In Vitro Biocompatibility of New PVA-Based Hydrogels as Vitreous Body Substitutes

In order to synthesize injectable hydrogels suitable as vitreous body substitutes, a new method based on the use of trisodium trimetaphosphate (STMP) to cross-link PVA was recently proposed. Hydrogels with different molar ratios between STMP and PVA were realised. The aim of the present study was the evaluation of the biocompatibility of the different STMP/PVA hydrogels synthesised by analysing the effects of their in vitro interaction with cultures of mouse fibroblasts NIH3T3, primary human microvascular endothelial cells adult (HMVECad) and human lens cells. Cytotoxicity of hydrogels was first evaluated by analysing cell density and proliferation. Morphological and morphometric analysis of cell in contact with hydrogels was then performed using light microscopy and scanning electron microscopy, respectively. Moreover, cell adhesion and growth onto the hydrogels surface was evaluated and correlated to the amount of adsorbed proteins. At last, the biocompatibility of the sheared STMP/PVA 1:8 hydrogel was tested. The in vitro data of all the STMP/PVA hydrogels demonstrated their good biocompatibility, and indicated that the 1:8 sample was the most promising as vitreous body substitute.     

On the tissue compatibility of poly(ether imide) membranes: an in vitro study on their interaction with human dermal fibroblasts and keratinocytes

Recently we have developed a novel type of membrane based on poly(ether imide) (PEI) which is considered for biomedical application. To improve its physical and biological performance it was modified by blending with poly(benzimidazole) (PBI). In the present study both membranes were characterized in terms of their physicochemical properties and in vitro tissue compatibility using human dermal fibroblasts and keratinocytes. The modified membrane (PEI*) was more hydrophilic, less porous and had an increased surface (zeta) potential. We further found that blending with PBI tends to promote cell contact, at least initially, as indicated by the improved overall cell morphology, adhesion and spreading of fibroblasts, and the development of focal adhesion complexes. The effects of fibronectin (FN) and serum coating were also beneficial when compared to pure PEI and tissue culture polystyrene (TCP), which correlates to a higher adsorption of both FN and vitronectin detected by ELISA. However, a clear tendency for homotypic cellular interaction particularly of keratinocytes was obtained in contact with membranes, which was much stronger pronounced on PEI*. Although the initial adhesion was greater on PEI*, a surprising decrease in cell growth was observed at later stages of incubation, which may be explained with the membrane-promoted cellular aggregation leading to an easier detachment from the substratum. Thus, membranes based on blends of PEI with PBI could provide a tissue compatible scaffold with lowered adhesive properties, which might be a useful tool for the transfer of cells, for example, to in vitro engineered tissue constructs.     

In vitro toxicity of spiroorthocarbonate monomers designed for non-shrinking dental restoratives

In development of photopolymerized expanding monomers with epoxy resin systems, there is a need for reactive expanding monomers that exert a good biocompatibility profile. The objective of this study was to evaluate the in vitro toxicology of new spiroorthocarbonates designed to be expanding monomers. The expanding monomers investigated were: trans/trans-2,3,8,9- di(tetramethylene)-1,5,7,11-tetraoxaspiro[5,5]undecane (DTM-TOSU), 5,5-diethyl-19-oxadispiro[1,3-dioxane-2,2′-1,3-dioxane-5′,4-bi (DECHE-TOSU); 3,9-diethyl-3,9-dipropionyloxy methyl-1,5,7,11-tetraoxaspiro[5.5]undecane (DEDPM-TOSU); and 3,9-diethyl-3,9- diacetoxy methyl-1,5,7,11-tetraoxaspiro[5.5]undecane (DAMDE-TOSU). The in vitro toxicology of these monomers measured their cytotoxicity and mutagenicity potential. Succinic dehydrogenase (SDH) activity in the MTT assay was used to assess the toxic dose that kills 50% of cells (TC₅₀) for all the monomers. Their mutagenic potential was measured in the Ames Salmonella assay with and without metabolic activation. Two solvents, DMSO and acetone, were used to validate effects. Appropriate controls included the solvents alone. All the expanding monomers in this study were less cytotoxic than BISGMA (p < 0.01), a commercial component of dental restoratives. The relative cytotoxicity of the expanding monomers in DMSO was defined in the following order: DTM-TOSU (more toxic) > DECHE-TOSU > DEDPM-TOSU > DAMDE-TOSU. Each was significantly different from the other (p < 0.05). Overall, the TC₅₀ values of all expanding monomers were significantly greater in DMSO than in acetone (p < 0.05). However, for BISGMA this trend was opposite. For mutagenicity results, the expanding monomers were non-mutagenic and there was no solvent effect on this outcome. The non-mutagenicity and low cytotoxicity profile of these expanding monomers suggests their potential for development of biocompatible non-shrinking composites.     

乳腺癌间质成纤维细胞的平滑肌分化及意义

目的　观察乳腺癌间质成纤维细胞平滑肌分化情况 ,评价临床病理学意义。方法　对69例乳腺癌标本进行α SMA免疫组织化学染色 ,以 8例无癌区域乳腺组织 (距癌肿边缘 5cm以上 )为对照 ,分析浸润性乳腺癌间质成纤维细胞平滑肌分化与年龄、肿瘤大小、淋巴结转移、组织学分级和雌激素受体状态的关系。结果　无癌区域乳腺组织和原位癌间质无成纤维细胞平滑肌分化现象 ,5 5 .5 %的浸润性乳腺癌间质成纤维细胞有α SMA阳性表达 (P <0 .0 5 ) ;68.3 % ( 2 8/ 41)的淋巴结转移病例出现间质成纤维细胞平滑肌分化 ,且明显高于无淋巴结转移者 ( 2 6.3 % ,P <0 .0 5 ) ;组织学Ⅱ ,Ⅲ级的浸润性乳腺癌间质成纤维细胞平滑肌分化 ( 2 6/ 3 9)明显多于组织学Ⅰ级 ( 7/ 2 1,P <0 .0 5 )。结论　间质成纤维细胞平滑肌分化与乳腺癌的浸润和恶性程度有关 ,肌成纤维细胞可能在乳腺癌的浸润和转移中发挥重要的旁分泌作用。     

The Role of Stroma in Breast Carcinoma Growth In Vivo

The malignant progression of tumors is thoughtto be related to the expression of oncogenes and loss ofexpression of tumor suppressor gene. These factors areintrinsic to the cancer cells themselves. However, carcinomas are also infiltrated by host cells(fibroblasts, endothelial cells, inflammatory cells) andsurrounded by an extracellular matrix which isextensively remodeled. The extracellular matrixcomponents and infiltrating host cells provide amicroenvironment that conditions both tumor progressionand the metastatic process. Transplantation of humantumors into athymic nude mice has become an importantexperimental approach to study the biology of human cancers.The different models developed so far are beginning toelucidate the role of matrix molecules, growth factorsand enzymes as well as fibroblasts in tumor progression. These animal models are likely toprovide a useful tool to evaluate new antitumortreatments.     

酶消化组织块法原代培养人牙周膜成纤维细胞的初步研究

目的 提高人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPIF)原代培养的成功率,快速建立体外细胞研究模型。方法 采取组织块与酶消化相结合的方法处理牙周膜组织块,获取细胞;用形态学、免疫细胞化学染色等方法鉴定细胞来源,通过生长曲线的测定研究体外细胞生长特性。结果 原代培养成功率为77．8％。获得的细胞呈梭形,抗波形丝蛋白染色呈阳性,抗角蛋白染色呈阴性,符合人牙周膜成纤维细胞的形态学特征和生物学特性,生长良好。结论 酶消化组织块法可显著提高人牙周膜成纤维细胞原代培养成功率,方法可行。     

In Vitro Wound Repair by Human Gastric Fibroblasts: Implications for Ulcer Healing

Fibroblasts modulate epithelial biologicalactivities and play a key role in the ulcer healingprocess. There is no information regarding thebiological response of human gastric fibroblasts toregulatory compounds. The aim of this study was to assessthe effects of growth factors and prostaglandins on anin vitro model of human gastric fibroblast wound repair.Subconfluent fibroblast cultures were used to study proliferative responses, determined by[³H]thymidine incorporation into DNA. Invitro wound repair was determined in confluentfibroblast monolayers after mechanical denudation. Thepresence of putative growth factors secreted by fibroblastswas studied in conditioned medium by heparin-affinitychromatography and immunodetection with specificantibodies. Serum and platelet-derived growth factor (PDGF)-BB induced a dramatic increase in bothgastric fibroblast proliferation and closure of woundedcell monolayers, whereas these activities were inhibitedby both transforming growth factor(TGF)-₁ and prostaglandin E₁. Basalactivities in unstimulated gastric fibroblasts werelower than those obtained in skin fibroblasts.Conditioned medium stimulated fibroblast proliferationand wound repair activity, which was inhibited by the addition of suramin,and was partially dependent on the presence of PDGF-likefactor. PDGF is a major, autocrine promotor of humangastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins andTGF-. These findings might be important for futuretherapeutic ulcer healing approaches.     

Dextran derivatives modulate collagen matrix organization in dermal equivalent

Dextran derivatives can protect heparin binding growth factor implied in wound healing, such as transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2). The first aim of this study was to investigate the effect of these compounds on human dermal fibroblasts in culture with or without TGF-β1. Several dextran derivatives obtained by substitution of methylcarboxylate (MC), benzylamide (B) and sulphate (Su) groups were used to determine the effects of each compound on fibroblast growth in vitro. The data indicate that sulphate groups are essential to act on the fibroblast proliferation. The dextran derivative LS21 DMCBSu has been chosen to investigate its effect on dermal wound healing process. Fibroblasts cultured in collagenous matrices named dermal equivalent were treated with the bioactive polymer alone or associated to TGF-β1 or FGF-2. Cross-sections of dermal equivalent observed by histology or immunohistochemistry, demonstrated that the bioactive polymer accelerates the collagen matrices organization and stimulates the human type-III collagen expression. This bioactive polymer induces apoptosis of myofibroblast, property which may be beneficial in treatment of hypertrophic scar. Culture media analyzed by zymography and Western blot showed that this polymer significantly increases the secretion of zymogen and active form of matrix metalloproteinase-2 (MMP-2), involved in granulation tissue formation. These data suggest that this bioactive polymer has properties which may be beneficial in the treatment of wound healing.     

Inorganic/Organic Biocomposite Cryogels for Regeneration of Bony Tissues

The present work focuses on the physical, mechanical and in vitro properties of porous inorganic/organic biocomposite scaffolds of polyvinyl alcohol–tetraethylorthosilicate–alginate–calcium oxide (PTAC). These scaffolds are prepared by means of cryogelation technology and are intended for bone tissue engineering applications. The biocomposite cryogels have much more favorable physical and biological properties compared to the previous work of our group on the same composition in the form of pellets and foams. The optimized and heat-treated PTAC biocomposite cryogels show homogenous porosity and good mechanical properties and also exhibit the formation of a hydroxyapatite-like layer on their surface on coming in contact with simulated body fluid (SBF). Furthermore, the biocomposite cryogels showed good biocompatibility with L929 fibroblasts. Also, the influence of pre-soaking in SBF to that of non-soaked scaffolds was compared in terms of proliferation of MG-63 osteoblast-like osteosarcoma cells on these scaffolds and it was found that the pre-soaking caused a decrease in cell proliferation. Finally, the response of human osteoblasts on these scaffolds was analyzed by MTT assay, scanning electron microscopy, energy dispersive X-ray spectroscopy and micro X-ray computing tomography. The cells revealed good biocompatibility with the biocomposite cryogels and were mostly present as cell sheets on the surface with thick bundles of collagenous extracellular matrix during initial period of incubation. During later phases, the formation of calcium phosphate-like mineral deposits was observed on the surface of the cryogels suggesting a high potential of the biocomposite cryogels towards bone regeneration. Therefore, the PTAC biocomposite cryogels, due to their favorable properties and high biocompatibility with human osteoblasts can be suggested as potential scaffolds for bone tissue engineering applications.     

Evaluation of functions and tissue compatibility of poly (D,L-lactic-co-glycolic acid) seeded with human dermal fibroblasts

In tissue engineering and wound-healing applications, dermal substitutes are used to provide fibroblasts with the mechanical support for their growth and then to facilitate the skin formation. In this study, three-dimensional porous poly(lactic-co-glycolic acid) (PLGA) 65/35 scaffolds were prepared and then the composites of the scaffolds and human fetal dermal fibroblasts were fabricated as a tissue-engineered dermal substitute. The function and tissue compatibility of the artificial dermal substitute were evaluated at the levels of gene expression (by RT-PCR) and protein expression (total collagen quantities), as well as by histological and immunohistochemical analysis. The PCR products indicated that the mRNA of type-I collagen, mainly secreted by the fibroblasts onto the PLGA scaffolds, was clearly expressed after 4 weeks. The amount of total collagen synthesized from the cells was shown to increase gradually during the initial culture period and slightly decreased afterwards. After 8 weeks of culture, the fibroblasts were well attached and migrated entirely throughout the pores of the PLGA scaffold with normal function. Furthermore, the positively stained type-I collagen was intensively detected throughout the pores. These results suggest that the function and tissue compatibility may be important criteria in evaluating an artificial tissue-engineered skin.