Acellular bi-layer silk fibroin scaffolds support functional tissue regeneration in a rat model of onlay esophagoplasty

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds.     

The use of bi-layer silk fibroin scaffolds and small intestinal submucosa matrices to support bladder tissue regeneration in a rat model of spinal cord injury

Adverse side-effects associated with enterocystoplasty for neurogenic bladder reconstruction have spawned the need for the development of alternative graft substitutes. Bi-layer silk fibroin (SF) scaffolds and small intestinal submucosa (SIS) matrices were investigated for their ability to support bladder tissue regeneration and function in a rat model of spinal cord injury (SCI). Bladder augmentation was performed with each scaffold configuration in SCI animals for 10 wk of implantation and compared to non-augmented control groups (normal and SCI alone). Animals subjected to SCI alone exhibited a 72% survival rate (13/18) while SCI rats receiving SIS and bi-layer SF scaffolds displayed respective survival rates of 83% (10/12) and 75% (9/12) over the course of the study period. Histological (Masson's trichrome analysis) and immunohistochemical (IHC) evaluations demonstrated both implant groups supported de novo formation of smooth muscle layers with contractile protein expression [α-smooth muscle actin (α-SMA) and SM22α] as well as maturation of multi-layer urothelia expressing cytokeratin (CK) and uroplakin 3A proteins. Histomorphometric analysis revealed bi-layer SF and SIS scaffolds respectively reconstituted 64% and 56% of the level of α-SMA+ smooth muscle bundles present in SCI-alone controls, while similar degrees of CK+ urothelium across all experimental groups were detected. Parallel evaluations showed similar degrees of vascular area and synaptophysin+ boutons in all regenerated tissues compared to SCI-alone controls. In addition, improvements in certain urodynamic parameters in SCI animals, such as decreased peak intravesical pressure, following implantation with both matrix configurations were also observed. The data presented in this study detail the ability of acellular SIS and bi-layer SF scaffolds to support formation of innervated, vascularized smooth muscle and urothelial tissues in a neurogenic bladder model.     

Therapeutic efficacy of antibiotic-loaded gelatin microsphere/silk fibroin scaffolds in infected full-thickness burns

Despite advances in burn treatment, burn infection remains a major cause of morbidity and mortality. In this study, an antibacterial silk fibroin (SF) scaffold for burn treatment was designed; gelatin microspheres (GMs) were impregnated with the antibiotic gentamycin sulfate (GS), and the GS-impregnated GMs were then embedded in a SF matrix to fabricate GS/GM/SF scaffolds. The developed GS/GM/SF scaffolds could serve as a dermal regeneration template in full-thickness burns. The average pore size and porosity of the GS/GM/SF scaffolds were 40–80 μm and 85%, respectively. Furthermore, the drug release rate of the scaffolds was significantly slower than that of either GS/GM or GS/SF scaffolds. And the composite scaffold exhibited stronger antimicrobial activities against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Hence, we evaluated the wound-healing effects and antibacterial properties of the GS/GM/SF scaffolds in a rat full-thickness burn infection model. Over 21 days, the GS/GM/SF scaffolds not only significantly reduced burn infection by P. aeruginosa but also accelerated the regeneration of the dermis and exhibited higher epithelialization rates than did GS/SF and SF scaffolds. Thus, GS/GM/SF scaffolds are potentially effective for treatment of full-thickness infected burns, and GS/GM/SF scaffolds are a promising therapeutic tool for severely burned patients.     

Effect of sterilization on structural and material properties of 3-D silk fibroin scaffolds

The development of porous scaffolds for tissue engineering applications requires the careful choice of properties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds is a prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methods used to provide sterility is as diverse as the possible effects they can have on the structural and material properties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffold materials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conventional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This study sought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-, ethanol- or antibiotic–antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphology, topography, crystallinity and short-term cell viability, the different sterilization methods showed only few effects. Nevertheless, mechanical properties were significantly decreased by a factor of two by all methods except for dry autoclaving, which seemed not to affect mechanical properties compared to the native control group. These data suggest that SF scaffolds are in general highly resistant to various sterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest.     

The promotion of functional urinary bladder regeneration using anti-inflammatory nanofibers

Current attempts at tissue regeneration utilizing synthetic and decellularized biologic-based materials have typically been met in part by innate immune responses in the form of a robust inflammatory reaction at the site of implantation or grafting. This can ultimately lead to tissue fibrosis with direct negative impact on tissue growth, development, and function. In order to temper the innate inflammatory response, anti-inflammatory signals were incorporated through display on self-assembling peptide nanofibers to promote tissue healing and subsequent graft compliance throughout the regenerative process. Utilizing an established urinary bladder augmentation model, the highly pro-inflammatory biologic scaffold (decellularized small intestinal submucosa) was treated with anti-inflammatory peptide amphiphiles (AIF-PAs) or control peptide amphiphiles and used for augmentation. Significant regenerative advantages of the AIF-PAs were observed including potent angiogenic responses, limited tissue collagen accumulation, and the modulation of macrophage and neutrophil responses in regenerated bladder tissue. Upon further characterization, a reduction in the levels of M2 macrophages was observed, but not in M1 macrophages in control groups, while treatment groups exhibited decreased levels of M1 macrophages and stabilized levels of M2 macrophages. Pro-inflammatory cytokine production was decreased while anti-inflammatory cytokines were up-regulated in treatment groups. This resulted in far fewer incidences of tissue granuloma and bladder stone formation. Finally, functional urinary bladder testing revealed greater bladder compliance and similar capacities in groups treated with AIF-PAs. Data demonstrate that AIF-PAs can alleviate galvanic innate immune responses and provide a highly conducive regenerative milieu that may be applicable in a variety of clinical settings.     

组织工程支架在神经修复中的应用

背景：组织工程支架能够营造适当的神经再生微环境,富集神经再生所需的营养因子,促进轴突生长。 目的：综述近年来组织工程材料在神经损伤修复方面的科研进展。 方法：应用计算机检索PubMed数据库2009至2014年关于组织工程材料修复神经损伤的文章,检索词为“nerve regeneration, prostheses and implants”,并限定为“Full text”。同时利用计算机检索中国知网数据库2004至2014年相关方面的文章,检索词为“神经修复,材料”。 结果与结论：目前用于神经损伤的支架材料主要有天然材料、天然衍生材料、合成材料与复合材料,不同种材料具备各自的优点与缺点。通过化学交联剂或化学修饰,将天然衍生聚合物与其他天然或合成材料复合,可提高其理化和生物学特性,即复合材料神经支架取得的神经再生效果比单一材料效果好,因此当前的研究热点是复合材料。在临床研究方面,胶原蛋白基的神经修复支架材料已开始进入临床研究阶段。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Vascularization of hollow channel-modified porous silk scaffolds with endothelial cells for tissue regeneration

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications.     

丝生物材料处理及其在组织工程中应用研究现状

丝纤维是一种天然的共聚物,其作为手术缝线等已在临床上应用多年.丝纤维由位于中间的丝素蛋白和包裹丝素蛋白的丝胶蛋白构成.近年来,丝纤维材料由于生物相容性良好,降解缓慢,而且具有非常优异的机械性能,因而其可以作为一种新的生物医学支架材料获得广泛应用.而且由于技术手段的发展,能够对丝纤维材料进行多种加工和处理将其加工成多种形态的支架材料和进行表面修饰,并且通过遗传工程和基因工程进行裁切和生产重组的丝蛋白类似物,这使其在生物医学工程领域有广阔的应用前景.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model

Although in vivo studies in small animal model show the ligament regeneration by implanting mesenchymal stem cells (MSCs) and silk scaffold, large animal studies are still needed to evaluate the silk scaffold before starting a clinical trial. The aim of this study is to regenerate anterior cruciate ligament (ACL) in pig model. The micro-porous silk mesh was fabricated by incorporating silk sponges into knitted silk mesh with lyophilization. Then the scaffold was prepared by rolling the micro-porous silk mesh around a braided silk cord to produce a tightly wound shaft. In vitro study indicated that MSCs proliferated profusely on scaffold and differentiated into fibroblast-like cells by expressing collagen I, collagen III and tenascin-C genes in mRNA level. Then the MSCs-seeded scaffold was implanted in pig model to regenerate ACL. At 24 weeks postoperatively, the MSCs in regenerated ligament exhibited fibroblast morphology. The key ligament-specific extracellular matrix components were produced prominently and indirect ligament–bone insertion with three zones (bone, Sharpey's fibers and ligament) was observed. Although there was remarkable scaffold degradation, the maximum tensile load of regenerated ligament could be maintained after 24 weeks of implantation. In conclusion, the results imply that silk-based material has great potentials for clinical applications.     

Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.     

Optimization and evaluation of silk fibroin-chitosan freeze-dried porous scaffolds for cartilage tissue engineering application

Silk fibroin/chitosan blend has been reported to be an attractive biomaterial that provides a 3D porous structure with controllable pore size and mechanical property suitable for tissue engineering applications. However, there is no systematic study for optimizing the ratio of silk fibroin (SF) and chitosan (CS) which seems to influence the scaffold property to a great extent. The present research, therefore, investigates the effect of blend ratio of SF and CS on scaffold property and establishes the optimum value of blend ratio. Among the various blends, the scaffolds with blend ratio of SF/CS (80:20) were found to be superior. The scaffold possesses pore size in the range 71–210 μm and porosity of 82.2 ± 1.3%. The compressive strength of the scaffold was measured as 190 ± 0.2 kPa. The cell supportive property of the scaffold in terms of cell attachment, cell viability, and proliferation was confirmed by cell culture study using mesenchymal stem cells derived from umbilical cord blood. Furthermore, the assessment of glycosaminoglycan secretion on the scaffolds indicates its potentiality toward cartilage tissue regeneration.     

Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase

In this study, we developed a method to fabricate large, perfusable, macroporous, cell-laden hydrogels. This method is suitable for efficient cell seeding, and can maintain sufficient oxygen delivery and mass transfer. We first loaded three types of testing cells (including NIH 3T3, ADSC and Huh7) into gelatin hydrogel filaments, then cross-linked the cell-laden gelatin hydrogel filaments using microbial transglutaminase (mTGase). In situ cross-linking by mTGase was found to be non-cytotoxic and prevented the scattering of the cells after delivery. The gelatin hydrogel constructs kept the carried cells viable; also, the porosity and permeability were adequate for a perfusion system. Cell proliferation was better under perfusion culture than under static culture. When human umbilical vein endothelial cells were seeded into the constructs, we demonstrated that they stably formed an even coverage on the surface of the hydrogel filaments, serving as a preliminary microvasculature network. We concluded that this method provides a viable solution for cell seeding, oxygen delivery, and mass transfer in large three-dimensional (3-D) tissue engineering. Furthermore, it has the potential for being a workhorse in studies involving 3-D cell cultures and tissue engineering.     

含种子细胞的丝素组织工程神经移植物修复大鼠脊髓损伤

目的 探讨体外构建含种子细胞的蚕丝丝素组织工程神经移植物(TENGs)的方法,评价其对大鼠脊髓损伤修复的影响。方法 分离大鼠皮肤前体细胞并向施万细胞诱导分化,S-100免疫荧光染色鉴定。将皮肤前体细胞诱导分化的施万细胞（SKP-SCs）作为种子细胞,联合蚕丝丝素神经导管和纤维支架共培养。共培养7d后将蚕丝丝素TENGs置入大鼠背侧T8~T10半横断损伤的脊髓中,于术后不同时间点利用BBB评分观察行为学的变化,术后8周取材,切片,免疫荧光染色观察脊髓损伤修复情况以及种子细胞的存活情况。 结果 相差显微镜下体外培养的SKP-SCs大部分细胞形态呈双极或3极,免疫荧光染色显示,SKP-SCs呈S-100阳性,将SKP-SCs与蚕丝丝素支架材料共培养,蚕丝丝素支架表面均匀贴附大量的细胞,生长状态良好。将该移植物移植入大鼠T8~T10半横断损伤脊髓处,术后BBB评分显示,从4周起至8周均优于对照组,且结果具有统计学差异;术后8周时取材切片仍能观察到大鼠体内有大量种子细胞存活。 结论 含种子细胞的蚕丝丝素组织工程神经移植物对于修复大鼠脊髓损伤具有一定的促进作用。     

皮肤替代模式的研究现状及发展前景

皮肤是维持机体内环境稳定，抵御外界环境刺激和病原菌入侵的重要屏障，同时也是烧创伤过程中最容易受损的器官。大面积皮肤缺损，皮源有限，无法自行恢复原有的结构和功能，而且易引起诸多严重并发症，直接危及患者生命。皮肤替代模式着力于研究修复、替代皮肤缺损的天然或人工合成的类皮肤结构，经历了从传统敷料到组织工程皮肤的演变。本文就皮肤替代模式的研究现状及发展前景进行综述。     

组织工程技术在创面修复中的应用

烧创伤等原因引起的大面积皮肤缺损是修复重建外科的一大难题。传统皮肤移植术往往组织来源受限,且存在供皮区损伤等缺点。组织工程技术的进步和商品化皮肤的临床应用,为大面积烧伤和难治性创面修复提供了新的治疗手段。本文回顾组织工程技术在创面修复领域的研究进展,并分析其在研发和转化过程中的关键问题,以期为人类皮肤的功能化构建提供新思路。     

纳米纤维支架的制备及其在组织工程皮肤中的应用

本文通过综述现阶段纳米纤维支架的制备方法、可用材料及制备后的生物功能修饰等方面的研究进展,为设计真正意义的组织工程皮肤纳米纤维支架提供理论帮助。多聚物纳米纤维支架能够提供三维空间结构,并且能够调节细胞行为,具有传递生物分子的潜能,因此它们在组织工程应用中具有广泛的前景。现在能应用多种方法及材料制备纳米纤维结构,但是通过现有的方法和材料还不能将纳米纤维的所有优点完全体现出来,并构建成功一个真正意义上的纳米纤维三维支架结构。所以对纳米纤维支架制备技术的不断改进和对应用的材料体系的深入了解,将对未来临床成功地应用聚合纳米纤维组织工程支架奠定坚实基础。     

Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique

Aiming to develop a scaffold architecture mimicking morphological and mechanically that of a blood vessel, a sequential multi-layering electrospinning (ME) was performed on a rotating mandrel-type collector. A bi-layered tubular scaffold composed of a stiff and oriented PLA outside fibrous layer and a pliable and randomly oriented PCL fibrous inner layer (PLA/PCL) was fabricated. Control over the level of fibre orientation of the different layers was achieved through the rotation speed of the collector. The structural and mechanical properties of the scaffolds were examined using scanning electron microscopy (SEM) and tensile testing. To assess their capability to support cell attachment, proliferation and migration, 3T3 mouse fibroblasts and later human venous myofibroblasts (HVS) were cultured, expanded and seeded on the scaffolds. In both cases, the cell-polymer constructs were cultured under static conditions for up to 4 weeks. Environmental-scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), histological examination and biochemical assays for cell proliferation (DNA) and extracellular matrix production (collagen and glycosaminoglycans) were performed. The findings suggest the feasibility of ME to design scaffolds with a hierarchical organization through a layer-by-layer process and control over fibre orientation. The resulting scaffolds achieved the desirable levels of pliability (elastic up to 10% strain) and proved to be capable to promote cell growth and proliferation. The electrospun PLA/PCL bi-layered tube presents appropriate characteristics to be considered a candidate scaffold for blood vessel tissue engineering.