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1.
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.  相似文献   

2.
This study investigated the biological response of fibroblasts cultured from uninjured skin and granulation tissue from different stages of healing wounds to the three isoforms of platelet-derived growth factor. Fibroblasts were derived by explant culture from the skin or the granulation tissue that formed within open mesh nylon Schilling-Hunt chambers (postoperative days 10, 20, 30, and 50) which had been implanted subcutaneously in the backs of domestic pigs. Cells were cultured under identical conditions in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Mitogenic activity was measured with (3)H-thymidine incorporation into DNA. Fibroblasts from normal skin responded equally well to all of the platelet-derived growth factor isoforms in the mitogenic assays. All of the wound fibroblasts exhibited a decreased response to platelet-derived growth factor compared with those from skin. Granulation tissue fibroblasts responded to platelet-derived growth factor BB, less to platelet-derived growth factor AB, and poorly to platelet-derived growth factor AA. These results correlated with a significantly decreased growth rate of fibroblasts in culture from both 30- and 50-day postsurgical wound tissue compared with normal skin. Western blot studies of cell membrane extracts showed that wound fibroblasts contained less than 20% as many platelet-derived growth factor-alpha receptors as found in fibroblasts cultured from normal skin. No significant difference in the amount of platelet-derived growth factor-beta receptor was detected. The decreases in platelet-derived growth factor-alpha receptors are sufficient to account for the diminished response of the wound fibroblasts to all platelet-derived growth factor isoforms and the differential loss of responsiveness to platelet-derived growth factor AA. These results show that fibroblasts derived from granulation tissue of pig skin wounds exhibit a decreased growth response to platelet-derived growth factor and a decreased growth rate in culture media as compared with fibroblasts derived from uninjured skin. How these differences may relate to the physiologic characteristics of normal and healing-impaired wounds is considered.  相似文献   

3.
Defensins are effector molecules of the innate host defense system with antimicrobial activity against a variety of pathogens, including microorganisms commonly found in burn units. beta-Defensins are variably expressed in the epithelia of skin and other organs. Cultured skin substitutes (CSS) grafted to burn wounds lack a vascular plexus and are therefore more susceptible to microbial contamination than split thickness skin autograft. To investigate whether beta-defensins can contribute to host defense in CSS, we examined expression of human beta-defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and CSS from uninjured donors and burn patients. HBD-1 was expressed in all keratinocyte strains analyzed. HBD-2 expression in keratinocyte monolayers was highly variable but did not correlate with burn injury. HBD-3 was expressed at variable levels in all but one keratinocyte strain. CSS were prepared from two donors that lacked expression of HBD-2 in keratinocyte monolayers. All three genes were readily detected in CSS from both donors, suggesting up-regulation of HBD-2 and HBD-3. In sections of CSS, HBD-1, HBD-2, and HBD-3 proteins were localized to distinct epidermal regions. We conclude that beta-defensins can potentially contribute to innate immunity in CSS, but their levels may be too low to prevent contamination after grafting.  相似文献   

4.
目的:评估人胚胎成纤维细胞(human embryonic fibroblasts,HEF)作为组织工程皮肤种子细胞的可行性和优越性。方法:取人流产胚胎皮肤、正常儿童皮肤,相同条件下分离培养成纤维细胞,比较两组细胞镜下、超微结构以及增殖特性,异体淋巴细胞混合实验对比其抗原性。以第三代细胞复合鼠尾胶原构建三维培养,ELASA法分别测定两组三维构建培养液中IL-6,TGF-β1含量。结果:与普通成纤维细胞相比,胚胎成纤维细胞扩增后具有更好的细胞形态和功能,生长速度快,分裂指数高,几乎不刺激异体淋巴细胞增殖。在鼠尾胶原支架中成纤维细胞生长状态良好,并且具有一定的组织强度。胎儿成纤维细胞组培养液中的TGF-β1和IL-6在各个时相上分别显著低于和高于普通成纤维细胞组。结论:胚胎成纤维细胞是组织工程皮肤较理想的种子细胞。  相似文献   

5.
目的观察不同创面血小板源性生长因子(PDGF)A及其受体(PDGFR)α的动态表达,探讨急性放射性皮肤溃疡难愈合的机制。方法应用γ射线单次照射Wistar大鼠,制作急性放射性皮肤溃疡模型(照射组,55只),以皮肤全层切割伤模型大鼠作为创伤对照(创伤组,55只),另取5只正常大鼠作为对照组。采用免疫组织化学及原位逆转录-聚合酶链反应(RT-PCR)等方法,观察不同时相点各组大鼠创面内PDGF-A、PDGFR-α和PDGF-AmRNA的表达。结果对照组大鼠皮肤中PDGF-A及PDGFR-α表达为阴性;创伤组大鼠炎症反应期及肉芽组织期(伤后3~9d)PDGF-A的积分吸光度(IA)值为20.0±1.6、28.3±1.0;照射组炎症反应期及肉芽组织期(伤后14~28d)其PDGF-A的IA值各为14.0±1.2、20.3±1.2,PDGF-A及PDGFR-α和PDGF-AmRNA的表达较创伤组明显减弱,至瘢痕形成期(伤后55d)时表达进一步减弱。结论急性放射性皮肤溃疡内PDGF-A及PDGFR-α的表达减弱,可能是创面难愈合的机制之一。  相似文献   

6.
Infection is the greatest problem in burn patients and topical antimicrobial agents must be chosen with great care, especially when cultured skin is grafted. We examined the cytotoxic effect of six antiseptics and six antibiotics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane® (chlorhexidine), Biseptine® (chlorhexidine + benzalkonium chloride + benzylic alcohol), Benzalkonium Chloride, Yellow Betadine® (polyvidone-iodine + nonoxinol), Betadine Scrub (polyvidone-iodine + quaternary ammonium) and Green Betadine® (polyvidone-iodine) and viability was determined using the MTT test. At therapeutic concentrations all the antiseptics are cytotoxic for fibroblasts and keratinocytes. Additionally the cells were exposed for 48 h to vancomycin, colistin, amikacin, imipeneme, pefloxaxin, piperacillin and cell viability was determined using the MTT test. The concentrations of antibiotics corresponding to the plasma peak obtained after therapeutic appliction were not cytotoxic to the tested cells. The CD50 was much higher than the MIC (from 125 to 875 times for keratinocytes and from 1400 to 5900 times for fibroblasts). These data suggest that commonly applied antiseptics must not be used before grafting cultured skin grafts. After grafting any infection can be controlled with topical applications of appropriate antibiotics.  相似文献   

7.
目的 探讨几丁质—胶原蛋白膜作为表皮细胞、成纤维细胞培养支架的可能性,体外构建包含双层细胞的复合皮。方法 取健康成人环切包皮,分离成纤维细胞、表皮细胞,分别制成单细胞悬液,将几丁质-胶原蛋白膜(有孔面向上)平铺于60mm培养皿中,首先接种成纤维细胞,培养2d后,将含10%小牛血清的DMEM更换成完全型DMEM,翻转膜,使光滑面朝上,再种植表皮细胞,每日换液。定期观察细胞与材料的粘附、细胞贴壁及其生长增殖情况。结果 几丁质—胶原蛋白膜对成纤维细胞、表皮细胞无明显毒性作用,成纤维细胞培养1d后,细胞贴附于材料支架,细胞胞体较大,呈典型的梭形。加入表皮细胞复合培养2d示表皮细胞贴壁生长,并分化、增殖。复合皮构建2周,网格支架及孔内均有大量细胞生长,膜表面细胞融合成片,表皮细胞分化形成复层。结论 几丁质—胶原蛋白膜对细胞无毒性,有利于培养细胞的粘附、生长。在体外可以构建成功类似生理性皮肤的人工皮肤。  相似文献   

8.
To test whether triglyceride-enriched low-density lipoprotein (LDL) obtained from subjects with diabetic hypertriglyceridemia is metabolized normally by cells, LDL was separated from seven healthy control subjects (fasting plasma glucose [FPG] 91 +/- 10 mg/dl [mean +/- SD], triglyceride [TG] 110 +/- 47 mg/dl), six diabetic normolipidemic patients (FPG 218 +/- 65 mg/dl; TG 139 +/- 75 mg/dl), six diabetic hypertriglyceridemic patients (FPG 214 +/- 71 mg/dl; TG 1915 +/- 1680 mg/dl), and five nondiabetic hypertriglyceridemic patients (FPG 92 +/- 8 mg/dl; TG 2013 +/- 1889 mg/dl). Binding of 125I-labeled LDL from hypertriglyceridemic subjects with and without diabetes to cultured skin fibroblasts was significantly decreased to 74 +/- 19% and 78 +/- 14% of that seen with LDL from normolipidemic nondiabetic subjects and diabetic normolipidemic controls (100 +/- 0%, 101 +/- 25%; P less than 0.005). Unlabeled LDL from hypertriglyceridemic subjects with and without diabetes failed to suppress LDL receptor activity and sterol synthesis from 14C-acetate as efficiently as unlabeled LDL from healthy subjects. The ability of LDL from hypertriglyceridemic subjects, whether diabetic or not, to suppress LDL binding was inversely related to the ratio of triglyceride to protein in LDL (r = 0.71, P less than 0.01) and showed a positive correlation with the LDL cholesterol/protein ratio (0.69, P less than 0.01). Thus, LDL from patients with hypertriglyceridemia, with or without coexistent diabetes, shows impaired binding to LDL receptors and less ability to downregulate LDL receptor activity and sterol synthesis than does LDL from normolipidemic diabetic and nondiabetic subjects. These findings suggest that factors associated with hypertriglyceridemia rather than with diabetes result in altered metabolism of LDL in these disorders.  相似文献   

9.
We have used an in vitro model of wound contraction, the fibroblast-populated collagen lattice, to examine the effect of platelet-derived growth factor BB (PDGF-BB) and PDGF-BB gene transfer by gene gun on the contraction of lattices composed of either diabetic or non-diabetic human fibroblasts. The area of collagen lattice and DNA synthesis were measured in 12 specimens. There were significant increases in lattice contraction with increasing doses of PDGF-BB and fibroblasts transfected with the PDGF-BB gene compared with control (p < 0.01). DNA synthesis of the non-diabetic and diabetic fibroblast lattices showed significantly increased incorporation of tritiated thymidine with increasing doses of PDGF-BB and fibroblasts transfected with the PDGF-BB compared with controls (p < 0.05). The effect of PDGF-BB gene transfer on diabetic and non-diabetic fibroblasts was similar to that of 20 ng/ml or less of PDGF-BB.  相似文献   

10.
BACKGROUND: Many cases of tendon rupture after glucocorticoid injections have been reported in the literature. Despite previous studies on the histological and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in causing tendon rupture still remains controversial. The objective of this study was to determine whether glucocorticoid has deleterious effects on the cellular metabolism and collagen production of cultured human tenocytes and the reversibility of these effects by platelet-derived growth factor-BB (PDGFBB). METHODS: Primary cultures of human tenocytes obtained from explants of healthy patellar tendon, harvested during anterior cruciate ligament reconstructions, were performed. The effects on cell viability, cell proliferation, and induction of apoptosis were measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, 5-bromo-deoxyuridine incorporation, and DNA fragmentation assay, respectively. The effect on collagen synthesis was measured by (3) H-proline incorporation assay. RESULTS: The number of viable cells was decreased, in a dose-dependent manner, by the administration of 10 (-9) to 10 (-4) -M dexamethasone. This dose range also suppressed cell proliferation. No apoptotic effect was detected. Treatment with 10 (-6) -M dexamethasone significantly reduced the amount of collagen synthesis. Co-incubation with 10 ng/mL of PDGFBB significantly reversed the effects caused by 10 (-6) -M dexamethasone. CONCLUSIONS: Dexamethasone significantly decreased cell viability, suppressed cell proliferation, and reduced collagen synthesis in cultured human tenocytes. The effects were reversed by the simultaneous administration of PDGFBB.  相似文献   

11.
Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6–9/group) were pulsed with 5‐bromo‐2'‐deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3–6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU‐positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long‐term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue‐engineered skin to natural skin, and contribute to more rapid and stable wound healing.  相似文献   

12.
BACKGROUND: Skin substitutes prepared from cultured skin cells and biopolymers may reduce requirements for donor skin autograft, and have been shown to be effective in treatment of excised burns, burn scars, and congenital skin lesions. DATA SOURCES: Cultured skin substitutes (CSS) generate skin phenotypes (epidermal barrier, basement membrane) in the laboratory, and restore tissue function and systemic homeostasis. Healed skin is smooth, soft and strong, but develops irregular degrees of pigmentation. Quantitative analysis demonstrates that CSS closes 67 times the area of the donor skin, compared to less than 4 times for split-thickness skin autograft. CONCLUSIONS: CSS reduce requirements for donor skin autograft for closure of excised, full-thickness cutaneous wounds, and demonstrate qualitative outcome that is not different from meshed, split-thickness autograft. These results offer reductions in morbidity and mortality for the treatment of burns and chronic wounds, and for cutaneous reconstruction.  相似文献   

13.
Abnormal scars result in distressing symptoms and disfiguring blemishes; an understanding of the molecular events that cause such scars, particularly keloids, would make possible the optimisation of both wound healing and treatment. Extracellular signal-regulated protein kinase (ERK) has a crucial role in distinct signalling pathways in different cells, but to date we know of no study on its signalling events in keloid fibroblasts. The purpose of this study was to characterise the expression of tyrosine phosphorylation kinases, particularly that of ERK, in keloids at the protein level by immunoblotting analysis. Studies on phosphorylation were made on cell lysates of three cultures of five different keloid fibroblasts (n = 5), their relatively 'normal' fibroblasts in adjacent skin (rNHDF, n = 5), and normal human dermal fibroblasts (n = 1, standard control). The result showed that ERK signalling molecular protein was more highly phosphorylated in keloid fibroblast culture than in the other two cultures.  相似文献   

14.
The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/10(6) cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm x 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 +/- 1.20 mm) compared with untreated wounds (14.66 +/- 0.591 mm), wounds treated with polyglycolic acid alone (14.80 +/- 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 +/- 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B-transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.  相似文献   

15.
16.
Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.  相似文献   

17.
含表皮细胞和成纤维细胞的复合皮构建及移植实验   总被引:16,自引:1,他引:16  
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。  相似文献   

18.
M L Cooper  J F Hansbrough 《Surgery》1991,109(2):198-207
In patients with extensive full-thickness burns, wound coverage may be accelerated if skin can be expanded to produce a skin replacement that reproducibly supplies blood to the wound and has good structural qualities. In addition, development of skin replacements may benefit patients who require reconstruction or replacement of large areas of abnormal skin. We have developed a composite skin replacement composed of cultured human keratinocytes (HK) and fibroblasts. Cultured human fibroblasts are seeded into the interstices, and cultured HKs are applied to the surface of a matrix composed of type I collagen crosslinked with a glycosaminoglycan, which has a defined physical structure. After HKs reach confluence on the matrix surface, the composite grafts are placed on full-thickness wounds on the dorsum of athymic mice. Graft acceptance, confirmed by positive staining with antibodies specific for human HLA-ABC antigens on HKs, is approximately 90%. A defined skin structure is present histologically by day 10 after grafting, with a differentiated epithelium and a subepidermal layer densely populated by fibroblasts and capillaries without evidence of inflammation. Fluorescent light microscopy to identify laminin and type IV collagen and electron microscopy confirm the presence of basement membrane components by 10 days after grafting. Attachment of the graft to the wound is similar with and without the addition of human basic fibroblast growth factor, a potent angiogenic agent, to the skin replacement before graft placement on wounds.  相似文献   

19.
Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.  相似文献   

20.
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