Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices

Healing of large open dermal wounds is associated with decreased values of the tensile strength even up to 6 months post-wounding. Results of previous studies have shown that healing is facilitated in the presence of a type I collagen sponge by promoting deposition of newly synthesized large-diameter collagen fibers parallel to the fibers of the sponge. In this study healing is evaluated in dermal wounds treated with a collagen sponge seeded with fibroblasts or coated with basic fibroblast growth factor (bFGF). Experimental results indicate that the presence of a collagen sponge results in increased wound tensile strength and increased collagen fiber diameters in the upper dermis 15 days post-wounding in an excisional guinea pig dermal wound model. In comparison, dermal wounds treated with collagen sponges seeded with fibroblasts or coated with bFGF showed increased tensile strengths 15 days postimplantation and increased degree of reepithelialization. These results indicate that fibroblast seeding and bFGF coating in conjunction with a type I collagen sponge matrix facilitate early dermal and epidermal wound healing.     

LED红光对糖尿病大鼠创面愈合的影响

目的探讨LED红光照射对糖尿病大鼠创面愈合的作用及相关机制。 方法将30只4周龄雄性SD大鼠按随机数字表法分为正常创面组、糖尿病创面组、红光治疗糖尿病创面组,每组10只。红光治疗糖尿病创面组及糖尿病创面组大鼠高脂饮食4周,正常创面组大鼠正常饮食。饲养4周后对红光治疗糖尿病创面组及糖尿病创面组大鼠按50 mg/kg的量腹腔注射10 mg/mL的链脲佐菌素(STZ)制作糖尿病大鼠模型,2组大鼠均造模成功。造模成功后在3组大鼠的背部两侧各制造2个1.5 cm×1.5 cm的全层皮肤缺损创面,每2 d对大鼠创面进行酒精消毒1次,红光治疗糖尿病创面组大鼠每次消毒后对创面进行LED红光照射5 min,能量密度为20 J/cm²,另外2组大鼠不进行LED红光照射。在观察第7、10、14、21天,观察红光治疗糖尿病创面组大鼠创面有无出现皮疹、红肿、水疱、烫伤等光照不良反应;肉眼观察3组大鼠创面愈合情况;统计3组大鼠创面愈合率。观察第10天从各组随机取2只大鼠,处死后取背部创面组织,固定后,进行苏木精-伊红染色观察创面新生血管情况及肉芽组织生长情况;采用免疫荧光法检测各组大鼠创面组织中CD34、血管内皮细胞生长因子(VEGF)表达情况。数据比较采用单因素方差分析和LSD-t检验。 结果观察第7、10、14、21天,红光治疗糖尿病创面组大鼠经LED红光照射后皮肤未见皮疹、红肿、水疱、烫伤等光照不良反应。在各个观察时间点,肉眼观察正常创面组及红光治疗糖尿病创面组创面愈合情况均优于糖尿病创面组,且正常创面组创面愈合情况略优于红光治疗糖尿病创面组;观察第7、10、14、21天,正常创面组的创面愈合率分别为(34.62±2.116)%、(53.83±7.92)%、(70.20±5.41)%、(95.65±2.58)%,红光治疗糖尿病创面组创面愈合率分别为(31.76±2.44)%、(50.48±4.54)%、(66.26±11.35)%、(93.96±2.80)%,糖尿病创面组创面愈合率分别为(23.67±4.18)%、(42.71±3.40)%、(53.77±7.74)%、(84.07±4.43)%,3组比较差异均有统计学意义(F＝34.69、10.35、10.32、34.40,P<0.05);观察第7、10、14、21天,正常创面组及红光治疗糖尿病创面组创面愈合率始终高于糖尿病创面组,差异均有统计学意义(P<0.05);观察第7、10天,正常创面组创面愈合率高于红光治疗糖尿病创面组,差异均有统计学意义(t＝2.80、3.26,P<0.05),观察第14、21天,正常创面组创面愈合率仍高于红光治疗糖尿病创面组,但差异均无统计学意义(t＝1.16、1.40,P>0.05)。观察第10天,创面组织苏木精-伊红染色显示,正常创面组内含大量新生毛细血管,肉芽组织内胶原及细胞排列紧密有序;红光治疗糖尿病创面组见较多新生毛细血管,肉芽组织内胶原及细胞较多,但少于正常创面组;而糖尿病创面组新生血管最少,肉芽组织内细胞及胶原稀疏。免疫荧光法检测创面组织中CD34、VEGF表达情况可见,正常创面组CD34、VEGF表达高于红光治疗糖尿病创面组,而红光治疗糖尿病创面组表达高于糖尿病创面组。 结论LED红光可促进糖尿病大鼠创面组织中CD34、VEGF表达,促进血管新生,进而促进创面愈合。     

CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue

Cardiac ankyrin repeat protein (CARP) was identified by subtractive hybridization as one of a group of genes that are rapidly modulated by acute wounding of mouse skin. Quantitative RT-PCR showed that CARP was strongly induced during the first day after wounding (157.1-fold), and the high level persisted for up to 14 days. Immunohistochemistry and in situ hybridization revealed that CARP was expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis in the wound area. To examine the effects of CARP on wound healing, we developed an adenoviral CARP vector to treat subcutaneously implanted sponges in either rats or Flk-1(LacZ) knock-in mice. Four days after infection, CARP-infected sponges in rats showed a remarkable increase in the vascular component in granulation tissue as compared to Ad-LacZ controls. This result was confirmed by CD34 immunostaining. By 7 days post-infection of sponge implants in Flk-1(LacZ) knock-in mice, granulation tissue showed many more LacZ-positive cells in Ad-CARP-infected sponges than in virus controls. Ad-CARP treatment also induced neovascularization and increased blood perfusion in rabbit excisional wounds in and ischemic rat wounds. These findings indicate that CARP could play a unique role in therapeutic angiogenesis during wound healing.     

Role of elevated plasma transforming growth factor-beta1 levels in wound healing

Transforming growth factor (TGF)-beta1 plays a central role in wound healing. Wounds treated with neutralizing antibody to TGF-beta1 have a lower inflammatory response, reduced early extracellular matrix deposition, and reduced later cutaneous scarring, indicating the importance of local tissue TGF-beta1. By contrast, increasing the local, tissue levels of TGF-beta1 increases the early extracellular matrix deposition but does not alter scar formation. Increased levels of plasma TGF-beta1 correlate with increased fibrogenesis in the lung, kidneys, and liver. The aim of the present study was to investigate the role of elevated systemic levels of TGF-beta1 on wound healing. We used transgenic mice that express high levels of active TGF-beta1 and have elevated plasma levels of TGF-beta1 and wild-type mice of the same strain as controls. Incisional wounds and subcutaneously implanted polyvinyl alcohol (PVA) sponges were analyzed. Surprisingly, cutaneous wounds in transgenic, TGF-beta1-overexpressing mice healed with reduced scarring accompanied by an increase in the immunostaining for TGF-beta3 and TGF-beta-receptor RII and a decrease in immunostaining for TGF-beta1 compared with wounds in control mice. By contrast, the PVA sponges showed the opposite response, with PVA sponges from transgenic mice demonstrating an enhanced rate of cellular influx and matrix deposition into the sponges accompanied by an increase in the immunostaining for all three TGF-beta isoforms and their receptors compared with PVA sponges from control mice. Together, the data demonstrate that increased circulating levels of TGF-beta1 do not always result in increased expression or activity in selected target tissues such as the skin. The two wound models, subcutaneously implanted PVA sponges and cutaneous incisional wounds, differ significantly in terms of host response patterns. Finally, the data reinforce our previous observations that the relative ratios of the three TGF-beta isoforms is critical for control of scarring.     

Water-soluble chitin as a wound healing accelerator.

Water-soluble chitin (WSC) was prepared by controlling degree of deacetylation (DD) and molecular weight of chitin through alkaline and ultrasonic treatment. Its accelerating effect on wound healing in rats was compared with those of chitin and chitosan. Full-thickness skin incision was made on the backs of the rats and then three kinds of powders (chitin, chitosan, WSC) and an aqueous solution of WSC were embedded in the wounds. The tensile strength and the hydroxyproline content of the wounded skins were measured and histological examination was performed. The WSC was found to be more efficient than chitin or chitosan as a wound-healing accelerator. The wound treated with WSC solution was completely reepithelialized, granulation tissues in the wound were nearly replaced by fibrosis and hair follicles were almost healed at 7 days after initial wounding. Also, the WSC-solution-treated skin had the highest tensile strength and the arrangement of collagen fibers in the skin was similar to normal skins. The WSC solution is considered to be a suitable wound-healing agent due to its easy application and high effectiveness.     

Effects of PerClot® on the healing of full-thickness skin wounds in rats

PerClot^® is a hemostatic material made of polysaccharide from modified starch and has been shown to assist in topical hemostasis. The principal goal in treating surgical and non-surgical wounds is the need for rapid closure of the lesion. This study investigated whether topical application of PerClot^® could improve impaired wound healing in Sprague-Dawley (SD) rats. Full-thickness skin wounds were created on the back of the rats. Immediately, PerClot^® was introduced into the wound bed, while wounds receiving starch or nothing served as controls. Wound closure was monitored using well-recognized wound-healing parameters: histological examination for inflammatory cells and fibroblast infiltration, newly formed capillaries, and collagen deposition. Meanwhile, transforming growth factor (TGF-β1) was measured by immunochemistry. Wound closure was significantly accelerated by local application of PerClot^®. Furthermore, PerClot^®-treated wounds showed significantly increased fibroblast numbers at 5 days post-wounding, and newly formed capillaries at 7 days post-wounding, and collagen regeneration at 7 and 14 days post-wounding. The number of infiltrating fibroblasts expressing TGF-β1 was significantly higher than that in the controls at 7 and 14 days post-wounding. PerClot^® can improve the wound healing and this effect might involve an increase in the activity of fibroblasts and increased release of TGF-β1.     

Basic fibroblast growth factor promotes apoptosis and suppresses granulation tissue formation in acute incisional wounds

Cytokines are thought to play an important role in cellular loss and apoptosis during the repair of granulation tissue. In order to investigate the role of apoptosis following the administration of basic fibroblast growth factor (bFGF) to a wound, the present study examined the relationship between the degree of granulation tissue formation and the level of apoptosis in rat skin incisional wounds, following treatment with an intradermal injection of bFGF (0.1 microg and 1 microg per cm of wound). Histological assessment of the width of the wound tissue showed that the degree of granulation tissue in the 1 microg-bFGF-treated group had increased by day 7, but then subsequently diminished by days 14 and 28. The TUNEL index increased rapidly from day 1, peaking on day 7, with the index being higher in the 1 microg-bFGF-treated group on days 4, 7, and 14, when compared with a control group. In parallel with a marked increase in the TUNEL index over the first 14 days, the number of cells positive for vimentin and CD3 in the 1 microg-bFGF-treated wounds had decreased by day 14. The number of PCNA-positive cells, an indicator of cell proliferation, peaked on day 4 in the bFGF-treated wounds and then declined rapidly. On the basis of these results, it is suggested that the suppression of granulation tissue formation in bFGF-treated wounds is mainly due to an early and persistent increase in apoptosis in the granulation tissue cells. The expression of both transforming growth factor (TGF)-beta1 and bFGF was also elevated in the bFGF-treated wounds on days 4 and 7, suggesting that fibroblast apoptosis was induced by bFGF treatment. Unexpectedly, on day 28, the wound breaking strength was not reduced in the bFGF-treated wounds. These results indicate that apoptosis regulation following bFGF administration to an incisional wound may lead effectively to granulation tissue formation and promote a scar-less repair process.     

Promotion of Wound Collagen Formation in Normal and Diabetic Mice by Quadrol

The rate of collagen deposition in implanted polytetrafluoroethylene (PTFE) tubing in non-diabetic and streptozotocin-induced (STZ) diabetic mice was measured during 14 days post-wounding. At the time of implantation, test groups received injections of either Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine], glucan, or buffer in an area adjacent to the wound site. The accumulation of collagen in the implants of Quadrol-treated non-diabetic animals was more than 200% above control on days 8 to 11 and was 50% above control on day 14. In Quadrol-treated STZ-diabetic mice, the collagen accumulation gradually increased from 50% above control on day 8 to 200% above control on day 14. Treatment with glucan increased the collagen accumulation in normal mice 200 to 300% above control from days 8 to 11 respectively and then 30% above control on day 14. Collagen accumulation in the implants of the glucan-treated STZ-diabetic mice was similar to the control group. These results indicate that Quadrol promotes in vivo collagen synthesis and that Quadrol may be effective as a stimulator of wound healing in diabetic and non-diabetic animals.     

Dynamics of the healing of skin wounds in the horse as compared with the rat.

The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of prolyl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.     

Topical pluronic F-127 gel application enhances cutaneous wound healing in rats

Pluronic F-127 gel is used as vehicle for various topical applications. In the present study, effects of topical application of pluronic F-127 gel were evaluated in cutaneous wound healing in Wistar rats. Normal saline solution and pluronic F-127 gel (25%) were applied topically on open excision wounds for 14 days. Photography, determination of percentage wound contraction, and collection of granulation tissue were done on days 3, 7, 11 and 14 post-wounding. Topical application of gel (once daily) significantly increased the wound closure on days 11 and 14. The gel application increased the expressions of vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-β₁) on days 3 and 7. Histopathologically, more leukocyte infiltration followed by well formed granulation tissue with marked fibroblast proliferation was evident in the gel-treated group, as compared to the saline-treated control group. Immunohistochemistry of CD31 on day 7 revealed significant higher microvessel density in gel-treated wounds. Picrosirius staining demonstrated higher collagen fraction in gel-treated wounds. Thus, from the results, it could be concluded that pluronic F-127 gel has a mild inflammatory nature and enhanced the healing by stimulating expression of VEGF and TGF-β₁.     

Macroscopic aspects of wound healing (contraction and epithelialisation) after topical administration of allicin in dogs

Macroscopical aspects of second-intention healing of full-thickness excisional wounds were studied in five normal male mixed-breed dogs. Test wounds were treated topically with allicin 0.5% in methyl cellulose gel, and control wounds were treated with methyl cellulose gel only. Wound treatment started 24 h after wounding. The wounds were evaluated over a 4-week period. At days 0, 3, 7, 10, 14, 17, 21, 24 and 28, digital photographs were taken of all wounds. Rulers were held vertically and horizontally close to the wound as a reference. The area of epithelialisation and granulation tissue were measured for each wound using Scion Image software. Percentage wound contraction, epithelialisation and healing were calculated for each wound. Initially, all wound areas increased in size. After the initial enlargement, wound areas decreased rapidly in size between days 7 and 17 in both the test and control groups. Epithelialisation was first noticed at day 3 in control and day 5 in the test wounds. No significant differences were observed in the percentage of wound contraction, epithelialisation and healing between the test and control wounds (P> 0.05).     

The reinnervation and revascularisation pattern of scarless murine fetal wounds

Fetal wounds can heal without scarring. There is evidence that the sensory nervous system plays a role in mediating inflammation and healing, and that the reinnervation pattern of adult wounds differs from that of unwounded skin. Ectoderm is required for development of the cutaneous nerve plexus in early gestation. It was hypothesised that scarless fetal wounds might completely regenerate their neural and vascular architecture. Wounds were made on mouse fetuses at embryonic day 16.5 of a 19.5-day gestation, which healed without visible scars. Immunohistochemical analysis of wound sites was performed to assess reinnervation, using antibodies to the pan neuronal marker PGP9.5 as well as to the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Staining for the endothelial marker von Willebrand factor (VWF) allowed comparison of reinnervation and revascularisation. Wounds were harvested at timepoints from day 1 after wounding to postnatal day 6. Quantification of wound reinnervation and revascularisation was performed for timepoints up to 6 days post-wounding. Hypervascularisation of the wounds occurred within 24 h, and blood vessel density within the wounds remained significantly elevated until postnatal day 2 (4 days post- wounding), after which VWF immunoreactivity was similar between wound and control groups. Wound nerve density returned to a level similar to that of unwounded skin within 48 h of wounding, and PGP9.5 immunoreactive nerve fibre density remained similar to control skin thereafter. CGRP and SP immunoreactivity followed a similar pattern to that of PGP9.5, although wound levels did not return to those of control skin until postnatal day 1. Scarless fetal wounds appeared to regenerate their nerve and blood vessel microanatomy perfectly after a period of hypervascularisation.     

MMP-9/TIMP-1表达在糖尿病鼠皮肤伤口愈合过程中的变化及意义初探

目的：观察基质金属蛋白酶-9（MMP-9）、组织金属蛋白酶抑制剂-1（TIMP-1）的表达和MMP-9/TIMP-1比值在糖尿病组和正常组大鼠皮肤伤口愈合过程中不同时点表达的动态变化，探讨其可能的作用机制。 方法:糖尿病大鼠形成6周后行皮肤伤口造模，采用HE染色、Masson染色和免疫组织化学方法评估伤口形成后3、7、14 d伤口组织的再上皮化、炎症细胞浸润、肉芽组织厚度、新生血管形成和胶原纤维密度的情况；通过逆转录-聚合酶链反应(RT-PCR)和Western印迹检测术后不同时点MMP-9、TIMP-1在伤口组织中的表达情况。结果:糖尿病大鼠伤口愈合明显迟缓。术后第3 d两组间胶原纤维、肉芽组织、新生血管和再上皮化没有差异，术后第7 d糖尿病组以上指标得分均低于正常组，第14 d这种趋势更加明显；而第3 d至14 d，糖尿病组的单核巨噬细胞浸润得分均低于正常组。术后第3 d两组MMP-9表达均达高峰，第7、14 d呈逐渐下降趋势，糖尿病组MMP-9表达水平在各时点均高于正常组；术后第3 d两组TIMP-1表达均达高峰，第7、14 d呈逐渐下降趋势，糖尿病组TIMP-1表达水平在各时点均低于正常组；正常组MMP-9/TIMP-1蛋白水平比值始终维持在一个动态平衡的稳定水平，而糖尿病组却长期处于高水平状态。结论:异常的胶原产生、新生血管重建、炎症反应、再上皮化、肉芽形成可能是糖尿病鼠创面愈合减慢的组织病理学基础；皮肤组织MMP-9/TIMP-1的平衡性改变可能是这种组织病理学异常的重要原因之一。     

2型糖尿病非收缩性难愈伤口模型的建立

背景：目前糖尿病创口的难愈性给临床的治疗增加了极大地困难。 目的：建立2型糖尿病状态下非收缩性难愈伤口的动物模型。 方法：高脂饮食喂养联合低剂量链脲佐菌素诱导形成大鼠2型糖尿病模型,在其背部造成圆形创面,用硅树脂固定伤口,模拟人类创面愈合过程,病理切片检查创面周缘肉芽组织皮肤厚度、微血管密度、胶原纤维及胶原蛋白水平的变化。 结果与结论：与正常组相比,链脲佐菌素注射72 h后高脂饮食喂养大鼠的空腹血糖、血清三酰甘油及胆固醇均明显增加,差异有显著性意义(P < 0.05~0.01);创伤后第1,3,5,7,14天,糖尿病组与正常组相比皮肤厚度、微血管密度、胶原纤维及胶原蛋白水平均有明显下降(P < 0.01)。提示高脂喂养联合链脲佐菌素诱导大鼠2型糖尿病的方法简便有效;创伤后检测指标均证明糖尿病大鼠比正常大鼠创面难愈。     

Impact of single-dose application of TGF-β, copper peptide, stanozolol and ascorbic acid in hydrogel on midline laparatomy wound healing in a diabetic mouse model

Despite numerous advances and improvements in surgical techniques the incidence of incisional hernias after laparotomy remains high. The aim of this study was to investigate possible effects of single application of ascorbic acid, stanozolol, a synthetic anabolic steroid, copper peptide and transforming growth factor-β (TGF-β) on laparotomy wound healing in an incisional wound model in diabetic mice. After diabetes induction with streptozotozin in Balb-c mice, midline laparatomies were carried out. Closure of the linea alba was followed by single-dose application of the agents dissolved in a hydrogel before skin closure. The functional outcome was assessed in terms of maximum tensile strength. In addition, vessel densities, collagen contents and proliferation, were measured. The breaking strength of the skin 14 days after surgery was significantly higher in ascorbic acid (ΑΑ)-treated incisional wounds, whereas the other agents did not show a significantly better functional outcome. No significant differences were seen in vessel densities. Collagen type III contents was higher in the ΑΑ-treated animals, whereas the percentage of Ki67-positive nuclei was lower compared to the other groups. These data underline the positive effect of topically applied ascorbic acid in wound healing.     

Effects of systemic and topical estrogen application on the healing of full-thickness skin wounds in diabetic rats

In this research, the possible effects of systemic and topical estrogen were investigated on wound healing in normal and diabetic male rats. One hundred and ten male Wistar rats were divided into two groups (normal and diabetic). After induction of diabetes by streptozotocin in rats, each group was divided into three subgroups (control, sham, and test). A round full-thickness skin excision with 1.5-cm diameter was performed on the dorsum of each rat. In systemic use, 10?μG/SC of estradiol benzoate was administered daily to test subgroups for 28?days. In topical use, the wounds in the test subgroup rats were treated with a daily topical dose of 0.5?mg estrogen. Sham subgroup was injected with placebo and the control subgroup received nothing. The area of the wounds was measured by using scion image software at 3, 5, 7, 14, 21, 28?days. Histopathologic evaluation was assessed semi-quantitatively for different parameters including re-epithelization, neoangiogenesis, and granulation tissue formation. In macroscopic study, estradiol subgroups (systemic and topical application) wound healing had considerable changes in day?7 (p?<?0.05). Histopathologic evaluation was revealed increased vascularization, re-epithelialization in estradiol subgroups. So, this research expressed that systemic and topical estrogen can improve the impaired healing of diabetic wounds.