability. While crosslink treatment always causes the shrinkage of the scaffolds, the fusion of the fibers and the destruction of the pores. At last, the 3D macroporous structure made by those methods would be damaged and the ideal effect could not be acquired. Moreover, most of the methods to improve the pore size and the thickness were limited to random scaffolds, methods to improve the pore size and thickness of aligned were few and with some limitations.3D macroporous electrospinning nanofiberous scaffolds made from natural polymers were studied in this research. Two kinds of natural polymers, gelatin and SF, were electrospun into fibers and collected by the crosslink ethanol, which were mainly studied. The gelatin and SF fibers were one-step crosslinked by the ethanol or by the crosslinker added to the ethanol. Aligned or random 3D macroporous nanofibrous scaffolds were prepared in this way. Then their physical, chemical and biological characteristics were preliminarily studied.Part one:3D macroporous nanofibrous scaffolds of gelatin prepared by one-step crosslink in ethanol bathObjectives:To discuss the 3D macroporous electrospinning nanofibrous scaffolds of water-soluble natural polymers prepared by one-step crosslink in ethanol bath.Material and methods:15% gelatin acetum was prepared for electrospinning. The glutaraldehyde was added in the ethanol as the crosslinker, fibers were collected and one-step crosslinked by the ethanol. The scaffolds were firstly collected by the pure ethanol then crosslinked by the crosslink solution to make two-step crosslink fibers. The physiochemical properties of scaffolds made by two different crosslink methods were compared by the following experiments:the scanning electron microscope (SEM) was used to observe the morphology of the fibers, the fiber diameter and pore size were analyzed by the photos taken by the SEM, the PBS uptake were taken to detect the water absorption, the stability was observed by degradation test and SEM of the morphology of the scaffolds immersed in the PBS for 14 days, the differential scanning calorimetry (DSC) was used to analyze the thermal stability of the scaffolds, and the 2,4,6-trinitro-benzene-sulfonic acid (TNBS) test was to detect the degree of crosslinking of the fibers.Results:The distribution of the fiber diameter showed, nanofibers could be made under those laboratory conditions. The fibers of ethanol\crosslink group were larger than those received by the crosslink-ethanol bath (P< 0.01), the fibers of crosslink-ethanol\uncrosslink group were the thinnest, the fiber diameter increased after the crosslinker solution treatment (P<0.05). The fibers of ethanol\uncrosslink group were dried by a different method, so the fibers were the largest among the four groups (P<0.001). The result of the pore size shown that ethanol\crosslink group had smaller pores than those collected in the crosslink-ethanol (P<0.001). The pore sizes of the fibers of crosslink-ethanol\crosslink group were smaller than those of crosslink-ethanol\uncrosslink group, while the difference was not significant (P> 0.05). The pore sizes of ethanol\uncrosslink fibers were quite different from the other groups for their different drying method, so their comparison was not significant. The result of water absorption showed that all of the three crosslinked fibers had high water absorption. Fibers collected by the crosslink-ethanol bath had higher water absorption (P<0.01). They had higher water absorption (P<0.001) after crosslink treatment. The result of degradation test showed:within 7 days, the fibers of three crosslinked groups had no significant change. On the day 14, two simples of the ethanol\crosslink group were obviously degraded, while the weight changes of different groups were still had no significant difference. The morphology of fibers immersed in PBS for 14 days showed by the SEM photos. Many fibers of ethanol\crosslink group merged together, some fibers of crosslink-ethanol\uncrosslink group merged together as well, while the merging of fibers of crosslink-ethanol\crosslink group was not obvious. The result of DSC showed an endothermic peak at 66.45 ℃ because of the degeneration and dehydration of gelatin happened at this temperature. However, the degeneration temperature of crosslink-ethanol\crosslink fibers and the crosslink-ethanol\uncrosslink fibers rose to 76.56℃ and 75.81℃ separately, and the endothermic peak became a little flatter. The ethanol\crosslink fibers didn’t form a typical endothermic peak, while heat absorption increased obviously at 66 ℃. The result of crosslinking degree showed that about 33% of the gelatin of ethanol\crosslink group were crosslinked, which was lower than the crosslink-ethanol\uncrosslink group (about 48%, P<0.01) and the crosslink-ethanol\crosslink group (about 56%, P<0.01).Conclusions:The one-step crosslink in ethanol bath is cost-effective and simplicity of operator. Electrospinning nanofibers could be made in the original electrospinnning conditions by this method. The required crosslinking degradation could be achieved by adjusting the time of crosslinking and the concentration of the crosslinker. When this technique applied on the preparation of gelatin nanofibers, the morphology of the fibers and the structure of the pores could be maintained better and the efficiency of the crosslinking could be improved, the ideal 3D macroporous nanofiberous scaffolds could be finally prepared.Part two:the conditions to prepare the one-step crosslinked 3D macroporous aligned nanofibrous scaffolds by the rotating mandrel in the ethanolObjectives:In this research, the technique of electrospinning was taken to study the preparation of the one-step crosslinked 3D macroporous aligned nanofibrous scaffolds collected by the rotating mandrel in the ethanol. They were compared with the scaffolds collected by the traditional method to analyze the effect on the fiber alignment and diameter of the collecting methods, the concentration of the SF, the speed of the rotating mandrel and the crosslinking method.Material and methods:The electrospinning aligned SF fibers were wet collected by the rotating mandrel in ethanol bath (AW). The aligned fibers dry collected by the traditional rotating mandrel were prepared as a control. The characteristics of the fibers were observed by the SEM. The distribution of the fiber diameter and the fiber alignment was analyzed by the SEM photos. The effect on morphology and alignment of the fibers collected by different ways, made in different concentrations of SF and picked up by different speed of the rotating mandrel were also discussed and compared.Results:The result of fiber alignment showed that no matter AW or AD, the fiber alignment was obviously affected by the speed of rotating mandrel when the concentration of SF was 8% or 10%. When the speed of wet collecting mandrel increased from 1.4m/s to 1.9m/s, or the speed of dry collecting mandrel increased from 2.6m/s to 5.2m/s, the fiber alignments all could be improved. While when the concentration of SF was 12%, the speed of rotating mandrel almost had nothing to do with the fiber alignment. The fibers received in all speed could be regarded as aligned. The alignment of AW was better than that of AD received when they were in the same speed levels. The distribution of the fiber diameter showed that the average fiber diameter increased from 0.44μm to 0.79μm and 3.04μm as the concentration of the SF increased from 8% to 10% and 12% accordingly. When the concentration of SF was 8% or 10%, the diameter of AW had no difference with that of the initial AD (P> 0.05), while the diameter of AD increased after crosslink, and became larger than that of AW (P<0.05). When the concentration of SF was 12%, the average diameter of AW was 2.84μm, which was a little higher than that of initial AD of 3.01μm (P< 0.05). The diameter of crosslinked AD was the largest among them (P<0.001). When the concentration of SF was 8%, the fibers received in the low and middle speed were smaller than those received in the high speed. When the concentration of SF was 10%, the fibers received in the low speed were larger than those received in the middle and high speed. When the concentration of SF was 12%, the fibers received in the low and middle speed were larger than those received in the high speed. The standard deviation of fiber diameter showed that the standard deviation of AW had no difference with AD when the concentration of SF was 8% or 10%. While the standard deviation of AW was lower than that of AD (P<0.05) when the concentration of SF was 12%. In addition, when the concentration of SF was 8%, most of the fibers were flat, and large plaques could be seen on the AD, which were hardly seen on the AW. Conclusions:The aligned one-step crosslink fibers prepared by the rotating mandrel in ethanol bath could get better alignment in wider conditions. Under the function of the one-step crosslink of ethanol, the initial diameter of fibers could be maintained, and the fibers and the structure of the fibers were more uniform. In condition that the concentration of SF was 10% and the speed of wet collected fibers and the dry collected fibers were 1.9m/s and 5.2m/s respectively, the fibers of both groups could be aligned with the premise of favorable properties of fibers, which would be used in the following physiochemical and biological studies.Part there:The properties of one-step crosslinked 3D macroporous aligned nanofibrous scaffolds prepared by the rotating mandrel in ethanolObjectives:In the condition that was discussed in part two, the 3D macroporous aligned nanofibrous scaffolds of SF were prepared by the rotating mandrel in the ethanol. They were compared with the scaffolds collected by the traditional method by analyzing of the physiochemical and biological properties.Material and methods:10% SF solution were prepared,1) the wet collected aligned fibers (AW) were made by the rotating mandrel in ethanol at the speed of 1.9m/s; 2) the dry collected aligned fibers (AD) were made by the traditional rotating mandrel at the speed of 5.2m/s; 3) the wet collected random fibers (RW) were prepared by the ethanol bath; 4) the dry collected random fibers (RD) were prepared by the electronic plate. The aligned scaffolds prepared by two different methods were particularly compared by the following experiments and tests. The properties of dimensionality and fluffiness were reflected by the thickness of scaffolds with the same weight/area ratio, the porosity was detected, the property of water absorption was reflected by the PBS up-take test, the stretch property was tested by the universal tension test machine, the effect on structure of fibers and conformation of the molecules of one-step crosslink were tested by the shrinkage test, SEM and FTIR. The pre-osteoblastes MC3T3-E1 were cultured on these scaffolds, the alignment and morphology of cells were observed by the fluorescence microscope and the SEM, the proliferation of the cells and the capacity of the scaffolds were tested by the MTT assay and the morphology and the infiltration of the cells were observed by the histological sections.Results:The thickness of scaffolds with the same weight/area ratio showed that the AD was paper-like and the thickness of AD was less than 1.0mm, while the thickness of the AW was about 2.5mm. The result of porosity and PBS up-take showed, the porosity of AW was higher than that of AD (P<0.05). The average weight of the scaffolds after absorbed the PBS was about 6 times of the initial weight, the PBS up-take of AW was higher than that of the AD as well (P<0.05). The result of shrinkage test showed, two aligned scaffolds were shrunk on both width and length, and the shrinkage of AD was more obvious than the AW (P<0.01). The AD further shrunk on both sides (P<0.001) after drying, while AW only shrunk on length (P<0.001). The result of FTIR showed, the absorption peaks of initial and crosslinked AW and the crosslinked AD were mainly at 1629,1627 and 1627 cm-1,1521,1523 and 1523cm-1, which could refer that the molecular conformations of the above scaffolds were mainly stable P-sheet. However, the absorption peaks of the initial AD was mainly at 1650 and 1541cm-1, which could refer that the molecular conformation of initial AD was almost random coil. The AW was already stable by the one-step crosslink as soon as it had been prepared, additional crosslink treatment was not needed. The fluorescence microscope and SEM could observe that the MC3T3-E1 grew well on the SF nanofibers, which showed elongate morphology on the aligned fibers. And they were more elongate and aligned on the AW than on AD. The result of MTT assay showed, the MC3T3-E1 had greater proliferation on the AW than on AD (P<0.01), and the AW could carry more cells (P<0.05). The result of the histological sections showed that cells can get elongate morphology and aligned arrangement on the aligned fibers. Meanwhile, they could infiltrate fast and orderly into the AW.Conclusions:The physical properties of the aligned electrospinning nanofibers were improved obviously when collected by the ethanol bath, which made a big difference on cell behavior. So ethanol collected aligned scaffolds might have a great potency to be applied in bone tissue engineering.