足底皮肤软组织支架的构建及其组织诱导功能的培育
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摘要
足是人体的一个重要器官,足底皮肤软组织的缺损在足部创伤中较为常见,其中烧伤、机械创伤以及慢性疾病导致的溃疡(如糖尿病)是造成足底皮肤软组织缺损及功能丧失的主要原因。
从组织学结构上看,足底皮肤软组织和人体其他部位的皮肤软组织存在一定的差异。足底皮肤致密,具有较厚的角质层,皮下组织结实并有脂肪垫,可缓冲足底压力对神经和血管的压迫。足底皮肤组织中有垂直走形的纤维与足底肌肉的腱膜相连,可有效地限制皮肤过度移动,形成“皮肤连接”。同时,足底皮肤组织中的脂肪垫内充满了散在的细小而强韧的结缔组织纤维束,可固定皮肤,限制脂肪移位,它们的排列方向与承受的主要压力方向相适应。因此,足底皮肤具有耐磨、耐压、承重的功能,并且有不打滑和不同部位厚度不同的特点,有利于行走和负重时的稳定。
目前国内、外对足底皮肤软组织缺损主要是通过皮瓣移植来进行修复。大量的国内外文献报道了皮瓣供区的选择、血供的恢复、感觉的建立等方面的研究。而这些研究,都是利用患者的健康部位来修复足底皮肤软组织的缺损,虽然对足底皮肤的感觉恢复、足底厚度及耐磨耐压方面有一定改善,但是移植皮瓣(除足底内、外侧皮瓣)愈合后缺少足底皮肤组织中特有的“皮肤连接”结构,造成移植修复后足底皮肤出现反复溃疡和移植皮瓣在行走负重时出现的“足底打滑”现象,至今没有很好的解决办法。另外对供区造成了人为的二次损伤,这种以“创伤修复创伤”的治疗模式必然会被组织工程学的“无损伤修复创伤”的新认识所取代。
国外从1975年开始,由Rheinwald和Green首先实现了表皮细胞的体外大量扩增,并于1979年获得了完整的表皮细胞膜片,使表皮层的移植成为可能。1981年,0'Connor首次成功地实现了体外培植的自体表皮细胞膜片在皮肤创面的移植。在上世纪80年代初由Burke和Yannas等研制成功无细胞型组织工程化真皮并于1996年成为商品化的产品—Integra (Integra life sciences),获得了美国FDA临床使用的许可。由Advanced tissue sciences公司生产的细胞复合型组织工程真皮Dermagraft治疗慢性糖尿病患者足部溃疡的愈合速度明显快于传统的覆盖物。
在我国,皮肤组织工程的研究也逐渐引起人们的重视,高等院校、科研院所与医疗机构也相继在组织工程化人工皮肤方面进行了大胆尝试与探索,取得了积极的成果。曹谊林等以聚乙交酯为主要材料,复合患者自体细胞,研制了一种组织工程化皮肤。马建标等用壳聚糖作为主要材料,开发了能促进人成纤维细胞生长的海绵状人工真皮。第四军医大的金岩等利用人成纤维细胞和表皮细胞在胶原凝胶中的复合种植,成功构建了一种类似Apligraf的组织工程双层皮肤替代物,并取得了国家食品药品监督管理局(SFDA)的临床使用许可。此外,夏照帆的脱细胞真皮支架、苏州大学蚕丝蛋白构建皮肤再生产品的研究都具有一定的特色。但是迄今为止,具有组织诱导再生特性的皮肤再生产品多处于实验室研究阶段。
虽然国内外学者利用组织工程学原理构建了人体皮肤替代物来解决皮肤缺损这一类问题,并取得了突破性进展,积累了有关皮肤组织工程方面的大量基础性数据。但是,在对足底皮肤软组织缺损的组织工程化修复方面的研究报道还很少。
本课题通过组织工程学的原理重新构建足底皮肤软组织。首先,通过显微技术、电镜扫描等技术分析足底纤维的结构特征,了解足底的生物力学性能。在此基础上,利用天然无毒高分子聚合物构建足底皮肤软组织支架,并对支架的各种性能进行测试,以达到足功能的生物学要求。同时进行细胞与支架的体外复合培养和支架植入动物体内,探索支架对细胞、组织体外及体内的诱导机制,为组织工程化足底皮肤缺损修复提供实验依据。本项目对重塑足功能的研究具有重要的科学和实践意义。
课题具体的研究内容包括以下几个方面：
1.足底的显微结构及支架构建
目的：了解足底软组织结构以及纤维、脂肪细胞在不同层次的配布、纤维的走形方向和纤维的组成情况；对足底厚度和生物力学指标进行测定,分析足底软组织的形貌特征,为构建足底软组织支架提供基础的生物学数据。方法：利用手术显微镜进行显微解剖；HE染色和VG染色、切片、光学显微镜观察；生物力学测定仪测量和电镜扫描等方法。结果：显微解剖观察可见足底皮下组织纤维隔分为浅、深两层,浅层的纤维隔小而密,深层的纤维隔大而疏。这些纤维隔分别与皮肤和足跟的筋膜紧密相连,其中有大量的纤维贯穿于纤维隔直接连接到皮肤和跟骨筋膜。HE染色显示,足底软组织由浅入深分为表皮、真皮、基膜和结缔组织四层,其中结缔组织层内有大量的纤维,呈纵行、横行和斜行排列编织呈立体网状结构,大量的脂肪细胞位于其中；VG染色证实这些纤维大部分是胶原纤维。电镜扫描发现,经过脱脂处理后足底内部可见纵横交错的纤维和脱落了脂肪细胞的蜂窝状小房。这些小房有规律的排列成管道状结构,内部有互相连通的孔隙,与HE染色相一致。对6只尸体足底软组织厚度进行了测量,平均厚度为18.6±1.7mm。利用生物力学测定仪对6只足底5个不同厚度的部位分别进行压力测试,利用二次方程进行曲线拟合,R2分别为0.982、0.977、0.993、0.992和0.989,说明拟合效果较好。B、 D、 F为不同厚度的足底皮肤结缔组织样本,其中B、 D拟合曲线特征基本相同,表明足底软组织越厚,吸收外力的能力越强。反之如H、J分别来自于足底的真皮层和表皮层,在受到外力的作用下,压力-位移拟合曲线较平缓,说明厚度越薄,吸收外力的能力越差。结论：足底软组织由浅、深两层纤维隔构成的,浅层致密,纤维紧张度高,深层疏松,纤维紧张度随外力增大逐渐增强,构成纤维隔的纤维主要是胶原纤维,其中部分胶原纤维直接连与皮肤和跟骨筋膜,限制足底皮肤的过度移动,保持了足弓的稳定性,维持了人体的平衡。同时,根据足底软组织的结构特点,研制设计了组织工程支架模型,利用高分子蛋白质制备了可降解的生物支架,该支架可诱导足底内部各种细胞的生长,尤其是成纤维细胞分泌的胶原纤维可按着支架内部的微米级(100-150μ m)通道定向生长；通道内表面粗糙,互相之间有微米级的小孔(20-40μ m)连通,可促进细胞的粘附和信息的传递。
2.支架的构建及细胞诱导功能的体外培育
目的：模拟足底软组织浅、深两层的结构特点,通过电纺丝法和自制模具制备接枝丝素蛋白的聚乳酸三维多孔支架和明胶-丝素蛋白聚合支架。分别测定支架的亲／疏水性、力学性能、降解性、表观形貌和细胞相容性等性能。构建符合足底生物力学要求,组织、细胞相容性良好的可降解高分子支架,了解细胞在支架内部的增殖、生长情况,为足底软组织创伤的修复提供体外培养数据。方法：首先利用自行研制的电纺系统和自制的多通道(直径100μ m)模具制备戊二醛接枝的聚乳酸-丝素蛋白(PLA-SP)纳米级电纺丝支架和戊二醛交联的多通道明胶-丝素蛋白(Gel-SP)聚合物支架。通过干燥法和质量损失法测定支架吸水率和降解率；生物力学测定仪测定电纺支架的拉伸力和聚合支架的压力；扫描电镜分析支架的形貌特征；细胞与支架的共培养对细胞粘附率、增殖率进行测定,通过MTT活性、免疫荧光等方法,评价细胞与支架的相容性。结果：运用电纺系统分别对0.11g/ml PLA/DMF+CH2Cl2和0.13g/ml PLA+SP(固体比1/2)/TFA在20kV电压下进行静电纺丝,得到直径分别为410±193.1nm和250.7±101.5nm分布均匀、无珠状物的纤维。力学性能测试,利用二次方程进行曲线拟合,R2分别为0.991和0.972,说明拟合效果较好,分析结果显示,PLA-SP与PLA两组之间差异均有统计学意义(F=181353.1,P<0.001)。说明PLA-SP电纺支架具有较好的抗拉性能和伸缩性能。降解性能测试,两组样本降解速度差异有统计学意义(F=20.506,P=0.011),PLA-SP组显著高于PLA组。由降解曲线可以看出,0.11g/ml PLA/DMF+CH2Cl2多孔纤维支架随时间延长质量损失加大,在30天到60天内降解速率趋于平缓。而对于以0.13g/ml (PLA-SP)(1:2)/TFA制备的多孔纤维支架在7天内与PLA质量损失相当,在随后的时间里质量损失大于PLA纤维支架,说明经过接枝改性的PLA纤维支架的亲水能力增强。不同固含量比的明胶-丝素蛋白聚合支架吸水率测定,三组支架的吸水率差异有统计学意义(F=2447.191,P<0.001),多重比较结果为,B、C两组吸水率差异无统计学意义(p>0.05),A与B、C之间差异有统计学意义(p<0.05),表明接枝改性后5：1支架的吸水率相对于10：1和1：0有明显提高,达到3.65g/cm2。压力测试,利用二次方程进行曲线拟合,R2分别为0.956、0.978和0.993,说明拟合效果较好。分析结果显示：三组支架力学性能差异有统计学意义(F=8972.991,P<0.001),多重比较结果为,A、B、C三组之间任意两组差异均有统计学意义(P<0.001)。说明不同固含量比的支架内部的孔隙率也不相同,5：1孔隙率最大,吸收外力的能力也最强,抗压能力最好。10：1次之,1：0最差。降解性能测试,三组样本降解速度差异有统计学意义(F=63.746,P<0.001),多重比较结果为,A、B、C三组任意两组之间差异均有统计学意义(P<0.001)。1：0降解的速度最快,10：1次之,5：1较慢。说明对于易溶于水的明胶和丝素蛋白,经过接枝改性后制备的支架,其表面亲水性能降低,降解速度就会变慢。细胞与支架的共培养,五组24h粘附率差异有统计学意义(F=108.651,P<0.001),多重比较结果显示,任意两组间差异都有统计学意义(P<0.05)；五组96h增殖率差异有统计学意义(F=40.076,P<0.001)。对Gel、Gel-SP和PLA、PLA-SP分别进行两组间的方差分析,时间因素、处理因素差异均有统计学意义(p<0.05)。说明种植24h成纤维细胞在Gel-SP支架上的粘附率高于Gel支架,培养96h后Gel-SP支架上的成纤维细胞的增殖速度明显比Gel支架快；上皮细胞在PLA-SP支架上的粘附率和增殖率均高于PLA支架。以上数据证明经过接枝改性的支架,细胞相容性有明显的提高。在PLA、PLA-SP中的MTT活性测定,三组支架MTT活性差异有统计学意义(F=2791.419,P<0.001),多重比较结果显示,任意两组间MTT活性差异有统计学意义(P<0.05),说明经丝素蛋白改性后的聚乳酸支架与上皮细胞的相容性明显好于单纯聚乳酸支架。上皮细胞在PLA和PLA-SP电纺支架上生长的电镜扫描图像显示,上皮细胞都沿着纤维方向进行生长,细胞在PLA-SP支架上的增殖速度比PLA快。共聚焦显微镜观察显示,细胞在未改性的PLA多孔支架中不易铺展,呈球状,并发生了聚集而分布不均匀,而在表面接枝丝素蛋白的多孔支架内部上皮细胞的铺展性有所提高,在支架中的分布也较均匀,细胞铺展良好。统计分析软件为spss13.0,支架吸水率、生物力学性能、降解性能、MTT活性的组间比较采用重复测量方差分析(Repeated Measures);24h粘附率、96h增值率的组间比较采用单向方差分析(One-WayANOVA);若总体差异有统计学意义,则进行多重比较(LSD)；以α=0.05为检验水准；检验均为双侧。结论：固含量比5：1的Gel-SP支架和PLA-SP电纺支架具有较好的生物力学特性和细胞相容性,可作为足底皮下软组织构建的支架材料。
3改性支架对细胞、组织诱导功能的体内培育
目的：通过将不同固含量比的Gel-SP支架植入SD大鼠皮下组织内,观察支架在体实验中的细胞相容性及诱导细胞、组织的生长情况。方法：选取6只体重为200-250g SD大鼠,将消毒过的支架(Gel-SP固含量比为1：0,5：1,10：1)植入同一只大鼠的皮下组织内,在不同时间处死大鼠取材,通过HE染色和电镜扫描进行观察,了解不同时间细胞在支架内部迁入情况和支架在动物体内的降解情况。结果：在植入支架7天后,没有引起大鼠出现明显的炎症反应,仅有少量的成纤维细胞浸入支架分泌细胞外基质,植入的支架没有明显的变化；17天后,大量炎症细胞(单核细胞、巨噬细胞等)迁入支架,并有部分新生血管生成；在21天后支架内部有大量的成纤维细胞团迁入,支架也逐步开始降解,炎症细胞开始减少；29天5：1比10：1支架降解速度快,并有脂肪细胞的迁入；在33天后,支架内部开始血管化,5：1支架比较明显；41天后,支架降解明显,血管大量长入支架,支架内部的组织大量成活。结论：Gel-SP固含量比5：1支架的细胞相容性较好,不会引起明显的炎症反应,并可诱导成纤维细胞和巨噬细胞的大量迁入,在巨噬细胞释放的细胞生长因子作用下,趋化内皮细胞在支架内部生成新小血管。
The foot is an important organ of the body, and the plantar skin soft tissue defects in foot trauma are common. Bums, mechanical trauma, and chronic diseases caused by ulcers (such as diabetes) are the main causes of the plantar skin soft tissue defects and loss of function.
From the perspective of histological structure, there are some differences between the plantar skin and other parts of the body. The plantar skin is dense with a thick cuticle and strong subcutaneous tissue and the fat pad so as to relieve the oppression of the buffer plantar pressure on the nerves and blood vessels. Plantar skin tissue in the vertical go-shaped fibers and the aponeurosis of the plantar muscles are connected to be effective in limiting excessive skin move, forming a "skin connection". At the same time, the fat pad in plantar skin tissue is full of scattered small and tough connective tissue fibers to fix the skin and limit fat to shift. Their orientation responds to the main pressure direction. Therefore, the plantar skin has the function of hard wearing, pressure wearing and load bearing, and has the characteristics of non-slipping and different thickness in different parts, which favors walking and weight-bearing stability.
The current domestic and foreign treatments of plantar skin soft tissue defects are mainly through the transplant of flaps. A number of reports in the literature concern the choice of the flap donor site, the restore of blood supply and the establishment of sense. These studies are about the transplant from the healthy parts of patients to repair defects of the plantar skin soft tissue. Although the sensory recovery, foot thickness and pressure wearing are improved to a certain extent, but the flaps (except the exterior and interior flaps) lack "skin connection"'structure unique to the plantar skin, causing recurring ulcers after flap transplant and the "plantar slipping" phenomena when the flaps bear weight in walking. There have been no good solutions till now. In addition, it does secondary damage to the donor site artificially. This treatment model of'wound healing wound" is bound to be replaced by the new approach of "no wound healing wound" of tissue engineering.
Abroad from1975onwards, Rheinwald and Green first achieved a large amplification of the epidermal cells in vitro, and in1979received a complete epidermal cell patch, so that the epidermis transplants become possible. In1981, O'Connor for the first time successfully cultivated in vitro with autologous epidermal cell patch in the transplantation of skin wounds. In the early1980s Burke and Yannas developed successful products of the cell tissue engineered dermal-Integra (Integra life sciences) and they were commercialized in1996and obtained the clinical use license by U.S. FDA. Cells in complex tissue engineering leather produced by the Advanced tissue sciences Dermagraft treats chronic diabetic foot ulcer significantly faster than traditional coverings
In China, skin tissue engineering research has attracted people's attention. Universities, research institutes and medical institutions have also made bold attempts to explore the tissue engineered skin and achieved positive results. Cao Yilin, etc. adopted Poly lactide as the main material, combined with patients' autologous cells, developed a tissue engineering skin. Ma Jian biao, etc. used chitosan as the main material to cultivate a sponge-like artificial leather which can promote human fibroblast growth. Jin yan of the Fourth Military Medical University utilized human fibroblasts and epidermal cells to plant in collagen gels, successfully constructing a similar Apligraf-like tissue-engineered bilayered skin substitute, which has been approved by the State Food and Drug Administration (SFDA) for clinical use. In addition, the acellular dermal scaffold of Xia Zhaofan and Suzhou University's study of building skin regeneration products from silk protein have certain characteristics. But so far, skin regeneration products with tissue induction features are still in the laboratory research stage.
Although scholars have used tissue engineering principles to build human skin equivalents to solve the problem of skin defects, and made some breakthroughs, and accumulated a lot of basic data on skin tissue engineering. However, reports on the tissue engineering restoration of plantar skin soft tissue defects are still very few.
This project rebuilds the plantar skin and soft tissue through the principles of tissue engineering. First, we use micrologicaltechnique and scanning electron microscope to analyze the structural characteristics of plantar fibers and understand the biomechanical properties of the foot. On this basis, we use natural non-toxic polymers to build the plantar skin soft tissue scaffold, and carry out performance tests on the functions of the scaffold to meet the biological requirements on the foot's function. Meanwhile we culture cells and stent in vitro and implant the stent into animals, and explore the induction mechanism of the bracket on the cells, tissues in vitro and in vivo, and provide experimental basis for the tissue engineering of plantar skin defects. This project has important scientific and practical significance on the study of reshaping the foot function.
The research includes the following aspects.
1. The microstructure of the plantar and the construction of scaffolds
Objective:To understand the plantar soft tissue structures as well as fiber and fat cells'distribution at different levels, the growing direction of the fiber and the composition of the fiber; to measure heel thickness and biomechanical indicators, analyze the plantar soft tissue morphology, provide fundamental biological data to construct plantar soft tissue scaffolds. Methods:using surgical microscope to conduct microsurgical anatomy; HE staining and VG staining, slice, optical microscopy; biomechanical test instrument measurements and scanning electron microscopy method.
Results:Through microsurgical anatomy observation, plantar subcutaneous tissue fibers are divided into superficial and deep layers. The superficial fibrous septa are small and dense while the deep fibrous septa are big and sparse. These fibrous septa and the skin and the fascia of plantar are closely linked, with a number of fibers impenetrating the fibrous septa to link skin and calcaneal fascia. HE staining showed that the plantar soft tissue from the surface to the depth consists of four layers:the epidermis, dermis, basement membrane and connective tissue. A lot of fibers in the connective tissue layer are in longitudinal, transverse and oblique rows, weaving a three-dimensional network structure, with a number of fat cells located in them; the VG staining showed that most of these fibers are collagen fibers. Scanning electron microscopy found that the fibers can be seen criss-crossing within the plantar after the degreasing and the honeycomb rooms shed of the fat cells. These small rooms are arranged in a regular pipe-like structure with interconnected pores, which is consistent with HE staining. For Six body plantars soft tissue thickness was measured. The average thickness is18.6±1.7mm. We use biomechanical analyzer to test stress on five parts of different thickness conduct quadratic curve fitting. R2are respectively0.982,0.977,0.993,0.992and0.989, indicating fitting well.B, D and F for the skin and connective tissue samples of different thickness of the plantar, where B, D, fitting curve is basically the same features, indicating that the thicker the plantar soft tissue, the stronger ability to absorb external forces. On the contrary, such as H, J, respectively, are from the dermis and epidermis of the plantar. By external forces, pressure-displacement fitting curve is relatively flat, indicating that the thinner, the poorer ability to absorb external forces.
Conclusion:Plantar soft tissue consist of the superficial and deep fibers, with the superficial layer being dense and of high tension while the deep layer loose and of gradually increasing tension responding to the increase of external force. The fibrous septa are mainly composed of collagen fibers, some of which are directly connected with the skin and calcaneal fascia to prevent heel skin from moving excessively and maintain the stability of the arch to keep the balance of the body. Meanwhile, according to the structural characteristics of plantar soft tissue, we design scaffolds for tissue engineering, and use polymer protein to make a biodegradable stent, which can induce internal growth of cells of the plantar. The collagen fibers grow along the micron (100-150μm) channel within the scaffold. The channel surface is rough, connected with micrometer-sized holes (20-40μm), which can promote cell adhesion and the transmission of message between each other.
2.Construction of stents and the cultivation in vitro of cell induction function
Objective:To simulate the structural feature of two layers of plantar soft tissue, via electrospinning method and self-made mold, we prepare grafted silk fibroin polylactide three-dimensional porous scaffolds and gelatin-silk fibroin polymer scaffold. We measured the stent pro/hydrophobicity. mechanical properties, degradability, morphology and cell compatibility performance. Constructed in line with the foot biomechanics, the organization has good cell compatibility of biodegradable polymer stents, understanding of cell proliferation in the stent within the growth of cultured in vitro data for the repair of the plantar soft tissue trauma.
Methods:First developed by the spinning system and self-made multi-channel (diameter100μm) mold preparation of glutaraldehyde-grafted PLA-silk fibroin (PLA-SP) nanoscale electrospinning stent and glutaraldehyde cross-linkingchannel gelatin-silk fibroin protein (Gel-SP) polymer scaffold. Determination of the stents water absorption and degradation rate of drying and mass loss; biomechanics was determined by measuring the tensile strength of electrospun scaffold and the pressure of the polymer scaffold; scanning electron microscopy analysis of the morphology of the stent; cells and scaffold co-cultured cells adhesion rate of proliferation rates were measured by MTT activity, immunofluorescence methods, evaluation of the compatibility of the cells and scaffold.
Results:the use of the electrospinning system0.11g/ml PLA/DMF+CH2Cl2and0.13g/ml the PLA+SP (solid ratio of1/2)/TFA in the electrospinning voltage of20kV, with diameters were410±193.lnm and250.7±101.5nm evenly distributed, no bead fiber. Mechanical tests, the use of the quadratic curve fitting, and R2were0.991and0.972, indicating that well fitted by analysis, difference of PLA-SP with PLA between the two groups were statistically significant (F=181353.1,P<0.001) The PLA-SP electrospun scaffold has better tensile performance and scalability. Degradation of the performance test, the degradation rate of the two samples was statistically significant (F=.20.506,P=0.011).PLA-SP group is higher significantly than PLA group.The degradation curves of the two samples,0.11g/ml PLA/DMF+CH2Cl2, porous fibers scaffolds over time to extend the mass loss increased, the degradation rate leveled off in30days to60days.0.13g/ml (PLA-SP)(1:2)/TFA porous fibrous scaffold mass loss in7days with PLA is quite at a later time, the mass loss is greater than the PLA fiber bracket, shows that, after grafting hydrophilic ability of the modified PLA fiber stent. Different solid content than gelatin-the silk fibroin polymer stents water absorption was statistically significant (F=2447.191, P<0.001). Do multiple comparisons test found that the B and C were water absorption, the difference was not statistically significant (p>0.05), the difference A and B. and C was statistically significant (p<0.05), showed that the graft modification of5:1water absorption of the stent relative to the10:1and1:0significantly improved to achieve3.65g/cm2. Stress tests, using quadratic curve fitting, and R2, respectively, for the0.956,0.978and0.993, indicating that the fitting better. The three groups were statistically significant (F=8972.991,P<0.001). Do multiple comparisons test, A, B, C groups differences were statistically significant differences between (P<0.001). Description of the different solid content than the porosity of the scaffolds is not the same,5:1porosity also the strongest ability to absorb external forces, compressive strength.10:1followed by1:0worst. Degradation of performance testing, three groups of sample degradation speeds were statistically significant (F=63.746,P<0.001), and further to do multiple comparisons test found that A, B, and C groups were statistically significant differences between (P<0.001).1:0degradation of the fastest, followed by10:1.5:1slow. Note for the water-soluble gelatin and silk fibroin, the preparation of the stent after graft modification, the surface hydrophilic properties reduce the degradation will slow down. Cells and scaffold training, five groups of samples,24h adhesion rate were statistically significant (F=108.651, P<0.001).Do multiple comparisons test found that five groups were statistically significant differences between (P<0.05). Five groups of samples,96h proliferation rate were statistically significant (F=40.076, P<0.001).Gel, Gel-SP and PLA, the PLA-SP analysis of variance between the two groups, the time factor to deal with factors that differences were statistically significant p<0.05. Description planting24h fibroblast Gel-SP stand adhesion rate the Gel bracket, culture96h after Gel-SP stand on the fibroblast proliferation rate significantly faster than the Gel scaffold; epithelial cells stand in the PLA-SP the adhesion rate and proliferation rate higher than that of the PLA scaffold. The above data demonstrate significantly improved after the grafted stent, cell compatibility. MTT activity in the PLA, PLA-SP, three groups of samples were statistically significant (F=2791.419, P<0.001). Do multiple comparisons test found that three groups were statistically significant differences between (P<0.05), indicating the compatibility of silk fibroin modified polylactic acid stents and epithelial cells significantly better than in the pure polylactide stent. The scanning electron microscope image shows the growth of epithelial cells in the PLA and PLA-SP spinning stand, the epithelial cells along the fiber direction of the growth of cells in the proliferation rate of PLA-SP stand faster than PLA. Confocal microscopy showed that cells in the unmodified PLA porous scaffolds is not easy spreading, globular, and aggregation and uneven distribution in the surface grafted silk fibroin porous scaffolds epithelial cells spreading someimproved the distribution of the stent more uniform cell spreading. Statistical analysis software SPSS13.0. scaffold-water absorption, bio-mechanical properties, degradability, MTT activity between the two groups were compared using repeated measures ANOVA (Repeated Measures).24h adhesion rate and96h proliferation rate between the two groups were compared using one-way varianceanalysis (One-Way ANOVA).If the overall difference was statistically significant, multiple comparisons (LSD).a=0.05for the test standards. Test were two-sided.
Conclusion:The solid content ratio5:1Gel-SP bracket and PLA-SP spinning stent has better biomechanical properties and cell compatibility can be used as the plantar soft tissue build scaffolds.
3.Modified bracket to cultivate cells, tissues and body of the induced function
Objective:Gel-SP by different solid content ratio stent implantation in SD rats subcutaneous tissue, and observe the stent cells in vivo compatibility and induced cells, the organization's growth.
Methods:6Weight200-250g SD rats, sterile stents (Gel-SP solid content ratio1:0,5:1,10:1) implanted in the subcutaneous tissue of the same rats. The rats were sacrificed at different times, subjects, were observed by HE staining and scanning electron microscopy to understand the different times of cells in the scaffolds to move and the degradation of stents in animals.
Results:7days after stent implantation did not cause rats had significant inflammatory response, only a few fibroblasts immersed in the bracket secretion of extracellular matrix, the implantation of the stent did not change significantly;17days, a large number of inflammatory cells(monocytes, macrophages, etc.) to move into the bracket, and part of angiogenesis; A large number of fibroblasts regiment moved to within the21days after stent, the stent gradually began to degrade, inflammatory cells started to decrease;29days5:1than10:1bracket degradation speed faster, and fat cells to move into; in33days later, the stent internal vascularization.5:1stent obvious;41days after stent degradation significantly, vascular many long into the stent, the stent within the organization a lot of survival.
Conclusion:Gel-SP solid content ratio5:1stent cells compatible with good, does not cause significant inflammatory response, and can be induced into fiber cells and macrophages to move into the release of macrophage cellsrole of growth factors, chemokines, endothelial cells in the scaffolds to generate new small blood vessels.
从组织学结构上看,足底皮肤软组织和人体其他部位的皮肤软组织存在一定的差异。足底皮肤致密,具有较厚的角质层,皮下组织结实并有脂肪垫,可缓冲足底压力对神经和血管的压迫。足底皮肤组织中有垂直走形的纤维与足底肌肉的腱膜相连,可有效地限制皮肤过度移动,形成“皮肤连接”。同时,足底皮肤组织中的脂肪垫内充满了散在的细小而强韧的结缔组织纤维束,可固定皮肤,限制脂肪移位,它们的排列方向与承受的主要压力方向相适应。因此,足底皮肤具有耐磨、耐压、承重的功能,并且有不打滑和不同部位厚度不同的特点,有利于行走和负重时的稳定。
目前国内、外对足底皮肤软组织缺损主要是通过皮瓣移植来进行修复。大量的国内外文献报道了皮瓣供区的选择、血供的恢复、感觉的建立等方面的研究。而这些研究,都是利用患者的健康部位来修复足底皮肤软组织的缺损,虽然对足底皮肤的感觉恢复、足底厚度及耐磨耐压方面有一定改善,但是移植皮瓣(除足底内、外侧皮瓣)愈合后缺少足底皮肤组织中特有的“皮肤连接”结构,造成移植修复后足底皮肤出现反复溃疡和移植皮瓣在行走负重时出现的“足底打滑”现象,至今没有很好的解决办法。另外对供区造成了人为的二次损伤,这种以“创伤修复创伤”的治疗模式必然会被组织工程学的“无损伤修复创伤”的新认识所取代。
国外从1975年开始,由Rheinwald和Green首先实现了表皮细胞的体外大量扩增,并于1979年获得了完整的表皮细胞膜片,使表皮层的移植成为可能。1981年,0'Connor首次成功地实现了体外培植的自体表皮细胞膜片在皮肤创面的移植。在上世纪80年代初由Burke和Yannas等研制成功无细胞型组织工程化真皮并于1996年成为商品化的产品—Integra (Integra life sciences),获得了美国FDA临床使用的许可。由Advanced tissue sciences公司生产的细胞复合型组织工程真皮Dermagraft治疗慢性糖尿病患者足部溃疡的愈合速度明显快于传统的覆盖物。
在我国,皮肤组织工程的研究也逐渐引起人们的重视,高等院校、科研院所与医疗机构也相继在组织工程化人工皮肤方面进行了大胆尝试与探索,取得了积极的成果。曹谊林等以聚乙交酯为主要材料,复合患者自体细胞,研制了一种组织工程化皮肤。马建标等用壳聚糖作为主要材料,开发了能促进人成纤维细胞生长的海绵状人工真皮。第四军医大的金岩等利用人成纤维细胞和表皮细胞在胶原凝胶中的复合种植,成功构建了一种类似Apligraf的组织工程双层皮肤替代物,并取得了国家食品药品监督管理局(SFDA)的临床使用许可。此外,夏照帆的脱细胞真皮支架、苏州大学蚕丝蛋白构建皮肤再生产品的研究都具有一定的特色。但是迄今为止,具有组织诱导再生特性的皮肤再生产品多处于实验室研究阶段。
虽然国内外学者利用组织工程学原理构建了人体皮肤替代物来解决皮肤缺损这一类问题,并取得了突破性进展,积累了有关皮肤组织工程方面的大量基础性数据。但是,在对足底皮肤软组织缺损的组织工程化修复方面的研究报道还很少。
本课题通过组织工程学的原理重新构建足底皮肤软组织。首先,通过显微技术、电镜扫描等技术分析足底纤维的结构特征,了解足底的生物力学性能。在此基础上,利用天然无毒高分子聚合物构建足底皮肤软组织支架,并对支架的各种性能进行测试,以达到足功能的生物学要求。同时进行细胞与支架的体外复合培养和支架植入动物体内,探索支架对细胞、组织体外及体内的诱导机制,为组织工程化足底皮肤缺损修复提供实验依据。本项目对重塑足功能的研究具有重要的科学和实践意义。
课题具体的研究内容包括以下几个方面：
1.足底的显微结构及支架构建
目的：了解足底软组织结构以及纤维、脂肪细胞在不同层次的配布、纤维的走形方向和纤维的组成情况；对足底厚度和生物力学指标进行测定,分析足底软组织的形貌特征,为构建足底软组织支架提供基础的生物学数据。方法：利用手术显微镜进行显微解剖；HE染色和VG染色、切片、光学显微镜观察；生物力学测定仪测量和电镜扫描等方法。结果：显微解剖观察可见足底皮下组织纤维隔分为浅、深两层,浅层的纤维隔小而密,深层的纤维隔大而疏。这些纤维隔分别与皮肤和足跟的筋膜紧密相连,其中有大量的纤维贯穿于纤维隔直接连接到皮肤和跟骨筋膜。HE染色显示,足底软组织由浅入深分为表皮、真皮、基膜和结缔组织四层,其中结缔组织层内有大量的纤维,呈纵行、横行和斜行排列编织呈立体网状结构,大量的脂肪细胞位于其中；VG染色证实这些纤维大部分是胶原纤维。电镜扫描发现,经过脱脂处理后足底内部可见纵横交错的纤维和脱落了脂肪细胞的蜂窝状小房。这些小房有规律的排列成管道状结构,内部有互相连通的孔隙,与HE染色相一致。对6只尸体足底软组织厚度进行了测量,平均厚度为18.6±1.7mm。利用生物力学测定仪对6只足底5个不同厚度的部位分别进行压力测试,利用二次方程进行曲线拟合,R2分别为0.982、0.977、0.993、0.992和0.989,说明拟合效果较好。B、 D、 F为不同厚度的足底皮肤结缔组织样本,其中B、 D拟合曲线特征基本相同,表明足底软组织越厚,吸收外力的能力越强。反之如H、J分别来自于足底的真皮层和表皮层,在受到外力的作用下,压力-位移拟合曲线较平缓,说明厚度越薄,吸收外力的能力越差。结论：足底软组织由浅、深两层纤维隔构成的,浅层致密,纤维紧张度高,深层疏松,纤维紧张度随外力增大逐渐增强,构成纤维隔的纤维主要是胶原纤维,其中部分胶原纤维直接连与皮肤和跟骨筋膜,限制足底皮肤的过度移动,保持了足弓的稳定性,维持了人体的平衡。同时,根据足底软组织的结构特点,研制设计了组织工程支架模型,利用高分子蛋白质制备了可降解的生物支架,该支架可诱导足底内部各种细胞的生长,尤其是成纤维细胞分泌的胶原纤维可按着支架内部的微米级(100-150μ m)通道定向生长；通道内表面粗糙,互相之间有微米级的小孔(20-40μ m)连通,可促进细胞的粘附和信息的传递。
2.支架的构建及细胞诱导功能的体外培育
目的：模拟足底软组织浅、深两层的结构特点,通过电纺丝法和自制模具制备接枝丝素蛋白的聚乳酸三维多孔支架和明胶-丝素蛋白聚合支架。分别测定支架的亲／疏水性、力学性能、降解性、表观形貌和细胞相容性等性能。构建符合足底生物力学要求,组织、细胞相容性良好的可降解高分子支架,了解细胞在支架内部的增殖、生长情况,为足底软组织创伤的修复提供体外培养数据。方法：首先利用自行研制的电纺系统和自制的多通道(直径100μ m)模具制备戊二醛接枝的聚乳酸-丝素蛋白(PLA-SP)纳米级电纺丝支架和戊二醛交联的多通道明胶-丝素蛋白(Gel-SP)聚合物支架。通过干燥法和质量损失法测定支架吸水率和降解率；生物力学测定仪测定电纺支架的拉伸力和聚合支架的压力；扫描电镜分析支架的形貌特征；细胞与支架的共培养对细胞粘附率、增殖率进行测定,通过MTT活性、免疫荧光等方法,评价细胞与支架的相容性。结果：运用电纺系统分别对0.11g/ml PLA/DMF+CH2Cl2和0.13g/ml PLA+SP(固体比1/2)/TFA在20kV电压下进行静电纺丝,得到直径分别为410±193.1nm和250.7±101.5nm分布均匀、无珠状物的纤维。力学性能测试,利用二次方程进行曲线拟合,R2分别为0.991和0.972,说明拟合效果较好,分析结果显示,PLA-SP与PLA两组之间差异均有统计学意义(F=181353.1,P<0.001)。说明PLA-SP电纺支架具有较好的抗拉性能和伸缩性能。降解性能测试,两组样本降解速度差异有统计学意义(F=20.506,P=0.011),PLA-SP组显著高于PLA组。由降解曲线可以看出,0.11g/ml PLA/DMF+CH2Cl2多孔纤维支架随时间延长质量损失加大,在30天到60天内降解速率趋于平缓。而对于以0.13g/ml (PLA-SP)(1:2)/TFA制备的多孔纤维支架在7天内与PLA质量损失相当,在随后的时间里质量损失大于PLA纤维支架,说明经过接枝改性的PLA纤维支架的亲水能力增强。不同固含量比的明胶-丝素蛋白聚合支架吸水率测定,三组支架的吸水率差异有统计学意义(F=2447.191,P<0.001),多重比较结果为,B、C两组吸水率差异无统计学意义(p>0.05),A与B、C之间差异有统计学意义(p<0.05),表明接枝改性后5：1支架的吸水率相对于10：1和1：0有明显提高,达到3.65g/cm2。压力测试,利用二次方程进行曲线拟合,R2分别为0.956、0.978和0.993,说明拟合效果较好。分析结果显示：三组支架力学性能差异有统计学意义(F=8972.991,P<0.001),多重比较结果为,A、B、C三组之间任意两组差异均有统计学意义(P<0.001)。说明不同固含量比的支架内部的孔隙率也不相同,5：1孔隙率最大,吸收外力的能力也最强,抗压能力最好。10：1次之,1：0最差。降解性能测试,三组样本降解速度差异有统计学意义(F=63.746,P<0.001),多重比较结果为,A、B、C三组任意两组之间差异均有统计学意义(P<0.001)。1：0降解的速度最快,10：1次之,5：1较慢。说明对于易溶于水的明胶和丝素蛋白,经过接枝改性后制备的支架,其表面亲水性能降低,降解速度就会变慢。细胞与支架的共培养,五组24h粘附率差异有统计学意义(F=108.651,P<0.001),多重比较结果显示,任意两组间差异都有统计学意义(P<0.05)；五组96h增殖率差异有统计学意义(F=40.076,P<0.001)。对Gel、Gel-SP和PLA、PLA-SP分别进行两组间的方差分析,时间因素、处理因素差异均有统计学意义(p<0.05)。说明种植24h成纤维细胞在Gel-SP支架上的粘附率高于Gel支架,培养96h后Gel-SP支架上的成纤维细胞的增殖速度明显比Gel支架快；上皮细胞在PLA-SP支架上的粘附率和增殖率均高于PLA支架。以上数据证明经过接枝改性的支架,细胞相容性有明显的提高。在PLA、PLA-SP中的MTT活性测定,三组支架MTT活性差异有统计学意义(F=2791.419,P<0.001),多重比较结果显示,任意两组间MTT活性差异有统计学意义(P<0.05),说明经丝素蛋白改性后的聚乳酸支架与上皮细胞的相容性明显好于单纯聚乳酸支架。上皮细胞在PLA和PLA-SP电纺支架上生长的电镜扫描图像显示,上皮细胞都沿着纤维方向进行生长,细胞在PLA-SP支架上的增殖速度比PLA快。共聚焦显微镜观察显示,细胞在未改性的PLA多孔支架中不易铺展,呈球状,并发生了聚集而分布不均匀,而在表面接枝丝素蛋白的多孔支架内部上皮细胞的铺展性有所提高,在支架中的分布也较均匀,细胞铺展良好。统计分析软件为spss13.0,支架吸水率、生物力学性能、降解性能、MTT活性的组间比较采用重复测量方差分析(Repeated Measures);24h粘附率、96h增值率的组间比较采用单向方差分析(One-WayANOVA);若总体差异有统计学意义,则进行多重比较(LSD)；以α=0.05为检验水准；检验均为双侧。结论：固含量比5：1的Gel-SP支架和PLA-SP电纺支架具有较好的生物力学特性和细胞相容性,可作为足底皮下软组织构建的支架材料。
3改性支架对细胞、组织诱导功能的体内培育
目的：通过将不同固含量比的Gel-SP支架植入SD大鼠皮下组织内,观察支架在体实验中的细胞相容性及诱导细胞、组织的生长情况。方法：选取6只体重为200-250g SD大鼠,将消毒过的支架(Gel-SP固含量比为1：0,5：1,10：1)植入同一只大鼠的皮下组织内,在不同时间处死大鼠取材,通过HE染色和电镜扫描进行观察,了解不同时间细胞在支架内部迁入情况和支架在动物体内的降解情况。结果：在植入支架7天后,没有引起大鼠出现明显的炎症反应,仅有少量的成纤维细胞浸入支架分泌细胞外基质,植入的支架没有明显的变化；17天后,大量炎症细胞(单核细胞、巨噬细胞等)迁入支架,并有部分新生血管生成；在21天后支架内部有大量的成纤维细胞团迁入,支架也逐步开始降解,炎症细胞开始减少；29天5：1比10：1支架降解速度快,并有脂肪细胞的迁入；在33天后,支架内部开始血管化,5：1支架比较明显；41天后,支架降解明显,血管大量长入支架,支架内部的组织大量成活。结论：Gel-SP固含量比5：1支架的细胞相容性较好,不会引起明显的炎症反应,并可诱导成纤维细胞和巨噬细胞的大量迁入,在巨噬细胞释放的细胞生长因子作用下,趋化内皮细胞在支架内部生成新小血管。
The foot is an important organ of the body, and the plantar skin soft tissue defects in foot trauma are common. Bums, mechanical trauma, and chronic diseases caused by ulcers (such as diabetes) are the main causes of the plantar skin soft tissue defects and loss of function.
From the perspective of histological structure, there are some differences between the plantar skin and other parts of the body. The plantar skin is dense with a thick cuticle and strong subcutaneous tissue and the fat pad so as to relieve the oppression of the buffer plantar pressure on the nerves and blood vessels. Plantar skin tissue in the vertical go-shaped fibers and the aponeurosis of the plantar muscles are connected to be effective in limiting excessive skin move, forming a "skin connection". At the same time, the fat pad in plantar skin tissue is full of scattered small and tough connective tissue fibers to fix the skin and limit fat to shift. Their orientation responds to the main pressure direction. Therefore, the plantar skin has the function of hard wearing, pressure wearing and load bearing, and has the characteristics of non-slipping and different thickness in different parts, which favors walking and weight-bearing stability.
The current domestic and foreign treatments of plantar skin soft tissue defects are mainly through the transplant of flaps. A number of reports in the literature concern the choice of the flap donor site, the restore of blood supply and the establishment of sense. These studies are about the transplant from the healthy parts of patients to repair defects of the plantar skin soft tissue. Although the sensory recovery, foot thickness and pressure wearing are improved to a certain extent, but the flaps (except the exterior and interior flaps) lack "skin connection"'structure unique to the plantar skin, causing recurring ulcers after flap transplant and the "plantar slipping" phenomena when the flaps bear weight in walking. There have been no good solutions till now. In addition, it does secondary damage to the donor site artificially. This treatment model of'wound healing wound" is bound to be replaced by the new approach of "no wound healing wound" of tissue engineering.
Abroad from1975onwards, Rheinwald and Green first achieved a large amplification of the epidermal cells in vitro, and in1979received a complete epidermal cell patch, so that the epidermis transplants become possible. In1981, O'Connor for the first time successfully cultivated in vitro with autologous epidermal cell patch in the transplantation of skin wounds. In the early1980s Burke and Yannas developed successful products of the cell tissue engineered dermal-Integra (Integra life sciences) and they were commercialized in1996and obtained the clinical use license by U.S. FDA. Cells in complex tissue engineering leather produced by the Advanced tissue sciences Dermagraft treats chronic diabetic foot ulcer significantly faster than traditional coverings
In China, skin tissue engineering research has attracted people's attention. Universities, research institutes and medical institutions have also made bold attempts to explore the tissue engineered skin and achieved positive results. Cao Yilin, etc. adopted Poly lactide as the main material, combined with patients' autologous cells, developed a tissue engineering skin. Ma Jian biao, etc. used chitosan as the main material to cultivate a sponge-like artificial leather which can promote human fibroblast growth. Jin yan of the Fourth Military Medical University utilized human fibroblasts and epidermal cells to plant in collagen gels, successfully constructing a similar Apligraf-like tissue-engineered bilayered skin substitute, which has been approved by the State Food and Drug Administration (SFDA) for clinical use. In addition, the acellular dermal scaffold of Xia Zhaofan and Suzhou University's study of building skin regeneration products from silk protein have certain characteristics. But so far, skin regeneration products with tissue induction features are still in the laboratory research stage.
Although scholars have used tissue engineering principles to build human skin equivalents to solve the problem of skin defects, and made some breakthroughs, and accumulated a lot of basic data on skin tissue engineering. However, reports on the tissue engineering restoration of plantar skin soft tissue defects are still very few.
This project rebuilds the plantar skin and soft tissue through the principles of tissue engineering. First, we use micrologicaltechnique and scanning electron microscope to analyze the structural characteristics of plantar fibers and understand the biomechanical properties of the foot. On this basis, we use natural non-toxic polymers to build the plantar skin soft tissue scaffold, and carry out performance tests on the functions of the scaffold to meet the biological requirements on the foot's function. Meanwhile we culture cells and stent in vitro and implant the stent into animals, and explore the induction mechanism of the bracket on the cells, tissues in vitro and in vivo, and provide experimental basis for the tissue engineering of plantar skin defects. This project has important scientific and practical significance on the study of reshaping the foot function.
The research includes the following aspects.
1. The microstructure of the plantar and the construction of scaffolds
Objective:To understand the plantar soft tissue structures as well as fiber and fat cells'distribution at different levels, the growing direction of the fiber and the composition of the fiber; to measure heel thickness and biomechanical indicators, analyze the plantar soft tissue morphology, provide fundamental biological data to construct plantar soft tissue scaffolds. Methods:using surgical microscope to conduct microsurgical anatomy; HE staining and VG staining, slice, optical microscopy; biomechanical test instrument measurements and scanning electron microscopy method.
Results:Through microsurgical anatomy observation, plantar subcutaneous tissue fibers are divided into superficial and deep layers. The superficial fibrous septa are small and dense while the deep fibrous septa are big and sparse. These fibrous septa and the skin and the fascia of plantar are closely linked, with a number of fibers impenetrating the fibrous septa to link skin and calcaneal fascia. HE staining showed that the plantar soft tissue from the surface to the depth consists of four layers:the epidermis, dermis, basement membrane and connective tissue. A lot of fibers in the connective tissue layer are in longitudinal, transverse and oblique rows, weaving a three-dimensional network structure, with a number of fat cells located in them; the VG staining showed that most of these fibers are collagen fibers. Scanning electron microscopy found that the fibers can be seen criss-crossing within the plantar after the degreasing and the honeycomb rooms shed of the fat cells. These small rooms are arranged in a regular pipe-like structure with interconnected pores, which is consistent with HE staining. For Six body plantars soft tissue thickness was measured. The average thickness is18.6±1.7mm. We use biomechanical analyzer to test stress on five parts of different thickness conduct quadratic curve fitting. R2are respectively0.982,0.977,0.993,0.992and0.989, indicating fitting well.B, D and F for the skin and connective tissue samples of different thickness of the plantar, where B, D, fitting curve is basically the same features, indicating that the thicker the plantar soft tissue, the stronger ability to absorb external forces. On the contrary, such as H, J, respectively, are from the dermis and epidermis of the plantar. By external forces, pressure-displacement fitting curve is relatively flat, indicating that the thinner, the poorer ability to absorb external forces.
Conclusion:Plantar soft tissue consist of the superficial and deep fibers, with the superficial layer being dense and of high tension while the deep layer loose and of gradually increasing tension responding to the increase of external force. The fibrous septa are mainly composed of collagen fibers, some of which are directly connected with the skin and calcaneal fascia to prevent heel skin from moving excessively and maintain the stability of the arch to keep the balance of the body. Meanwhile, according to the structural characteristics of plantar soft tissue, we design scaffolds for tissue engineering, and use polymer protein to make a biodegradable stent, which can induce internal growth of cells of the plantar. The collagen fibers grow along the micron (100-150μm) channel within the scaffold. The channel surface is rough, connected with micrometer-sized holes (20-40μm), which can promote cell adhesion and the transmission of message between each other.
2.Construction of stents and the cultivation in vitro of cell induction function
Objective:To simulate the structural feature of two layers of plantar soft tissue, via electrospinning method and self-made mold, we prepare grafted silk fibroin polylactide three-dimensional porous scaffolds and gelatin-silk fibroin polymer scaffold. We measured the stent pro/hydrophobicity. mechanical properties, degradability, morphology and cell compatibility performance. Constructed in line with the foot biomechanics, the organization has good cell compatibility of biodegradable polymer stents, understanding of cell proliferation in the stent within the growth of cultured in vitro data for the repair of the plantar soft tissue trauma.
Methods:First developed by the spinning system and self-made multi-channel (diameter100μm) mold preparation of glutaraldehyde-grafted PLA-silk fibroin (PLA-SP) nanoscale electrospinning stent and glutaraldehyde cross-linkingchannel gelatin-silk fibroin protein (Gel-SP) polymer scaffold. Determination of the stents water absorption and degradation rate of drying and mass loss; biomechanics was determined by measuring the tensile strength of electrospun scaffold and the pressure of the polymer scaffold; scanning electron microscopy analysis of the morphology of the stent; cells and scaffold co-cultured cells adhesion rate of proliferation rates were measured by MTT activity, immunofluorescence methods, evaluation of the compatibility of the cells and scaffold.
Results:the use of the electrospinning system0.11g/ml PLA/DMF+CH2Cl2and0.13g/ml the PLA+SP (solid ratio of1/2)/TFA in the electrospinning voltage of20kV, with diameters were410±193.lnm and250.7±101.5nm evenly distributed, no bead fiber. Mechanical tests, the use of the quadratic curve fitting, and R2were0.991and0.972, indicating that well fitted by analysis, difference of PLA-SP with PLA between the two groups were statistically significant (F=181353.1,P<0.001) The PLA-SP electrospun scaffold has better tensile performance and scalability. Degradation of the performance test, the degradation rate of the two samples was statistically significant (F=.20.506,P=0.011).PLA-SP group is higher significantly than PLA group.The degradation curves of the two samples,0.11g/ml PLA/DMF+CH2Cl2, porous fibers scaffolds over time to extend the mass loss increased, the degradation rate leveled off in30days to60days.0.13g/ml (PLA-SP)(1:2)/TFA porous fibrous scaffold mass loss in7days with PLA is quite at a later time, the mass loss is greater than the PLA fiber bracket, shows that, after grafting hydrophilic ability of the modified PLA fiber stent. Different solid content than gelatin-the silk fibroin polymer stents water absorption was statistically significant (F=2447.191, P<0.001). Do multiple comparisons test found that the B and C were water absorption, the difference was not statistically significant (p>0.05), the difference A and B. and C was statistically significant (p<0.05), showed that the graft modification of5:1water absorption of the stent relative to the10:1and1:0significantly improved to achieve3.65g/cm2. Stress tests, using quadratic curve fitting, and R2, respectively, for the0.956,0.978and0.993, indicating that the fitting better. The three groups were statistically significant (F=8972.991,P<0.001). Do multiple comparisons test, A, B, C groups differences were statistically significant differences between (P<0.001). Description of the different solid content than the porosity of the scaffolds is not the same,5:1porosity also the strongest ability to absorb external forces, compressive strength.10:1followed by1:0worst. Degradation of performance testing, three groups of sample degradation speeds were statistically significant (F=63.746,P<0.001), and further to do multiple comparisons test found that A, B, and C groups were statistically significant differences between (P<0.001).1:0degradation of the fastest, followed by10:1.5:1slow. Note for the water-soluble gelatin and silk fibroin, the preparation of the stent after graft modification, the surface hydrophilic properties reduce the degradation will slow down. Cells and scaffold training, five groups of samples,24h adhesion rate were statistically significant (F=108.651, P<0.001).Do multiple comparisons test found that five groups were statistically significant differences between (P<0.05). Five groups of samples,96h proliferation rate were statistically significant (F=40.076, P<0.001).Gel, Gel-SP and PLA, the PLA-SP analysis of variance between the two groups, the time factor to deal with factors that differences were statistically significant p<0.05. Description planting24h fibroblast Gel-SP stand adhesion rate the Gel bracket, culture96h after Gel-SP stand on the fibroblast proliferation rate significantly faster than the Gel scaffold; epithelial cells stand in the PLA-SP the adhesion rate and proliferation rate higher than that of the PLA scaffold. The above data demonstrate significantly improved after the grafted stent, cell compatibility. MTT activity in the PLA, PLA-SP, three groups of samples were statistically significant (F=2791.419, P<0.001). Do multiple comparisons test found that three groups were statistically significant differences between (P<0.05), indicating the compatibility of silk fibroin modified polylactic acid stents and epithelial cells significantly better than in the pure polylactide stent. The scanning electron microscope image shows the growth of epithelial cells in the PLA and PLA-SP spinning stand, the epithelial cells along the fiber direction of the growth of cells in the proliferation rate of PLA-SP stand faster than PLA. Confocal microscopy showed that cells in the unmodified PLA porous scaffolds is not easy spreading, globular, and aggregation and uneven distribution in the surface grafted silk fibroin porous scaffolds epithelial cells spreading someimproved the distribution of the stent more uniform cell spreading. Statistical analysis software SPSS13.0. scaffold-water absorption, bio-mechanical properties, degradability, MTT activity between the two groups were compared using repeated measures ANOVA (Repeated Measures).24h adhesion rate and96h proliferation rate between the two groups were compared using one-way varianceanalysis (One-Way ANOVA).If the overall difference was statistically significant, multiple comparisons (LSD).a=0.05for the test standards. Test were two-sided.
Conclusion:The solid content ratio5:1Gel-SP bracket and PLA-SP spinning stent has better biomechanical properties and cell compatibility can be used as the plantar soft tissue build scaffolds.
3.Modified bracket to cultivate cells, tissues and body of the induced function
Objective:Gel-SP by different solid content ratio stent implantation in SD rats subcutaneous tissue, and observe the stent cells in vivo compatibility and induced cells, the organization's growth.
Methods:6Weight200-250g SD rats, sterile stents (Gel-SP solid content ratio1:0,5:1,10:1) implanted in the subcutaneous tissue of the same rats. The rats were sacrificed at different times, subjects, were observed by HE staining and scanning electron microscopy to understand the different times of cells in the scaffolds to move and the degradation of stents in animals.
Results:7days after stent implantation did not cause rats had significant inflammatory response, only a few fibroblasts immersed in the bracket secretion of extracellular matrix, the implantation of the stent did not change significantly;17days, a large number of inflammatory cells(monocytes, macrophages, etc.) to move into the bracket, and part of angiogenesis; A large number of fibroblasts regiment moved to within the21days after stent, the stent gradually began to degrade, inflammatory cells started to decrease;29days5:1than10:1bracket degradation speed faster, and fat cells to move into; in33days later, the stent internal vascularization.5:1stent obvious;41days after stent degradation significantly, vascular many long into the stent, the stent within the organization a lot of survival.
Conclusion:Gel-SP solid content ratio5:1stent cells compatible with good, does not cause significant inflammatory response, and can be induced into fiber cells and macrophages to move into the release of macrophage cellsrole of growth factors, chemokines, endothelial cells in the scaffolds to generate new small blood vessels.
引文
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