Decellularized skeletal muscle as an in vitro model for studying drug-extracellular matrix interactions

Several factors can affect drug absorption after intramuscular (IM) injection: drug solubility, drug transport across cell membranes, and drug metabolism at the injection site. We found that potential interactions between the drug and the extracellular matrix (ECM) at the injection site can also affect the rate of absorption post-injection. Using decellularized skeletal muscle, we developed a simple method to model drug absorption after IM injection, and showed that the nature of the drug-ECM interaction could be investigated by adding compounds that alter binding. We validated the model using the vitamin B₁₂ analog cobinamide with different bound ligands. Cobinamide is being developed as an IM injectable treatment for cyanide poisoning, and we found that the in vitro binding data correlated with previously published in vivo drug absorption in animals. Commercially available ECM products, such as collagen and GelTrex, did not recapitulate drug binding behavior. While decellularized ECM has been widely studied in fields such as tissue engineering, this work establishes a novel use of skeletal muscle ECM as a potential in vitro model to study drug-ECM interactions during drug development.     

22 week assessment of bladder acellular matrix as a bladder augmentation material in a porcine model

Previous studies on the reconstruction of porcine bladder using bladder acellular matrix allograft (BAMA) have indicated positive preliminary results with respect to graft shrinkage and cellular repopulation. The current study was conducted to investigate the feasibility of using BAMA in a similar model of bladder reconstruction out to longer time frames (22 weeks). At predetermined time points, the macroscopic, histological and mechanical properties of explanted native and BAMA tissues were evaluated and compared. Macroscopically, contracture of the BAMA was observed. The peripheral regions of the grafts experienced extensive cellular repopulation. Towards the centre however, all grafts were consistently devoid of organized smooth muscle bundles and a well-developed urothelium. An alteration in both the amount and organization of collagen was also observed within this region. Significant differences (p < 0.05) in the rupture strain and the elastic modulus of the BAMA compared to native bladder tissue appear to correlate with macroscopic graft contracture as well as the fibroproliferative tissue response of the matrix.     

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2?wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.     

Development and characterisation of a full-thickness acellular porcine bladder matrix for tissue engineering

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (+/-0.1) microg/mg compared to 0.1 (+/-0.1) microg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.     

Characterization of a novel composite scaffold consisting of acellular bladder submucosa matrix,polycaprolactone and Pluronic F127 as a substance for bladder reconstruction

The bladder is an organ susceptible to a variety of congenital anomalies, injuries and disorders. To address the clinical limitations of existing scaffolds, we fabricated a novel scaffold that can be applied to morphological and functional bladder reconstruction. As a first step to prove the benefit of the scaffold, intensive in vitro and in vivo analyses were conducted. The novel composite scaffold was fabricated using polycaprolactone/Pluronic F127 (PCL/F127) and variable proportions (1, 3, 5 and 10 wt.%) of porcine acellular bladder submucosa matrix (BSM). Physicochemical properties and biocompatibilities of the scaffolds were characterized. For cell-mediated analysis, upper-urinary-tract-derived urine stem cells were used. Observations of tensile strength, modulus, porosity, cell adhesion, viability and proliferation characteristics of scaffolds indicated that the optimum proportion of BSM in the composite scaffolds was 3 or 5 wt.%. Based on comparison of 3 and 5 wt.% BSM/PCL/F127 scaffolds with respect to degradability, hydrophilicity, surface properties and functional group presence, the 3 wt.% BSM was chosen for in vivo studies. 8 weeks after kidney-subcapsular implantation of the 3 wt.% BSM/PCL/F127 scaffold, cells remained attached to the surface and there was no evidence of teratomas. A BSM content of 3 wt.% was the optimum proportion for fabrication of the neo scaffold. We predict that the 3 wt.% BSM/PCL/F127 composite scaffold could act as an ideal matrix after cystectomy based on its favorable physicochemical properties and biocompatibilities.     

Serum stimulation, cell-cell interactions, and extracellular matrix independently influence smooth muscle cell phenotype in vitro.

Vascular injury profoundly alters the vessel wall microenvironment, and smooth muscle cells respond with cell cycle re-entry, loss of contractile elements, extracellular matrix remodeling, and altered signaling by endogenous growth factors and their receptors. Environmental cues include stimulation by exogenous mitogens and both cell-cell and cell-matrix interactions. Modeling this process in smooth muscle cells in vitro, these environmental determinants were varied independently and the phenotypic consequences assessed. Mitogenic stimulation with serum promoted the synthesis of collagen and fibronectin and the expression of fibroblast growth factor receptor-1 and suppressed the content of smooth muscle alpha-actin, myosin heavy chain, and basic fibroblast growth factor. Low cell density (reduced cell-cell contact) was also associated with enhanced extracellular matrix protein production, increased fibroblast growth factor receptor-1 expression, and reduced contractile protein and basic fibroblast growth factor content. The influence of serum stimulation and reduced cell-cell contact were independent and additive. Provision of a type I collagen matrix blunted the influence of serum and cell-cell contact on collagen synthesis but had minor effects on other measures of phenotype. Environmental factors thus independently influence smooth muscle cell phenotype, including endogenous growth factor expression and responsiveness, which can in turn influence the microenvironment of the vessel wall after injury.     

Effects of hyaluronan and SPARC on fibroproliferative events assessed in an in vitro bladder acellular matrix model

Bladder acellular matrix (BAM) is a promising candidate for urinary biomaterials development. In the current work we have modified the BAM construct to include two biologically active components; hyaluronan (HA) and a peptide (SP4.2) derived from secreted protein, acidic, rich in cysteine (SPARC), a matricellular glycoprotein. In order to assess the potential of an HA/SP4.2 modified BAM to influence cellular functions associated with bladder healing, experiments were conducted to evaluate the individual and combined effects of these molecules on in vitro fibroproliferative endpoints within a co-culture model. Thiol-modified HA (246 kDa, 15 mg/ml)+/-SP4.2 (200 microm) was incorporated and cross-linked into BAM disks through disulfide bond formation. The following scaffolds compositions were then evaluated in a bladder smooth muscle cell (SMC)-urothelial (UEC) cell co-culture model: BAM unmodified; BAM+HA, BAM+SP4.2 (media addition); BAM+HA+SP4.2 (media addition); BAM+HA+SP4.2 (matrix incorporated). At 3, 7 and 14 days post-seeding, SMC-mediated matrix contraction and gelatinolytic activity were evaluated. HA-modified BAM exhibited a significantly higher degree of contraction and gelatinase activity compared to unmodified BAM. In contrast, addition of SP4.2 to BAM produced a negligible effect on contraction, while significantly reducing gelatinase activity. Matrices containing both molecules displayed significant increases in contraction, while gelatinase activity was dependent upon the method of peptide delivery. These results demonstrate that both HA and SP4.2 have significant, yet distinct effects on the contractile and proteolytic activity of bladder SMCs and suggest that a modified BAM may be capable of modulating processes associated with post-surgical graft contracture and scar formation.     

Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth: in vitro interactions with human cells

Silk fibroin (SF)-based or -coated biomaterials are likely to be endowed with structural and surface properties that render them particularly apt for biomedical applications. In this work we investigated the behavior of four different strains of normal human adult fibroblasts that had been seeded onto membranes made up of poly(carbonate) urethane (PCU), the surfaces of which had or had not been homogeneously coated with SF. Cell adhesion within 3h to the SF-coated PCU films was 2.2-fold that to their uncoated homologues. After 30 days of incubation in vitro, 2.5-fold more cells had grown on the SF-coated specimens than on the uncoated ones. This enhanced cell adherence and hence growth on the SF-coated surfaces was coupled with higher cumulative rates of D-glucose (but not L-glutamine) uptake and of both lactate and interleukin-6 (IL-6) cumulative secretion. Conversely, human fibroblasts cultured on either type of PCU scaffolds never secreted any ELISA-assayable amount of three main proinflammatory cytokines, namely interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1). Finally, when the metabolic activities were compared on a per 10(5) cells basis, it became clear that the adhesion to SF favored an initially higher consumption of D-glucose, a late higher release of IL-6, and an at-first more intense, but declining, extracellular assembly of type I collagen fibers. Overall, these results show that SF-coated PCU membranes represent a novel type of biomaterial that favors the adhesion, the growth and performance of specific metabolic tasks by normal human adult fibroblasts without eliciting any concurrent secretion of some of the chief proinflammatory cytokines.     

无细胞基质移植物在动物膀胱重建中的应用

膀胱扩大术和替代术是治疗膀胱疾病的常用术式。通常用胃肠道作为替代材料 ,但常会带来营养、代谢和感染等方面的并发症。人们试图寻找其它材料 ,包括自体的和人工合成的材料 ,但均可出现包括感染、排斥、结石形成等方面的问题。最近 ,一种新型生物材料——无细胞基质作为膀胱的替代物被成功应用于实验动物模型 ,取得了满意结果。本文将目前国外无细胞基质移植物的制备、应用及结果作一介绍。     

组织工程用猪膀胱无细胞基质的体外细胞毒性检测

生物相容性是评价组织工程支架材料的重要指标之一,是组织工程产品进行临床应用的前提和基础.本研究参照医疗器械生物学评价标准,对本实验室制备的猪膀胱无细胞基质进行体外细胞毒性测定,为其合理应用于组织工程提供实验依据.     

异体脱细胞真皮基质作为组织工程皮肤真皮支架的可行性*

背景：组织工程皮肤是目前研究皮肤损伤修复重建的重要手段之一,异体脱细胞真皮基质不存在免疫原性,在异体移植时不会发生排斥反应,是比较理想的真皮替代物。 目的：观察异体脱细胞真皮基质的组织相容性。 方法：以正常人体真皮组织作为对照,通过体外、体内细胞毒性实验检测异体脱细胞真皮基质的组织相容性,以膨胀度、饱和含水量及生物力学分析检测异体脱细胞真皮基质的亲水性及机械性能。 结果与结论：真皮基质中未见任何细胞成分,其网孔直径介于100~180 μm 之间。脱细胞真皮基质组饱和含水量为(69.6±3.97)%,膨胀度2.30±0.42,最大断裂力为(3.082±0.046) N,与对照组相比,差异无显著性意义(P > 0.05)。体内外细胞毒性检测,未见明显细胞生长抑制及免疫排斥反应。提示异体脱细胞真皮基质机械性能接近正常皮肤,组织相容性好,免疫排斥反应小,是构建组织工程皮肤理想的真皮材料。     

乙酰肝素酶-细胞与基质间相互作用的新型调节剂

乙酰肝素酶——细胞与基质间相互作用的新型调节剂@吴建秋$第二军医大学长海医院血管外科!上海200433 @景在平$第二军医大学长海医院血管外科!上海200433细胞外基质;;硫酸乙酰肝素蛋白多糖~~     

脱细胞天然骨基质与诱导性成骨细胞的体外相容性

背景：前期工作表明TritonX-100处理的脱细胞骨基质已满足组织学和免疫学方面的修复要求。如果细胞能在材料表面很好地生长,将利于进一步进行体内动物实验。 目的：采用细胞培养法在体外评估脱细胞骨基质与诱导后成骨细胞的生物相容性。 方法：第3代骨髓基质干细胞经成骨诱导分化培养液诱导分化为成骨细胞,接种于TritonX-100处理的脱细胞骨基质及羟基磷灰石表面,检测成骨细胞的碱性磷酸酶表达并用扫描电镜观察材料表面的细胞生长情况。 结果与结论：碱性磷酸酶活性分析均表明,TritonX-100处理的脱细胞骨基质在培养48 h之后比羟基磷灰石更利于诱导成骨细胞生长;扫描电镜下可见,成骨细胞在脱细胞骨基质表面呈现立体生长方式,细胞呈球形,并且聚集成簇。体外实验结果显示成骨细胞与脱细胞天然骨基质有较好的生物相容性。     

Lectins and antibodies as tools for studying cellular interactions

Specific interactions between multiple cell types are critical for a variety of processes central to the development, homeostasis and immune defense of multicellular organisms. Studies designed to elucidate how cells communicate through physical encounters have exploited exogenously supplied factors to bypass intrinsic recognition mechanisms and facilitate cellular conjugation. In this review, we compare the relatively nonspecific agglutinating properties of lectins and the selective cell targeting capabilities of antibodies and bispecific antibody constructs for studying cell-cell interactions in immunobiology. In addition, we discuss a novel system for inducing cellular interactions which closely resembles native receptor-mediated conjugation. In this system, surrogate receptors promote specific cell-cell interactions without hindering endogenous receptor-ligand interactions at the cell-cell interface which may be important in mediating physiologic cellular responses.     

In vitro engineering of a palatal mucosa equivalent with acellular porcine dermal matrix

The objective of this study was to develop a palatal mucosa equivalent composed of multilayered oral keratinocytes grown on the acellular porcine dermal matrix. Acellular porcine dermal matrix was prepared through a series of procedures and assessed by histological, immunohistochemical, and scanning electron microscopy examination. The palatal mucosa equivalent was fabricated by seeding oral keratinocytes, which cultured from human palate mucosa, onto the acellular dermal matrix. After 4 days submerged in medium, this composite was raised to the air-liquid interface for another 7 or 14 days of cultivation. The results demonstrated the processed porcine dermal matrix was totally cell-free. The resultant palatal mucosa equivalent showed a multilayered oral epithelium that had been formed, and the number of cell layers was correlated with the culture period at the air-liquid interface. Oral keratinocytes infiltrated into the empty hair follicles of the acellular porcine dermal matrix and formed an anchor-like structure, which exhibited resemblance to the rete ridges of the native palate mucosa. Immunohistochemical staining for CK10/13, CK19, Ki-67 nuclear antigen, and Heparan sulphate indicated the cultured palatal mucosa equivalent shared the same characteristics with that of the native palate mucosa. In conclusion, our fabricated palatal mucosa equivalent exhibited the characteristics of the native counterpart, and this equivalent might be useful for recovery of the wounds in the palate secondary to palatoplasty.     

平滑肌细胞在旋转生物反应器内的微载体培养与快速扩增

为获取足量数量活性良好的膀胱平滑肌种子细胞，本实验探索在旋转生物反应器内应用微载体技术快速扩增膀胱平滑肌细胞的方法。将培养的第二代新西兰兔膀胱不滑肌细胞应用Cytodex-3微载体在旋转生物反应器（RCCS）内进行动态培养，观测平滑肌细胞的生长情况和细胞代谢率，进行a-肌动蛋白免疫组化染色，并与常规方法培养平滑肌细胞进行比较。结果显示膀胱平滑肌细胞在Cytodex-3微载体上生长迅速、细胞代谢率高、生长倍增时间缩短。在培养第九天，细胞数量可达最初接种的23倍。免疫组化显示平滑肌细胞活性良好。结果表明利用微载体细胞培养技术可简便快速地在体外扩增平滑肌细胞，可为构建组织工程化人造膀胱提供大量活性良好的平滑肌细胞。     

In vitro systems for investigating group B streptococcal: host cell and extracellular matrix interactions

Streptococcus agalactiae or group B streptococci (GBS) are gram-positive diplococci and are the leading bacterial cause of pneumoniae, sepsis, and meningitis in neonates. Neonatal GBS infections may occur prior to or during birth. GBS have been cultured from the chorioamnionic membrane of pregnant women and have therefore been associated with chorioamnionitis and premature labor. A potential route for GBS to establish infection of a neonate would be to penetrate the placental membrane of colonized pregnant women. In our laboratory, we have constructed in vitro systems to emulate certain events during the colonization and invasion of host epithelial cell tissues by GBS. By utilizing techniques to grow primary cultures of both chorion cells and amnion cells isolated from human C-section placentas, we have established a relevant model to investigate certain aspects of GBS adherence and invasion into the placental membrane. To identify relevant molecules required for GBS to colonize the multiple tissues it encounters during an infection, we have applied a variety of biochemical approaches with host cell membrane preparations as well as purified extracellular matrix proteins. These techniques are enabling us to further characterize the pathogenic mechanisms utilized by GBS.     

Construction of varying porous structures in acellular bovine pericardia as a tissue-engineering extracellular matrix

In the study, a cell extraction process was used to remove the cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase and subsequently fixed with genipin. Biochemical analyses found that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, mechanical properties, and capability against enzymatic degradation of the bovine pericardial tissue remained unaltered after cell extraction. However, following further treatment with acetic acid and collagenase, the thermal stability and capability against enzymatic degradation of the acellular tissues declined. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid- and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered.     

Cyclodextrin, a probe for studying adhesive interactions

In this short communication, we introduce alpha-cyclodextrin as a new probe to study mechanisms of adhesive interactions. We show that this cyclic polysaccharide, that consisting of six glucosyl residues linked by alpha-1,4 bonds, was the only sugar of 22 tested that dramatically blocked a specific cellular interaction in the sea urchin embryo (p<0.001 compared with non-sugar controls). A total of 150-400 embryos were sampled for each concentration of each sugar tested. Mechanisms of cellular interactions have been studied for almost a century and they still remain poorly understood. Cyclodextrin is an exciting new tool that can be utilized for investigating these mechanisms.