Placenta growth factor in diabetic wound healing: altered expression and therapeutic potential

Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process.     

Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing

Nonhealing wounds of the lower equine limb represent a challenging model. The platelet is a natural source of a myriad of growth factors and cytokines that promote wound healing. This study evaluates the potential of platelet derived factors to enhance wound healing in the lower equine limb. Platelets were isolated from horse blood and activated with thrombin, a process known to induce growth factor release. This produced a platelet gel composed of platelet-rich plasma (PRP). To test this all-natural wound healant, 2.5-cm(2) full thickness cutaneous wounds were created below the knee and hock of a thoroughbred horse. Wounds were treated with PRP gel or left untreated. Sequential wound biopsies collected at Days 7, 36, and 79 postwounding permitted comparison of the temporal expression of differentiation markers and wound repair. To test the hypothesis that wounds treated with PRP gel exhibit more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls, tissues were stained for cytokeratin 10, a suprabasal differentiation marker, and the reestablishment of collagen was evaluated by trichrome staining. PRP gel-treated wounds at Day 7 expressed intense cytokeratin 10 staining near the wound junction in suprabasal epidermal layers, while staining in control tissues was less intense and restricted to apical epidermal layers distal to the wound junction. By Day 79, the staining was equal in both groups. However, PRP gel-treated wounds at Day 79 contained abundant, dense collagen bundles oriented parallel to each other and to the overlying epithelium, whereas control tissues contained fewer collagen fibers that were oriented randomly. Thus, treatment of wounds with PRP gel induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. This study reveals that this novel all-natural wound healant induced wound repair in injuries previously deemed untreatable.     

Both constitutive and inducible prostaglandin H synthase affect dermal wound healing in mice

In an attempt to define the roles of prostaglandin H synthase 1 (PGHS-1, cyclooxygenase-1, COX-1) and prostaglandin H synthase 2 (PGHS-2, cyclooxygenase-2, COX-2) in wound healing, we investigated the healing of incisional dermal wounds in wild-type, PGHS-1 null, and PGHS-2 null mice. We measured tensile strength of the wounds, levels of PGHS-1 and PGHS-2 mRNA in the wound site, and histologic markers for the inflammatory, proliferative, and remodeling phases of wound healing. Although no gross visible differences were noted among healed wounds of the different mouse types, measurement of tensile strength showed that both PGHS-1 and PGHS-2 null wounds were weaker (75% and 70%, respectively) than wild-type wounds at 12 days after incision. At Day 8 the endothelial staining was 70% greater in the wounds of PGHS-2 null mice compared with their wild-type counterparts. In contrast at Day 12, staining for macrophages and myofibroblasts was less in PGHS-1 null wounds compared with wild-type and PGHS-2 null tissue. Compensatory expression of the alternate PGHS mRNA could be demonstrated by RT-PCR in the wounds of PGHS null mice on Days 1 and 4. We conclude that both PGHS-1 and PGHS-2 genes play distinct roles in the process of dermal wound healing.     

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.     

富林密伤口愈合凝胶在Ⅱ度烧伤及供皮区创面的应用

目的观察富林密伤口愈合凝胶（Flamigel）对Ⅱ度烧伤创面和供皮区创面愈合的影响。方法2003年2月～2007年2月选择70例浅Ⅱ度烧伤、50例深Ⅱ度烧伤和40例供皮区创面作为治疗组，局部创面清创后外用富林密伤口愈合凝胶，无菌敷料覆盖包扎。同期选70例浅Ⅱ度烧伤、50例深Ⅱ度烧伤和40例供皮区创面作为对照组进行常规治疗，观察创面愈合时间、创面局部及全身反应情况。结果70例浅Ⅱ度烧伤创面的愈合时间治疗组和对照组分别为7．9±3．5天和11．3±2．4天；50例深Ⅱ度烧伤创面的愈合时间治疗组和对照组分别为16．4±2．2天和21．4±3．1天；40例供皮区创面的愈合时间治疗组和对照组分别为12．3±3．3天和16．3±2．5天；显示治疗组创面愈合时间明显提前，经统计学处理，有显著性差异（P〈0．05）。结论富林密伤口愈合凝胶有明显促进烧伤创面和供皮区创面愈合的作用。     

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.     

Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair

The bone marrow provides inflammatory cells and endothelial progenitor cells to healing cutaneous wounds. To further explore the bone marrow contribution to skin and healing wounds, we used a chimeric mouse model in which the bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice is transplanted into normal C57BL mice. We found that normal skin is a target organ for bone marrow-derived cells from both the hematopoietic and the mesenchymal stem cell pool. We present evidence that the bone marrow contribution to normal skin and the healing cutaneous wound is substantially greater than the previously recognized CD45+ subpopulation, where 15%-20% of the spindle-shaped dermal fibroblasts were bone marrow-derived (EGFP+). Furthermore, the bone marrow-derived cells were able to contract a collagen matrix and transcribe both collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. Whereas endothelial progenitor cells were found early during the wound repair process, bone marrow-derived endothelial cells were not seen after epithelialization was complete. Our data show that wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow-derived cells and the adjacent uninjured dermal mesenchymal cells for reconstituting the dermal fibroblast population.     

Accelerated wound closure in neutrophil-depleted mice

The infiltration of neutrophils into injured tissue is known to protect wounds from invading pathogens. However, more recent studies suggest that neutrophils might inhibit the wound repair process. To investigate the role of neutrophils in wounds, mice were neutrophil-depleted by injection with rabbit anti-mouse neutrophil serum. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in neutropenic than control mice (77.7+14.2% vs. 41.2+0.9%, P<0.02 at day 2). Dermal healing was not affected by neutrophil depletion, as neither collagen deposition nor wound-breaking strength was significantly different between neutropenic and control mice. As the delayed repair of diabetic individuals exhibits robust inflammation, the effect of neutrophil depletion on diabetic wound healing was investigated. Similar to the observations in wild-type mice, wound closure was accelerated by nearly 50% in neutropenic, diabetic mice. The results suggest that although neutrophils may provide protection against infection, they may retard wound closure.     

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).     

Effects of granulocyte-macrophage colony-stimulating factor on wound contraction

The effect of topical recombinant murine and human GM-CSF, 1 or 10 µg/cm² for one to ten days, on the contraction and healing of acute and chronic granulating wounds infected withEscherichia coli was studied in Sprague-Dawley rats. Bacterial contamination of wounds produced significant inhibition of wound contraction. Application of GM-CSF at either dose level to infected wounds markedly increased the rate of wound closure compared to the rate in infected untreated controls. Ten days treatment was found to be more effective than a single application. An advanced stage of wound healing was observed at ten days in the GM-CSF-treated rats compared with controls. Bacterial counts decreased in the GM-CSF-treated wounds which may suggest bactericidal activity. Topical treatment with GM-CSF was shown to effectively inhibit the retardation of wound closure produced by bacterial contamination and may therefore be useful in the management of patients with infected wounds.     

Effects of granulocyte-macrophage colony-stimulating factor on wound contraction

The effect of topical recombinant murine and human GM-CSF, 1 or 10 µg/cm² for one to ten days, on the contraction and healing of acute and chronic granulating wounds infected withEscherichia coli was studied in Sprague-Dawley rats. Bacterial contamination of wounds produced significant inhibition of wound contraction. Application of GM-CSF at either dose level to infected wounds markedly increased the rate of wound closure compared to the rate in infected untreated controls. Ten days treatment was found to be more effective than a single application. An advanced stage of wound healing was observed at ten days in the GM-CSF-treated rats compared with controls. Bacterial counts decreased in the GM-CSF-treated wounds which may suggest bactericidal activity. Topical treatment with GM-CSF was shown to effectively inhibit the retardation of wound closure produced by bacterial contamination and may therefore be useful in the management of patients with infected wounds.     

Effects of subcutaneous injection of ozone during wound healing in rats

Fibroblast growth factor 2 (FGF2) regulates the wound repair process and it is secreted by inflammatory and endothelial cells, and by myofibroblasts. This study aimed to establish the expression patterns of FGF2 and myofibroblastic differentiation during wound healing in rats treated with subcutaneous ozone injection. We created full-thickness excisional wounds in rats, and the healing process was analyzed through morphometric analyses and digital quantification of immunoreactivity of smooth muscle actin and FGF2. Ozone therapy-treated wounds presented granulation tissue with a reduced number of inflammatory cells and greater dermal cellularity, and intense collagen deposition. FGF2 immunoreactivity, microvessel density, and amount of myofibroblasts were significantly higher in treated wounds compared to controls. In conclusion, it was demonstrated that subcutaneous injections of ozone accelerate and ameliorate wound repairing process. Moreover, injectable ozone therapy’s action mechanism may be associated with FGF2 overexpression.     

Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/-) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/- and -/-) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 post-wounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.     

Effects of Zn-7® on open wound healing in dogs

Analysis of the accelerating effects of open wound healing by Zn-7 gel compared with normal saline was carried out in dogs. Five normal male, mixed breed dogs approximately 3 years old were selected. Histological and macroscopic aspects of second-intention wound healing were studied. Two rectangular, full-thickness skin wounds (20 × 30 mm) were created symmetrically on each dog at both sides of the dorsal midline at 0, 1, 2, 3, and 4 weeks. Left wounds were treated with Zn-7 gel (test group) and right wounds were treated with normal saline (control group) twice daily. Photographs were taken twice a week. Rulers were held vertically and horizontally close to the wound as a reference. The area of epithelialization and granulation tissue were measured for each wound, using the Scion Image software. Percentage wound contraction, epithelialization, and healing were calculated for each wound. No significant differences were observed in wound contraction, epithelialization and healing in the test and control groups (P > 0.05). At day 35 after initial wounding, biopsies were taken from wounds for histological examination. There were no significant differences in the number of inflammatory cells, fibrocyte, or amount of the fibrin and collagen (P > 0.05) between the test and control wounds.     

Role of collagen cross-linking on equine wound contraction and healing

The present study was carried out to evaluate the effect of collagen cross-linking inhibition on equine wound contraction and healing. In five male adult donkeys, two full-thickness skin wounds (20 × 20 mm in diameter) were created on the lateral aspect of forelimbs, at the mid-point between the carpal and fetlock joints under general anesthesia. Two other wounds were created on the neck of each donkey symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate, 5 mg/ml, added to methyl cellulose gel and only methyl cellulose gel, respectively. Treatment of wounds were started at 24 h after wounding and continued every other day for ten successive days. The wounds were evaluated over a 3-week period. On days 0, 1, 3, 5, 7, 9, 12, 15, 18, and 21, digital photographs were taken of all wounds after careful shaving to visualize the wound margin. Rulers were held vertically and horizontally close to the wound as a reference. Epithelialization and granulation tissue formation were measured for each wound using Scion Image software. Percentage of the wound contraction, epithelialization, and healing were calculated for each wounds. At the end of the study, biopsy was taken from the center of each wound for hydroxyproline measurement and the same corner of each wound for histopathological examination. Macroscopic evaluation revealed significant differences in wound contraction and healing process between test and control groups in wounds located in neck (P < 0.05), but there was no significant difference in percent of epithelialization at the same area (P > 0.05). Significant differences were observed in the percent of wound contraction and healing between the test and control groups in wounds located in the forelimb (P < 0.05), but no significant difference was observed in percent of epithelialization at this area (P > 0.05). There were no significant differences between median of hydroxyproline levels of left and right wounds in forelimb and neck (P > 0.05). Histopathological examination revealed no significant differences between median of epithelialization, inflammatory infiltration, presence of dermal granulation tissue, fibroblast proliferation, arrangement of fibroblasts, collagen deposition, and collagen bundle formation scores in the specimens prepared from left and right wounds in forelimb and neck (P > 0.05). Our data demonstrated that collagen cross-linking could play a key role in equine wound contraction and healing at the limb and neck area.     

Effect of collagen cross-linking inhibition by local application of beta-aminopropionitrile fumarate on wound contraction and healing (macroscopic study)

Wound contraction is a major component of second-intention wound healing. The mechanism of this process is not completely understood. Two theories have been described for the mechanism of the wound contraction. To evaluate the collagen cross-linking inhibition on wound contraction, the present study was carried out. Macroscopical aspects of second-intention healing of full-thickness, excisional wounds were studied in five normal male mixed-breed dogs. Under general anesthesia, two full-thickness skin wounds (20 × 20 mm) were created on the back of each dog symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate 5 mg/ml in methyl cellulose gel and methyl cellulose gel, respectively. Wounds were treated starting at 24 h after wounding and continued for ten successive days. The wounds were evaluated over a 4-week period. At the 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 the epithelialization and granulation tissue were measured for each wound using Scion Image software. Percent of the wound contraction, epithelialization, and healing were calculated for each wounds. Wound contraction, epithelialization, and healing were significantly decreased in the wounds treated by beta-aminopropionitrile fumarate (P < 0.05). Our data demonstrated that the collagen and collagen cross-linking play a key role in the wound contraction and healing during the first 10 days of the wound healing.     

纳米银－猪脱细胞真皮基质敷料治疗浅Ⅱ度烧伤创面的临床观察

目的观察纳米银－猪脱细胞真皮基质敷料在临床上治疗浅Ⅱ度烧伤创面的临床疗效。 方法2014年1月到2015年12月,选取北京军区总医院烧伤整形科收治的浅Ⅱ度烧伤患者90例,按入院顺序依次编码,采用随机排列数字表法将90例患者分为纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组,每组30例。患者入院当天,拍照计算创面面积,并用咽拭子取创面分泌物作细菌培养,行创面清创术,分别在创面上敷以纳米银敷料、猪脱细胞真皮基质敷料以及纳米银－猪脱细胞真皮基质敷料。于治疗后第5天,用咽拭子取创面分泌物作细菌培养;采用痛觉评分标准,通过询问与观察患者换药时的痛觉情况,评估患者换药时痛觉评分。于治疗后第7天,拍照计算创面面积,计算创面愈合率。记录创面最终愈合时间。数据比较采用单因素方差分析、χ²检验及SNK－q检验。 结果纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组在治疗后第5天创面细菌培养阳性数结果分别为2例(6.6%)、9例(30.0%)、1例(3.3%),3组结果比较,差异有统计学意义(χ²＝10.962,P＝0.004);纳米银－猪脱细胞真皮基质敷料组的细菌培养阳性数结果显著优于猪脱细胞真皮基质敷料组,差异有统计学意义(χ²＝7.680,P＝0.006)。纳米银敷料组、猪脱细胞真皮基质敷料组及纳米银－猪脱细胞真皮基质敷料组治疗后第5天的痛觉评分[(8.6±0.5)、(6.6±0.8)、(0.6±1.3)分],治疗后第7天的创面愈合率[(61.67±18.22)%、(86.77±15.32)%、(99.80±0.56)%],创面愈合时间[(11.5±1.3)、(10.3±0.7)、(7.3±0.7)d],组间差异均有统计学意义差异(F＝201.7、19.9、55.7,P值均小于0.05);纳米银－猪脱细胞真皮基质敷料组治疗后第5天的痛觉评分显著低于其余两组,差异均有统计学意义(P值均小于0.05);治疗后第7天的创面愈合率明显优于其余两组,差异均有统计学意义(P值均小于0.05);创面愈合时间显著短于其余两组,比较差异均有统计学意义(P值均小于0.05)。 结论纳米银－猪脱细胞真皮基质敷料具有抗感染、促进创面愈合及减轻换药痛觉的作用。     

PDGF and FGF stimulate wound healing in the genetically diabetic mouse.

To examine the effects of recombinant growth factors in vivo, impaired wound healing was studied in genetically diabetic C57BL/KsJ-db/db mice. Large full-thickness skin wounds made on the backs of these mice exhibited significant delays in the entry of inflammatory cells into the wound, the formation of granulation tissue, and in wound closure when compared to their nondiabetic littermates. Recombinant human platelet-derived growth factor (rPDGF-BB, 1 or 10 micrograms), recombinant human basic fibroblast growth factor (rbFGF, 1 micrograms), or combinations of both were applied topically to the wounds for 5 to 14 days after wounding. Diabetic mouse wounds treated with rPDGF-BB or rbFGF had many more fibroblasts and capillaries in the wound bed at 10 and 21 days than did wounds treated with the vehicle alone. The animals treated with growth factors also had significantly greater wound closure at 21 days than those treated with the vehicle. Combinations of rPDGF-BB and rbFGF improved all parameters of healing but not to a greater extent than either growth factor alone. The effectiveness of rPDGF-BB and rbFGF suggest that recombinant growth factors may be useful in the treatment of patients with deficient wound repair.     

Accelerated wound closure in mice deficient for interleukin-10

The impact of the local inflammatory response on the process of wound healing has been debated for decades. In particular, the question whether infiltrating macrophages and granulocytes promote or impede tissue repair has received much attention. In the present study, we show that wound healing is accelerated in mice deficient for the anti-inflammatory cytokine interleukin (IL)-10. IL-10-/- mice closed excisional wounds significantly earlier compared with IL-10-competent control littermates. This effect was attributable to accelerated epithelialization as well as enhanced contraction of the wound tissue in the mutant animals. Increased alpha-smooth muscle actin expression in IL-10-deficient mice suggests that augmented myofibroblast differentiation is responsible for the enhanced contraction of wounds in mutant mice. The number of macrophages infiltrating the wound tissue was significantly increased in IL-10-/- mice compared with control littermates suggesting that this cell type mediates the accelerated tissue repair. These results show for the first time that IL-10 can impede wound repair.