Endogenous lectins (galectins-1 and -3) as probes to detect differentiation-dependent alterations in human squamous cell carcinomas of the oropharynx and larynx

Expression of glycan determinants for in situ binding is the prerequisite for a productive protein (lectin)-carbohydrate recognition. Labeled tissue lectins as tools are preferable to plant lectins to assess this parameter, because plant and animal lectins with identical saccharide specification can well differ in their profiles of oligosaccharide binding pattern. Due to their relevance in growth control and matrix adhesion the family of galectins (galactoside-binding metal ion independent animal lectins) is receiving increasing utilization in human biology. Employing biotinylated galectin-1 and galectin-3 we studied the expression of binding sites for these galectins in normal human squamous epithelium and human carcinomas from the oropharyngeal region and larynx in relation to the expression of LP-34+ cytokeratins by the procedure of double labeling. Tissue sites accessible for galectin-1 were located in all layers of normal epithelium and in tumor cells. In contrast, galectin-3 binding was suprabasal in the normal epithelium and in tumor cells exhibiting signs of keratinization. These results reveal differences in the localization of accessible sites for the two galectins. Relating to cell development galectin-3 appeared to display affinity to areas with increased extent of differentiation.     

Regulatory mechanisms of galectin-9 and eotaxin-3 synthesis in epidermal keratinocytes: possible involvement of galectin-9 in dermal eosinophilia of Th1-polarized skin inflammation

BACKGROUND: Skin eosinophilia is a common feature of allergic skin diseases, but it is unclear how epidermal and dermal eosinophil infiltration is controlled. To investigate regulation of localization of eosinophils in skin, we examined the regulatory mechanisms of expression of eosinophil-specific chemoattractants in dermal fibroblasts and epidermal keratinocytes. METHODS: We analyzed production of eotaxin, eotaxin-2, eotaxin-3 and galectin-9 by dermal fibroblasts and epidermal keratinocytes in response to several stimuli in vitro. RESULTS: Dermal fibroblasts produced eotaxin and eotaxin-3 in response to stimulation by interleukin (IL)-4 and/or tumor necrosis factor-alpha. Similarly, IL-4 stimulated epidermal keratinocytes to secrete eotaxin-3. However, we did not detect eotaxin mRNA expression or protein secretion by keratinocytes stimulated in vitro. Interferon (IFN)-gamma induced galectin-9 expression on dermal fibroblasts. Conversely, expression of galectin-9 on epidermal keratinocytes was dose-dependently inhibited by IFN-gamma. The immunohistochemical assays revealed that dermal fibroblasts (but not epidermal keratinocytes) in the lesional skin of psoriasis vulgaris (a Th1-polarized disease) express significant levels of galectin-9. CONCLUSION: Eotaxin-3 contributes to dermal and epidermal eosinophil infiltration in Th2-polarized skin inflammation in which IL-4 is produced. In contrast, IFN-gamma-dominated inflammation appears to mediate eosinophil extravasation into the dermis and eosinophil adhesion to dermal fibroblasts via galectin-9 in association with decreased chemoattractant activity of epidermal galectin-9. The present results reveal a novel mechanism of dermal eosinophilia in IFN-gamma-mediated skin inflammation, and reflect concerted chemoattractant production involving dermal and/or epidermal eosinophilia during changes in the local cytokine profile.     

脱细胞异体真皮基质与人脂肪干细胞的生物相容性

背景：脱细胞异体真皮基质具有优良的生物相容性和组织细胞诱导功能。 目的：评价人脂肪干细胞与脱细胞异体真皮基质的生物相容性。 方法：取健康成年人吸脂术后的脂肪组织分离脂肪干细胞,并行原代与传代培养,传至第3代,将细胞与脱细胞异体真皮基质联合体外培养3,7 d,倒置相差显微镜和扫描电镜观察细胞在支架材料上的黏附、生长及增殖情况,并计算细胞在材料上的黏附率;XTT比色法检测细胞的生长增殖情况。 结果与结论：脂肪干细胞在支架材料上分布均匀,24 h内细胞开始伸展、黏附,二三天完全伸展变形,以梭形为主,呈网状排列;随着培养时间延长,支架上的细胞逐渐增多;人脂肪干细胞细胞与脱细胞异体真皮基质混合培养后平均黏附率为95.03%,并保持正常的生长增殖速度,表明支架对细胞具有良好的黏附性;脱细胞异体真皮基质材料与人脂肪干细胞复合后相容性良好。     

Primary human dermal fibroblast interactions with open weave three-dimensional scaffolds prepared from synthetic human elastin

We present an elastic, fibrous human protein-based and cell-interactive dermal substitute scaffold based on synthetic human elastin. Recombinant human tropoelastin promoted primary human dermal fibroblast attachment, spreading and proliferation. Tropoelastin was cross-linked to form a synthetic elastin (SE) hydrogel matrix and electrospun into fibrous SE scaffolds. Fibroblasts attached to and proliferated across SE hydrogel scaffold surfaces for at least 14 days and deposited the extracellular matrix proteins fibronectin and collagen type I. To allow for the benefit of greater cell infiltration, SE was electrospun into open weave, fibrous scaffolds that closely mimic the fibrous nature of the skin dermis. 3D SE scaffolds were robust and consisted of flat, ribbon-like fibers with widths that are similar to native dermal elastic fibers. The scaffolds displayed elasticity close to that of natural elastin. 3D SE retained the ability to interact with primary human dermal fibroblasts, which consistently attached and proliferated to form monolayers spanning the entire scaffold surface. The open weave design, with larger spaces between individual fibers and greater fiber diameters beneficially allowed for substantial cell infiltration throughout the scaffolds.     

An in vitro examination of an extracellular matrix scaffold for use in wound healing

This paper describes evidence that an extracellular matrix (ECM) secreted by human umbilical vein endothelial cells (HUVECs) assembled on gelatin coated plates overlaid by a mixed matrix secreted by human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts provides a viable acellular scaffold for use in wound healing. Trypsinized epidermal keratinocytes or colonies from Dispase-digested fresh and cadaver skin tissue adhered and proliferated on either HUVECs ECM/gelatin or mixed matrix overlaid on HUVECs ECM/gelatin. An epithelial-mesenchymal interaction, previously thought to be tissue-specific, was exposed as well as concomitant integrin versatility. Furthermore, heterologous HDMECs and dermal fibroblasts attached and proliferated on the mixed matrix as well as HUVECs ECM. The conditioned medium from HUVECs (HUVECs CM) was found to neutralize the lingering after effects of Dispase, and could be used for the tissue culture of epidermal keratinocytes, HDMECs and dermal fibroblasts, which share related extracellular secretions. Taken together, these results indicate that cultured epithelial autografts can be redesigned to include both epithelial and dermal elements, and advances the acellular 'sandwich' ECM scaffold as a possible structural replacement for the lamina densa and lamina lucida, damaged or completely missing in some wounds and burns.     

Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration

In order to obtain an abundant supply of autologous dermal fibroblasts for the manufacture of engineered autologous dermal substitutes, we fabricated the micronized acellular dermal matrix (MADM) microcarriers and expanded human fibroblasts on them. This novel approach eliminated the need for the repeated trypsinizations that may disrupt cell–extracellular matrix interactions and impair cell viability. This cell expansion protocol simultaneously formed an engineered particulate dermal substitute (EPDS) avoiding cell reseeded on the scaffolds process. We further tested its feasibility and effectiveness in athymic murine subcutaneous injection and full-thickness cutaneous wound model. Our results showed that MADM microcarriers retained the ultrastructure of the acellular dermal matrix, had good biocompatibility, and supported human fibroblast expansion either as a direct culture substrate or through culturing cells in conditioned medium prepared from them. In the animal study, EPDS formed a thick layer of tissue below the subcutaneous muscle tissue at 3 weeks following EPDS injection into subcutaneous tissue. In full-thickness cutaneous wound, the degree of wound healing with EPDS implantation was better than that without EPDS although healing rates were not significantly different between wounds implanted with or without EPDS. This demonstrates the potential utility of MADM not only as a cell culture substrate to expand fibroblasts but also as a cell transplantation vehicle for skin regeneration, with several advantages over current expansion–transplantation protocols for skin regeneration. In addition, EPDS may be used for cosmetic or reconstructive soft tissue augmentation in a minimally invasive fashion.     

IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes

Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.     

Evaluation of ultra-thin poly(epsilon-caprolactone) films for tissue-engineered skin

Various natural and synthetic polymeric materials have been used as scaffold matrices for tissue-engineered skin. However, the commercially available skin replacement products pose problems of poor mechanical properties and immunological rejection. We have thus developed a film of 5 microm thickness, via biaxial stretching of poly(epsilon-caprolactone) (PCL), as a potential matrix for living skin replacements. The aim of this study was to evaluate the feasibility of using biaxially stretched PCL films as matrices for culturing human dermal fibroblasts. For this purpose, we cultured human dermal fibroblasts for 7 days on the films. Glass cover slips and polyurethane (PU) sheets were used as controls. The data from phase contrast light, confocal laser, and scanning electron microscopy suggested that biaxially stretched PCL films support the attachment and proliferation of human dermal fibroblasts. Thymidine-labeling results showed quantitatively that cell proliferation on the PCL films was superior to that on the PU samples. These results indicated that biaxially stretched PCL films supported the growth of human dermal fibroblasts and might have potential to be applied in tissue engineering a dermal equivalent or skin graft.     

Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.     

Dermal fibroblasts cultured on small intestinal submucosa: Conditions for the formation of a neotissue

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial that has been used extensively as a soft tissue replacement, as a scaffold for tissue engineering, and as a substrate for the study of cells in 3D culture. The aim of this study is to define culture parameters that promote neotissue formation with the use of dermal fibroblasts and SIS. SIS sheets were seeded with dermal fibroblasts and cultured for 4 weeks. The resultant cell-scaffold composites (CSCs) were cultured with media alone, media supplemented with ascorbic acid, or fibronectin-pretreated SIS and ascorbic acid. CSCs were analyzed for cellular invasion into the scaffold, the rate of type I collagen production, MMP gelatinolytic activity, thickness, and ultrastructural morphology. CSCs treated with fibronectin and ascorbate showed an increase in Type I collagen production, no change in the MMP gelatinolytic activity, an increase in CSC thickness, and an organized neotissue on the surface of the SIS. Minimal cellular invasion was noted, suggesting that fibroblasts use the SIS as a template for neotissue growth rather than as a scaffold. These results indicate that fibronectin-treated SIS cultured with dermal fibroblasts in the presence of ascorbic acid will promote true neotissue formation for future cardiovascular tissue engineering efforts.     

A two-component pre-seeded dermal–epidermal scaffold

We have developed a bilayered dermal–epidermal scaffold for application in the treatment of full-thickness skin defects. The dermal component gels in situ and adapts to the lesion shape, delivering human dermal fibroblasts in a matrix of fibrin and cross-linked hyaluronic acid modified with a cell adhesion-promoting peptide. Fibroblasts were able to form a tridimensional matrix due to material features such as tailored mechanical properties, presence of protease-degradable elements and cell-binding ligands. The epidermal component is a robust membrane containing cross-linked hyaluronic acid and poly-l-lysine, on which keratinocytes were able to attach and to form a monolayer. Amine–aldehyde bonding at the interface between the two components allows the formation of a tightly bound composite scaffold. Both parts of the scaffold were designed to provide cell-type-specific cues to allow for cell proliferation and form a construct that mimics the skin environment.     

Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.     

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-beta1 (TGF-beta1) 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-beta1. 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-beta1 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.     

Biodegradable honeycomb collagen scaffold for dermal tissue engineering

Tissue engineering requires a mechanically stable, biocompatible, and biodegradable scaffold that permits cell adherence and proliferation, allows preservation of cell-specific properties, and suitable for surgical implantations. In this study, honeycomb collagen sheet was used for three-dimensional (3D) cultures of human skin fibroblasts and characterized as an effective and suitable scaffold for dermal tissue engineering. About 1-mm-thick honeycomb collagen sheets, prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation, and sterilized by heat, were placed on the proliferating fibroblasts on day 3 of the culture. The cells attached quickly to the collagen scaffold, proliferated inside the honeycomb pores, and formed a structure similar to dermis within 60 days. On day 60, total cellular DNA content of the 3D cultures was 12-fold higher when compared with the 2D control cultures without the scaffold. Measurement of procollagen type I in the media demonstrated a 20-fold increase. Scanning electron microscopy of the 3D cultures showed a well-formed structure similar to dermis and biodegradation of the honeycomb collagen scaffold. Our study proved that honeycomb collagen sheet is a mechanically stable, biocompatible and biodegradable scaffold for dermal tissue engineering, and also potentially useful for other cell-based therapies and tissue engineering applications.     

Transdifferentiation of Human Dermal Fibroblasts to Smooth Muscle‐Like Cells to Study the Effect of MYH11 and ACTA2 Mutations in Aortic Aneurysms

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC‐like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC‐like cells within a 2‐week duration using 5 ng/ml TGFβ1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC‐like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.     

In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.     

Interaction of dermal fibroblasts with electrospun composite polymer scaffolds prepared from dextran and poly lactide-co-glycolide

A highly porous electrospun scaffold was prepared by physically blending Dextran and poly lactide-co-glycolide (PLGA). The interaction between dermal fibroblasts and the Dextran/PLGA scaffold such as viability, proliferation, attachment, migration, extracellular matrix deposition, cytoskeleton organization and the functional gene expressions were characterized. The results indicated that cells interacted favorably with the scaffold. Moreover, cells migrated into the highly porous three dimensional matrix of the scaffold and organized into dense multi-layered structures that resembled dermal structure. The results of collagen gel assay also revealed that gel contraction was enhanced by the presence of the scaffold. The additional mechanical strength provided by the scaffold could enhance the binding of the seeded fibroblasts. These findings suggested that Dextran/PLGA scaffold can potentially be useful in enhancing the healing of chronic or trauma wounds.