An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse

The revascularization of human skin transplanted onto the nude mouse was studied by performing double-labeling immunofluorescence microscopy on human skin before transplantation and at different stages, ranging from one week to six months after grafting. With a crossreacting anti-factor-VIII antigen antibody used to identify the endothelial cells, and with human-specific monoclonal antibodies directed against vimentin or HLA-DR antigen, it appeared that the original human endothelial cells of transplanted skin progressively disappear, while murine endothelial cells invade the graft. Moreover, in double-labeling experiments with a crossreacting anti-factor-VIII antibody and a human-specific anti-type-IV-collagen antibody, anastomosis between host and graft vessels and a constant codistribution of graft endothelial cells with human type IV collagen were observed. Finally, double staining with species-specific antibodies directed against murine or human type IV collagen showed that mouse type IV collagen appeared progressively in the graft and was constantly colocalized with human type IV collagen. From these observations, it was concluded that revascularization of human skin transplanted onto the nude mouse proceeds as follows: inoculation; disappearance of human endothelial cells and migration of mouse endothelial cells into the graft over the basement membrane of preexisting human vessels; and production of a new vascular basement membrane by mouse endothelial cells on the original basement membrane of human graft vessels.     

含鼠胚胎成纤维细胞的组织工程皮肤体外构建及大鼠移植研究

目的 利用鼠胚胎成纤维细胞(mouse embryonic fibrobalsts,MEFs)三维培养修复大鼠全层皮肤缺损,并初步探讨其机制.方法 培养MEFs和普通成纤维细胞(fibroblasts,FBs),复合鼠尾胶原形成三维构建,ELASA法测定其分泌功能.制作大鼠全层皮肤缺损,按移植物不同分为3组:MEFs组(实验组)、FBs组(对照组1)和空白胶原组(对照组2).观察愈合时间并计算愈合面积比率,定期取创面组织行CD31、波形蛋白免疫组化检测,并以Hoechst33342荧光标记MEFs,示踪其转归.结果 与FBs组相比,MEFs组的IL-6分泌更加旺盛,而TGF-β1分泌量少(P＜0.05).实验组创面愈合快(P＜0.05),微血管密度高(P＜0.05),创面中FBs排列有序,移植的MEFs随时间减少.结论 MEFs三维构建可加速创面愈合,减轻瘢痕形成,其机制可能与胚胎细胞对创面愈合的诱导有关.

Abstract：

Objective To investigate the application and mechanism of tissue-engineered skin with mouse embryonic fibroblasts(MEFs)for the full-thickness skin defects on mice.Methods The MEFs and fibroblasts were cultured and seeded in scaffold made of rat tail collage.ELISA method was used for detection of secretory function.The full-thickness skin defects were created on mice and covered by MEFsscaffold complex(experimental group),or FBs-scaffold complex(control group 1),or scaffold only(control group 2).The process of wound healing was evaluated by observation of the re-epithelization rate.Microvessal density(MVD)and vimentin within the wound sites were also detected with immunohistochemistry staining technique to describe the characteristics of wound healing.Hoechst 33342 staining was performed to trace MEFs'fate.Results MEFs scaffold group had higher level secretion of IL-6 and lower of TGF-β1 than FBs scaffold group(P＜0.05).Compared with wounds in control groups,the wounds in MEFs group healed markedly fast(P＜0.05)and the MVD was significantly higher(P＜0.05).The fibroblasts in the wounds of MEFs group were arranged regularly and the MEFs decreased during the healing process.Conclusions The MEFs-scaffold complex can promote wound healing with less scar formation.MEFs may have an inducing effect on the wound healing.     

Free transplantation of skin onto the face

Basing on their experiences the authors describe principal technical methods of free transplantation of skin in different areas of the face. Their personal observations are used as illustrations.     

The biology of human malignant melanoma. Preliminary findings in the nude mouse.

The nude mouse model was explored as a means of predicting the behavior of human cutaneous malignant melanoma. Subcutaneous implants from 20 patients were observed for external growth, microscopic appearance, and autoradiographic analyses following tritiated thymidine labeling. Normal epithelial and dermal components were maintained in the six low-risk primary implants, but none had any external growth or tumor cell preservation. Four of the five metastatic implants grew throughout the observation periods, with 30% of the counted tumor cells taking up the radioisotope. There was a slower growth rate in four of nine high-risk primary implants, with tumor cell preservation in eight. Only 6% of the counted tumor cells incorporated tritiated thymidine. This model appears to be capable of discriminating among the behavior of various forms of human malignant melanoma.     

Interleukin-10 suppresses proliferation and remodeling of extracellular matrix of cultured human skin fibroblasts

When we previously examined the participation of local expression of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNFalpha) in wound healing of an intestinal anastomosis under septic conditions in mice, we found that IL-10 and TNFalpha expressions were markedly enhanced around the anastomosis and that wound healing was impaired in this animal model. The purpose of the present study was to investigate the combined effect of IL-10 on proliferation and remodeling of the extracellular matrix (ECM) of cultured human skin fibroblasts. Human skin fibroblasts were cultured for 48 h with IL-10 and/or TNFalpha at various concentrations, then the proliferation rates were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The concentration of transforming growth factor-beta1 (TGFbeta1) in cell culture supernatants was measured by enzyme-linked immunosorbent assay, and type I collagen protein and matrix metalloproteinase-I (MMP-I) were detected by indirect immunofluorescence in cultured cells incubated for 48 h with 10 ng/ml of IL-10 and/or 10 ng/ml of TNFalpha. IL-10 itself had no effect on fibroblast proliferation, but reduced TNFalpha-induced fibroblast proliferation. The concentration of TGFbeta1 in cell culture supernatants was significantly lower in the presence of TNFalpha and IL-10 than in the presence of TNFalpha alone. Immunolabeling of fibroblasts for type I collagen protein was decreased in cells incubated with IL-10 and/or TNFalpha compared to controls. MMP-I immunolabeling was increased in cells incubated with IL-10, IL-10 and TNFalpha compared to control and cells incubated with TNFalpha. It is suggested that IL-10 is an inhibitory factor for the remodeling of the ECM during wound healing.     

Novel model for evaluation of epidermal preservation and dermal collagen remodeling following photorejuvenation of human skin

BACKGROUND AND OBJECTIVES: In order to optimize photorejuvenation of human skin, a method must be developed to reliably compare the potential for epidermal preservation and dermal fibroblast stimulation of different laser devices and irradiation parameters. We describe a novel human skin tissue culture model developed for this purpose. MATERIALS AND METHODS: An artificial skin model, consisting of human keratinocytes in the epidermis and human fibroblasts and rat-tail collagen in the dermis, was cultured using the floating collagen gel (RAFT) method. Repetitive low-fluence Er:YAG laser irradiation was applied to test the applicability of our RAFT model for characterization of epidermal preservation and dermal fibroblast stimulation post-laser treatment. RESULTS: Histopathologic evaluation revealed a thin layer of epidermal keratinocyte preservation immediately after low fluence sub-ablative Er:YAG laser irradiation. One-week post-laser irradiation, the average increase in number of dermal fibroblasts as compared to control was statistically significant (P < 0.01). CONCLUSIONS: The RAFT model can be used to assess the potential for epidermal preservation and dermal fibroblast stimulation of different photorejuvenation devices and irradiation parameters and offers several advantages over traditional animal and human skin models.     

The role of altered fatty acid in pathological scars and their dermal fibroblasts

PurposeThe proposed pathological mechanism for scar formation is controversial, and increased attention has been paid to the fatty acids (FAs) in the formation of pathological scars. Notably, FAs are known to be important in inflammation and mechanotransduction, which is closely related to scar formation. Therefore, it is necessary to clarify the roles of FA in scar formation.MethodsHypertrophic scar and keloid formed for more than a year and without other treatment, as well as normal skin samples were obtained from patients who underwent plastic surgery. Finally, keloids (n = 10), hypertrophic scars (n = 10), and normal skin samples (n = 10) were collected under informed consent. Primary dermal fibroblasts were isolated and cultured. The amount and variety of FAs were detected by lipid chromatography-mass spectrometry. Immunohistochemistry, real-time PCR, and western blotting were used to verify the expression of sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FASN) in the samples and their fibroblasts. Student's t-test, ANOVA, and orthogonal partial least square discriminant analysis were performed for statistical analysis (1p < 0.05, 7p < 0.01, 71p < 0.001, 77p < 0.0001).ResultsCompared with full-thickness normal skin, there were 27 differential FAs in keloids and 15 differential FAs in hypertrophic scars (1p < 0.05 and variable influence on projection >1.0). The expression of SREBP1 and FASN was lower in pathological scars both at mRNA and protein levels (all 1p < 0.05). However, the mRNA levels of SREBP1 (71p = 0.0002) and FASN (71p = 0.0021) in keloid-derived fibroblasts were higher than that in normal skin fibroblasts (NFBs), while the expression in hypertrophic scar-derived fibroblasts was lower than that in NFBs (both 1p < 0.05). Whereas there was no significant difference in FASN protein expression between keloid-derived fibroblasts and NFBs (p > 0.05).ConclusionFAs involved in pathological scars are abnormally changed in scar formation. Thus, fatty acid-derived inflammation and de novo synthesis pathway of FA may play a key role in the formation of pathological scars.     

Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte-fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.     

皮肤移植免疫研究

Skin grafting has been one of the most important approaches for covering burn wounds, however long-term survival of allogeneie or xenogeneic skin graft is currently not successful. How to induce immune tolerance for life-time survival of allogeneic or xenogeneie skin graft is still remote objective to be solved. However, clinicians and scientists in China have worked very hard and made great contribution to this field during the past 50 years, no matter how diffieult it is. They are the re spected pioneers in the understanding of immunological change in "Chinese Method" skin grafting, its local immune tolerance, immunology of pre-treatment of skin graft, etc. Herein, the most outstanding and impressive progresses in immunological responses after skin grafting in the past 50 years in China have been re viewed and presented for memory, for future and for extanding a salute.     

Collagen-dependent growth suppression and changes in the shape of human dermal fibroblasts

This study was designed to investigate the effects of collagen on the growth and cellular shape of cultured human fibroblasts. The fibroblasts grown in the presence of collagen assumed a slim, rod-like form with abundant cytoplasmic protrusions, while the cells on a plain dish showed flat forms with fewer cytoplasmic protrusions. Incorporation of [3H]thymidine was remarkably suppressed in the cells grown on collagen when compared with the fibroblasts on the plain dish. Specific cellular shape and suppressed DNA synthesis showed dependence on the steric conformation of native collagen, because collagen-specific effects on fibroblasts were completely abolished when 60% of the helix content was lost by heat denaturation. These collagen-dependent features of the cells disappeared when the collagen-containing medium was treated with bacterial collagenase. Growth of fibroblasts in contact with collagen was suppressed as compared to that on the plastic dish. These results indicate the importance of the native conformation of collagen in the interaction of collagen and fibroblasts.     

Host-donor interactions in healing of human split-thickness skin grafts onto nude mice: in situ hybridization, immunohistochemical, and histochemical studies.

The behavior of host and donor cell lines in human split-thickness skin grafts onto nude mice was studied by in situ hybridization (ISH) using genomic DNAs as probes, and immunohistochemically with species-specific or cross-species specific antibodies, at different stages ranging from day 3 to more than 1 year following grafting. Changes in the graft vascular and interstitial extracellular matrix were also assessed using species-specific or cross-species specific antibodies to human or murine type I, III, and IV collagens. Finally, transplant reinnervation was investigated using antibodies to various nerve cytoplasmic antigens and the thiocholine method to demonstrate acetylcholinesterase. Using these methods we were able to show the following: (1) the graft epidermis that is not replaced by mouse keratinocytes is progressively colonized by recipient Langerhans cells (LCs); (2) revascularization of the grafts begins soon by inoculation of the graft vessels with the host microcirculatory bed, and mouse endothelial cells growing into preexisting human capillary tubes produce a new basement membrane, prior to the replacement of the original one; (3) within 3-5 days following grafting, mouse fibroblasts migrate into the graft dermis. The density of the human and murine fibroblast populations then progressively increases. Characterization of the interstitial collagens identifies both human and murine type I and III collagens. Production of type III collagens decreases during the progression of fibrogenesis while human type I collagen becomes the predominant matrix protein; (4) transplant reinnervation is deficient, and neurites growing into severed graft nerve trunks were never detected.     

Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation

BACKGROUND CONTEXT

Increasing evidence suggests transplanting viable cells into the degenerating intervertebral disc (IVD) may be effective in treating disc degeneration and back pain. Clinical studies utilizing autologous or allogeneic mesenchymal stem cells to treat patients with back pain have reported some encouraging results. Animal studies have shown that cells injected into the disc can survive for months and have regenerative effects. Studies to determine the advantages and disadvantages of cell types and sources for therapy are needed.

Grafting of large pieces of human reconstructed skin in a porcine model.

Present techniques can save about 25% of patients burnt over more than 90% of their body surface. However, problems of functional and aesthetic repair arise, which are often resolved only by major therapeutic procedures. Current advances in skin substitutes permit the cultivation, from a skin biopsy, of large surfaces of in vitro human reconstructed skin (HRS). Our model, obtained by the co-culture of fibroblasts and keratinocytes on a dermal substrate composed of collagen-glycosaminoglycan-chitosan, reproduces, in vitro, a tissue close to human skin, which could play a role in reconstructive surgery. The objectives of this experiment were to assess whether it is possible to perform large HRS grafts and to evaluate the preliminary cosmetic results. We used four immunosuppressed female pigs. Full-thickness skin resections of 50-100 cm(2)were performed on the dorsa of the animals. The defects were grafted with between one and six pieces of HRS under tied-over dressings. At day 14, we found a soft and smooth surface of good transparent healthy pink skin, which was very easy to distinguish from the surrounding tissues. The junctions between different pieces of living skin were not visible. Immunohistological studies with specific anti-human keratin 14 antibodies confirmed the graft take: 7 days after grafting the human epidermis was attached to the living dermis and showed good organisation with a basal cell layer and suprabasal cells; 28 days after grafting the human epidermis seemed to be replaced by pig epidermis. This study highlights the possibility of grafting large surfaces with HRS using a routine operating technique.     

人膀胱癌裸鼠移植瘤模型的建立及其血管生成的观察

目的:观察人膀胱癌裸鼠移植瘤模型的病理形态特征和血管生成情况,并为今后的研究奠定基础。方法:常规培养T24细胞,通过皮下接种建立人膀胱癌裸鼠移植肿瘤模型,并于肿瘤生长至直径0.2~0.3cm、0.5~0.8cm、1.0~1.5cm时切除肿瘤,利用HE染色观察肿瘤病理形态,利用免疫组织化学技术标记CD31阳性细胞进行微血管计数。结果:动物接种后7~10天可见肿瘤生长,4周时肿瘤直径可达1cm以上,此后肿瘤生长速度下降。切片HE染色可见肿瘤形态与人体肿瘤存在一定差异;较大肿瘤表面可见明显的血管增生及中心坏死液化。直径0.2~0.3cm肿瘤微血管密度(Microvessel density,MVD)小于较大体积肿瘤(P<0.01)。结论:动物模型肿瘤与人体肿瘤之间尚存在一定差异,有待深入研究,使其能更好地模拟人体肿瘤。本研究结果为后续研究奠定了实验基础。     

Long-term remodeling of a bilayered living human skin equivalent (Apligraf®) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix

Type I collagen is a clinically approved biomaterial largely used in tissue engineering. It acts as a regenerative template in which the implanted collagen is progressively degraded and replaced by new cell-synthesized tissue. Apligraf, a bioengineered living skin, is composed of a bovine collagen lattice containing living human fibroblasts overlaid with a fully differentiated epithelium made of human keratinocytes. To investigate its progressive remodeling, athymic mice were grafted and the cellular and the extracellular matrix components were studied from 0 to 365 days after grafting. Biopsies were analyzed using immunohistochemistry with species-specific antibodies and electron microscopy techniques. We observed that this bioengineered tissue provided living and bioactive cells to the wound site up to 1 year after grafting. The graft was rapidly incorporated within the host tissue and the bovine collagen present in the graft was progressively replaced by human and mouse collagens. A normal healing process was observed, i.e., type III collagen appeared transiently with type I collagen, the major collagen isoform present at later stages. New molecules, such as elastin, were produced by the living human cells contained within the graft. This animal model combined with species-specific immunohistochemistry tools is thus very useful for studying long-term tissue remodeling of bioengineered living tissues.     

Effect of hyperbaric oxygen on human skin cells in culture and in human dermal and skin equivalents

A critical stage of cutaneous wound healing is the development and maturation of the epidermis. In the aged, and in certain pathologies, this repair process is compromised due to a variety of deficiencies, one of which is tissue oxygenation. Several phases of wound healing are dependent on adequate tissue oxygen levels, and hyperbaric oxygenation has been shown to transiently elevate these levels. The use of human cell monolayers, dermal equivalents and human skin equivalents provide excellent opportunities for studying wound healing using in vivo relevant models. The goal of this study was to examine the effect of hyperbaric oxygen on cell proliferation, differentiation, and matrix biosynthesis in monolayer cultures and epidermopoiesis in the developing skin equivalent. Normal human dermal fibroblasts, keratinocytes and melanocytes, dermal equivalents and skin equivalents were exposed to hyperbaric oxygen at pressures up to three atmospheres, for up to 10 consecutive daily treatments lasting 90 minutes each. Increase in fibroblast proliferation (cf., 30% at 1 atmosphere after 10 days treatment), was observed without a significant effect on proliferation of normal human melanocytes and glycosaminoglycan synthesis. Stimulation of collagen synthesis after two days of treatment was only significant at 1 atmosphere (about 20% increase) but this differential was not observed after 5 days of treatment. Hyperbaric oxygenation above 2 atmospheres, inhibited proliferation of fibroblasts and keratinocytes in cell monolayer cultures (e.g., a 10 day treatment at 3 atmospheres appeared cytostatic to keratinocytes). In contrast, hyperbaric treatment up to 3 atmospheres dramatically enhanced keratinocyte differentiation, and epidermopoiesis in the complete human skin equivalent. These results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue. The results also show the utility of the human skin equivalent as a model for evaluation of parameters involved in wound healing.     

含表皮细胞和成纤维细胞的复合皮构建及移植实验

目的 探讨成纤维细胞在构建复合皮中的作用，并观察复合皮对全层皮肤缺损创面的修复效果。方法 将表皮细胞与成纤维细胞种植于无细胞真皮替代物表面，于体外培养构建复合皮，观察种植成纤维细胞对表皮细胞与无细胞真皮替代物间粘附性的影响。然后将复合皮移植于裸鼠（16只）全层皮肤缺损创面，观察存活率及新生皮肤组织结构。结果 表皮细胞种植于无细胞真皮表面可形成复合皮，当种植少量成纤维细胞修饰无细胞真皮表皮面后，表皮细胞与真皮基质粘附紧密，在移植等操作过程中，表皮细胞膜片不易脱落。复合皮移植可封闭全层皮肤缺损创面，完全存活者最高达10只（62.5%)，新生皮肤基底膜结构完整，可见层粘连蛋白、Ⅳ型胶原形成。结论 以无细胞真皮替代物为载体的复合皮可修复全层皮肤缺损创面，种植成纤维细胞可增强表皮细胞的粘附性，有利于移植存活率的提高。