Liposomal gene transfer of keratinocyte growth factor improves wound healing by altering growth factor and collagen expression

BACKGROUND: Growth factors affect the complex cascade of wound healing; however, interaction between different growth factors during dermal and epidermal regeneration are still not entirely defined. In the present study, we thought to determine the interaction between keratinocyte growth factor (KGF) administered as liposomal cDNA with other dermal and epidermal growth factors and collagen synthesis in an acute wound. MATERIALS AND METHODS: Rats received an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and Lac-Z gene (0.22 microg). Histological and immunohistochemical techniques were used to determine growth factor, collagen expression, and dermal and epidermal structure. RESULTS: KGF cDNA increased insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3), and fibroblast growth factor (FGF), decreased transforming growth factor-beta (TGF-beta), while it had no effect on platelet-derived growth factor (PDGF) levels in the wound. KGF cDNA significantly increased collagen Type IV at both the wound edge as well as the wound bed, while it had no effect on collagen Type I and III. KGF cDNA increased re-epithelialization, improved dermal regeneration, and increased neovascularization. CONCLUSIONS: Exogenous administered KGF cDNA causes increases in IGF-I, IGF-BP3, FGF, and collagen IV and decreases TGF-beta concentration. KGF gene transfer accelerates wound healing without causing an increase in collagen I or III.     

Effect of platelet-derived growth factor on rabbit corneal wound healing

Human recombinant platelet-derived growth factor was evaluated with the use of wound healing models in New Zealand albino rabbits. The efficacy of the platelet-derived growth factor dimers, AA, AB, and BB, was determined in corneal reepithelialization and anterior keratectomy models which examined the healing response in the presence or absence of the basement membrane. All dimers increased the rate of wound healing in both models at 100 µg/ml when compared with control; however, the platelet-derived growth factor-BB isoform showed the most dramatic increase in both studies. The strength of the healing stroma after incision was evaluated by means of a tensile strength model. Histologic evaluation of the stromal wound area after 9 days of healing showed a marked increase in the number of keratocytes within the wound bed of the corneas treated with platelet-derived growth factor-BB when compared with control corneas. In addition, at 9 days, the epithelial plug was still present in the control corneas but had been extruded to the surface by the granulation tissue in the platelet-derived growth factor-BB—treated corneas. These results are indicative of a more advanced stage of healing in treated versus control wounds at 9 days after the operation. A 30% increase in corneal tensile strength versus control was noted after 21 days of healing. Finally, in an in vitro gel contraction assay, platelet-derived growth factor exhibited a dose-dependent effect on the contraction of fibroblasts for doses ranging from 0.01 to 10 ng/ml. These results indicate that platelet-derived growth factor is active in the corneal wound healing process.     

重组人角质细胞生长因子2涂膜剂对家兔损伤创面愈合的影响

目的观察重组人角质细胞生长因子2(rhKGF-2)涂膜剂对家兔损伤创面愈合的影响。方法用预热至100℃的100 g砝码在家兔背部脱毛部位压烫8 s,造成背部4个直径为2．5 cm 的深Ⅱ度烫伤创面;手术切除家兔背部部分皮肤,制作2个5．5 cm×4．0 cm的全层皮肤缺损创面。伤后第2天将家兔分成对照组、基质组、rhKGF-2溶液组和不同剂量rhKGF-2涂膜剂组,共12个组 (两种创面各6组),每天分别在创面涂抹等渗盐水、涂膜剂基质、rhKGF-2溶液及3种剂量的rhKGF-2 涂膜剂,连续给药15 d。每4天测量1次伤口面积并计算愈合率,第16天切取各组家兔伤口及其周围正常皮肤进行组织形态学观察,利用Masson三色法染色和MOTIC Advanced 3．2形态分析软件统计创面新生上皮面积、厚度和上皮细胞迁移距离。结果 30．0 μg rhKGF-2涂膜剂给药4、12 d时, 家兔的烫伤创面愈合率[(20±4)％、(65±6)％]明显高于对照组[(10±4)％、(56±9)％]和基质组 [(20±5)％、(59±7)％];160．0 μg rhKGF-2涂膜剂给药4、12 d时,家兔的皮肤缺损创面愈合率 [(33±6)％、(71±20)％]明显高于对照组[(23±4)％、(53±6)％]和基质组[(27±11)％、(58± 9)％]。组织形态学定量检测结果显示,rhKGF-2涂膜剂尤其是大剂量组能明显增加两种创面新生上皮面积、平均厚度和上皮细胞移行距离。结论 rhKGF-2涂膜剂能明显加快家兔皮肤烫伤和皮肤全层切除创面周围上皮细胞的增生与迁移,缩短其愈合过程。     

Insulin-like growth factor I and truncated keratinocyte growth factor accelerate healing of left-sided colonic anastomoses

BACKGROUND: Human full-length keratinocyte growth factor (KGF) promotes healing of colon anastomoses in rats through mechanisms other than enhancement of collagen synthesis. Since insulin-like growth factor (IGF) I increases matrix synthesis, the aim of this study was to evaluate the effect of systemic truncated KGF (tKGF), IGF-I and combined tKGF-IGF-I administration on the healing of colonic anastomoses in rats. METHODS: Rats underwent laparotomy, division of the left colon, and sigmoidosigmoidostomy. tKGF (1 mg/kg), IGF-I (1 mg/kg), tKGF-IGF-I (both 1 mg/kg) or vehicle was administered intraperitoneally in four groups (n = 18 per group) 12 h before surgical intervention, and then once daily until killing (six animals per group; 2, 4 and 6 days after surgery). Bursting pressure measurements, histological evaluation, morphometric analysis, mucin and collagen staining, and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry of the anastomotic site were undertaken. RESULTS: Administration of tKGF, IGF-I and the combination of both growth factors significantly increased anastomotic bursting pressure at postoperative day 2 (63, 71 and 113 per cent respectively), day 4 (68, 83 and 80 per cent) and day 6 (48, 43 and 43 per cent) compared with the control group. No intergroup differences were found. Histological examination, mucin and BrdU staining, and measurement of colonic crypt depth indicated less inflammation, increased acidic mucin content, a higher crypt cell proliferation rate and thickened mucosal layer in the growth factor-treated animals than in controls. Enhanced collagen staining was observed only in IGF-treated animals. CONCLUSION: tKGF and IGF-I markedly accelerate the healing of colonic anastomoses in rats. However, combined administration of the two growth factors does not show additional benefit. Both growth factors may be acting to accelerate host reparative processes as well as to enhance protection of the anastomotic wound bed.     

Effect of vascular endothelial growth factor on laryngeal wound healing in rabbits.

OBJECTIVE: Study the effects of vascular endothelial growth factor (VEGF) on laryngeal wound healing in a rabbit model. STUDY DESIGN: Prospective, randomized, blinded. METHODS: The anterior cricoid cartilage of 10 rabbits was split and a VEGF-soaked collagen sponge was sewn between the cut edges. In 10 control animals, the collagen sponge was soaked with phosphate-buffered saline solution. The larynx was harvested on day 10. The degree of epithelial closure, the degree of soft tissue closure, and the presence of inflammatory cells was graded. RESULTS: There was complete epithelial closure in the control group. There was a slightly higher, but not statistically significant, grade of soft tissue closure in the experimental group. The experimental group had a lower but not statistically significant acute inflammatory response score. CONCLUSIONS: The topical application of VEGF through an implanted collagen sponge to an anterior, subglottic incision in a rabbit has no significant effect on tracheal luminal epithelial closure, acute inflammatory response, or soft tissue repair at postsurgical day 10.     

生物型人工皮肤及复合bFGF对兔创面愈合的影响

目的：探讨生物型人工皮肤复合碱性成纤维生长因子对兔创面愈合的影响。方法：以动物膜组织为基材,经环氧交联固定及表面透明质酸修饰制备生物型人工皮肤,复合碱性成纤维生长因子形成生物活性人工皮肤。光镜观察生物型人工皮肤的结构,进行物理及生物性能测定水蒸汽透过量、液体吸收量、力学性能及细胞毒性。最后将两种人工皮肤植入兔全层皮肤创伤模型,观察术后4周内创面愈合情况,进行病理组织学检查评估创面的修复质量。结果：光镜下生物型人工皮肤为无细胞的胶原结构。贴附性好,无免疫排斥反应,有一定韧性,水蒸汽透过量：372±45g/（m2×24h）,液体吸收量：269±38%,无细胞毒性。复合少量碱性成纤维生长因子后,明显促进创面愈合,减少瘢痕形成。结论：生物型人工皮肤复合少量碱性成纤维生长因子使创面快速愈合,可作为烧伤科、整形外科进行皮肤重建的良好替代物。     

Recombinant basic fibroblast growth factor accelerates wound healing

Basic fibroblast growth factor (bFGF) stimulates extracellular matrix metabolism, growth, and movement of mesodermally derived cells. We have previously shown that collagen content in polyvinyl alcohol sponges increased after bFGF treatment. We hypothesized that bFGF-treated incisional wounds would heal more rapidly. After intraperitoneal pentobarbital anesthesia, male, 200- to 250-g, Sprague-Dawley rats (n = 27) each underwent two sets of paired, transverse, dorsal incisions closed with steel sutures. On Day 3 postwounding, 0.4 ml of bFGF (recombinant, 400 ng. Synergen) or normal saline was injected into one of each paired incisions. Animals were killed with ether on postwounding Days 5, 6, and 7 and their dorsal pelts were excised. Fresh or formalin-fixed wound strips were subjected to tensile strength measurements using a tensiometer. Breaking energy was calculated. Wound collagen content (hydroxyproline) was measured in wound-edge samples following hydrolysis using high-performance liquid chromatography. There was an overall significant increase in fresh wound tensile strength (13.7 +/- 1.06 vs 19.1 +/- 1.99 g/mm, P less than 0.01) and wound breaking energy (476 +/- 47 vs 747 +/- 76 mm2, P less than 0.001) in bFGF-treated incisions. There was an increase in wound collagen content which was not statistically significant and there was no difference in fixed incisional tensile strength. Histologic examination showed better organization and maturation in bFGF wounds. Recombinant bFGF accelerates normal rat wound healing. This may be due to earlier accumulation of collagen and fibroblasts and/or to greater collagen crosslinking in bFGF-treated wounds.     

Effect of substance P released from peripheral nerve ending on endogenous expression of epidermal growth factor and its receptor in wound healing

Objective: To explore the relationship between substance P (SP) released from peripheral nerve endings and the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) during wound bealing. Methods: Fifty Wistar rats were randomly divided into 2 groups, injury group and capsaicin group. In the injury group, a full-thickness skin wound on the back of the rat was taken. The wound edge and granulation tissues were taken on the 1st, 3rd, 6th, 9th, 12th days after injury, respectively. In the capsaicin group, capsaicin was injected subcutaneously on the back of the rats to destroy the sensory nerve to prevent the secretion of SP, then a wound and sample was made in the same way.Immunohistochemistry and in situ hybridization were employed to detect the expression of SP, EGF/EGFR, and EGF mRNA/EGFR mRNA in the granulation tissues.Results: In the injury group,immunohistochemical stain of SP and EGF/EGFR was located on the hair follicles and sebaceous glands at the 1st day. And the stain of SP was obvious at the 3rd day in the granulation tissues, then decreased gradually. EGF/EGFR was at low level at the 3rd day, then increased gradually and reached the peak at the 9th day, then declined. In the capsaicin group, the stain of SP and EGF/EGFR was faint and without obvious change during the wound healing process. The tendency of the EGF mRNA/EGFR mRNA expression was similar to that of EGF/EGFR. Conclusions: During wound healing, SP may promote the healing process by affecting the expression of EGF/EGFR in the erunuation tissues.     

The effect of epidermal growth factor on wound healing in mice

The data presented in this paper focus attention on the possible evolutionary advantages of communal licking, based upon the delivery of wound healing factors in the saliva to an immediate local injury. It is suggested that epidermal growth factor (EGF) is one of these factors, as topical application of EGF to a standardized back wound in mice caged separately enhanced wound closure in both control and sialectomized animals. A sex difference in the wound closing response was evident from these studies. The testosterone dependence of EGF synthesis and its action on wound closure as well as its release upon α-adrenergic stimulation, make teleological sense, in a context of an acute response to injury caused by fighting. It is also suggested that prostaglandins released in injured tissue may modulate these acute effects of EGF, as prostaglandin inhibitors prevented EGF-induced closure. Since EGF is known to be a potent mitogen for murine fibroblast and epithelial cell lines, it may also participate in longer term effects integral to wound healing.     

In vivo characterization of keratinocyte growth factor-2 as a potential wound healing agent

Human keratinocyte growth factor-2 exerts a proliferative effect on epithelial cells and mediates keratinocyte migration. It has also been shown to increase both deposition of granulation tissue and collagen and maturation of collagen. Because these properties should affect the healing trajectory of wounds, this study set out to investigate the effects of keratinocyte growth factor-2 on the healing of three different types of wounds. Human meshed skin grafts explanted to athymic "nude" rats, surgical incisions in Sprague-Dawley rats, and acute excisional rat wounds inoculated with Escherichia coli were used. Two concentrations of recombinant human keratinocyte growth factor-2 were compared to a vehicle control and keratinocyte growth factor-1. Keratinocyte growth factor-2 significantly accelerated the rate of epithelialization in the meshed skin graft model and effected a modestly more rapid gain in breaking strength of surgical incisions than keratinocyte growth factor-1 or the vehicle control treatment. Neither keratinocyte growth factors accelerated wound closure by contraction of the excisional wounds. Based on these data, keratinocyte growth factor-2 may be useful in accelerating healing in wounds healing mainly by the process of epithelialization such as venous stasis ulcers, partial thickness burn wounds, and skin graft donor sites. It might also accelerate the gain in incisional wound strength in acute surgical or traumatic wounds.     

The role of vascular endothelial growth factor in wound healing

Revascularization of damaged tissue is a necessary part of wound healing. With unregulated or insufficient vessel growth, healing is delayed or pathological. Angiogenesis is regulated by expression of a variety of vascular growth factors and modulators, the most widely expressed and critical of which is vascular endothelial growth factor (VEGF). This protein is secreted by tissues in response to ischemic and inflammatory stimuli and results in endothelial migration, proliferation, and increased vascular permeability. The regulation of VEGF expression during wound healing is of considerable importance since angiogenesis appears to be disturbed in abnormally healing wounds. This paper describes the current state of knowledge of VEGF expression in wounds, regulation of expression, control of isoform specificity, and the effects of VEGF expression on blood vessels as they grow in wound healing, as understood from many different pathological paradigms.     

Autologous keratinocyte suspensions accelerate epidermal wound healing in pigs

BACKGROUND: Tissue culture techniques enable in vitro expansion of keratinocytes that can be used to treat burns and chronic wounds. These keratinocytes are commonly grafted onto the wounds as differentiated sheets of mature epithelium. Less is however known about the effects of transplanting the cells as suspensions. This study evaluated epidermal regeneration in fluid-treated skin wounds treated with suspensions of cultured and noncultured autologous keratinocytes. MATERIALS AND METHODS: Eighty-seven full-thickness excisional skin wounds were created on the back of 6 pigs and then transplanted with either cultured or noncultured autologous keratinocytes. The wounds were enclosed with liquid-tight chambers containing saline to provide a hydrated and standardized environment. RESULTS: Keratinocyte transplantation resulted in several cell colonies within the granulation tissue of the wound. These colonies progressively coalesced and contributed to a new epithelium. The origin of the transplanted keratinocytes was confirmed by histochemical staining of wounds transplanted with transfected keratinocytes expressing beta-galactosidase. Transplantation of 0.125 x 10(6), 0.5 x 10(6), and 2.0 x 10(6) cultured keratinocytes, and 0.5 x 10(6) and 5.0 x 10(6) noncultured keratinocytes, increased reepithelialization dose dependently over saline-treated controls. The epithelial barrier function recovered faster in transplanted wounds as demonstrated by less protein leakage over the wound surface on Days 7-10 as compared to control wounds. Wound reepithelialization and the number of keratinocyte colonies observed in granulation tissue were significantly less in wounds transplanted with noncultured keratinocytes compared to wounds seeded with cultured keratinocytes. CONCLUSION: Our study demonstrates successful transplantation of keratinocyte suspensions and their dose-dependent acceleration of wound repair. Selection of proliferative cells during culture and higher colony-forming efficiency may explain the greater effects observed with cultured keratinocytes.     

Mathematical model for wound healing following autologous keratinocyte transplantation

In times of increasing economical pressure on the health care systems, it is important to optimise the outpatient treatment of chronic wounds. Another aim of wound healing research is to discover agents to accelerate healing. Wound healing trajectories or healing velocities can provide information to demonstrate the endpoints for wound healing. A great problem in clinical trials is to specify these parameters. Therefore, we developed a mathematical model for more transparency. In this initial project, we observed 19 wounds to construct the wound healing trajectories after transplantation of autologous keratinocytes, and the results are so encouraging that investigation in this area will continue. The developed mathematical model describes the clinical observed healing process. It was possible to find parameters to distinguish between old and young patients, retrospectively or prospectively calculate the healing rates and to determine exactly the endpoint of healing. Therefore, our model might be very useful in practices or for studies.     

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.     

Growth factor impact on wound healing

Sixty thousand to 118,000 lower extremity amputations are performed each year in the United States. The combination of peripheral vascular disease and diabetes mellitus accounts for most cases, with diabetic patients representing 45% to 70% of all nontraumatic, lower extremity amputations. The 3-year survival rate after amputation is only 50%. As podiatric physicians, we are directly involved in limb preservation. Progress has occurred in both the diagnosis and treatment of lower extremity, chronic, nonhealing ulcers. An aggressive, comprehensive amputation intervention program is critical to those patients with refractory wounds to prevent the emotional, functional, and economic costs of limb loss. Recent developments in recombinant growth factors are making it possible to decrease the morbidity and mortality associated with defective angiogenesis, fibroblastic proliferation, collagen remodeling, and epithelial regeneration. Widespread use of growth factors will first occur in topical applications. Absorbable sutures, as well as impregnated bandages, are a likely method of delivering the growth factors to the wound site. Biotechnology companies are developing a stable formulation for bFGF topical application. Clinical trials have begun at various teaching hospitals across the United States for treatment of venous stasis ulcers. U.S. and European firms are collaborating to conduct the clinical studies required to obtain regulatory approvals leading to the sale of topical recombinant bFGF. Although U.S. approval is pending, European use of EFG in the healing of corneal incisions began several years ago. In the future, use of recombinant EGF topically with burn patients may permit earlier reharvesting of healed donor sites as well as coverage of larger graft areas. As some growth factors affect specific processes of healing and cell types, it may be necessary to combine growth factors for complex wounds. PDGF application in combination with other growth factors to incisional wounds may decrease postoperative complications with wound dehiscence while mediating inflammation and repair. In vivo experimental findings suggest that combinations of PDGF and insulin applied topically to wounds may increase the rate of wound repair in diabetics. It is also possible that even the normal healing process may be accelerated, thereby shortening postsurgical convalescence. Approval for internal administration of growth factors will require additional research and thorough clinical trials. The ability of TGF-beta to promote collagen formation may also relate to a metabolic condition such as osteoporosis, in which inadequate formation of collagen or other components of the bone matrix may contribute to pathogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

TGF-β亚型在创伤愈合及瘢痕形成中的作用

<正>创伤愈合有两种形式,完全性修复(即再生)和瘢痕性愈合。转化生长因子-β(transforming growth factorβ,TGF-β)是迄今为止与创伤愈合关系最为密切的生长因子,TGF-β至少有6个亚型,各亚型之间有64%~82%的同源性[1],在成人皮肤组织中,TGF-β1、β2、β3三种异构体均有阳性表达。TGF-β1、β2、β3参与创伤愈合的诸多步骤:炎症反应、促血管新生、成纤维细胞增殖、胶原合成与分解以及新的ECM重建。以往研究大都聚焦于TGF-β亚型对创伤愈合的促进作用,但TGF-β亚     

神经生长因子促进骨折愈合研究进展

神经生长因子(NGF)是神经系统中一类重要的生物活性分子,可由不同类型细胞所分泌。近年研究发现,NGF不仅能促进神经元生长和神经损伤修复,还能促进新生骨形成,从而加速骨折愈合过程。该文就NGF生物学特性、促进骨折愈合作用机制、促进骨折愈合方法等作一综述。     

Nerve growth factor accelerates wound healing in diabetic mice.

Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full-thickness 6-mm punch biopsy wound was created on the dorsum of C57BL/6J-m+ Leprdb mice (db/db) and heterozygous (db/-) littermates and treated daily with normal saline or 2.5S NGF (1 microg/day or 10 microg/day) on post-injury days 0-6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t-test. Color subtractive-computer-assisted image analysis was used to quantify immunolocalized nerves in wounds. Non-overlapping (20x) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline-treated mice to 26 days in mice treated with 1 microg/day NGF (p<0.05) and 24 days in mice treated with 10 microg/day NGF (p<0.02). A similar trend in db/- mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p<0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF-treated wounds at 14, 21, and 35 days (p<0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.