Physicochemical properties of extracellular matrix proteins in post-burn human granulation tissue

Wound healing is a finely controlled biological process involving a series of complex cellular interactions. Following inflammation, the wound bed matrix is gradually replaced by granulation tissue followed by the long slow process where collagen accumulates and restores tensile strength. The studies revealed that human granulation tissue varied in many aspects in comparison with normal skin. In granulation tissue the molecular organization of collagen showed an increased amount of type III collagen resembling embryonic tissue. The presence of type V collagen with three distinct chains was the characteristic feature of granulation tissue. The physicochemical properties of collagen extracted from granulation tissue showed the influence of proteoglycans during collagen aggregation and these proteoglycans from the major non-collagenous proteins during the proliferative phase of healing.     

封闭负压引流对兔糖尿病溃疡创面组织愈合影响的观察

目的:探讨封闭负压引流(VAC)对兔糖尿病溃疡创面组织愈合的影响及其可能机制。方法:采用四氧嘧啶法建立兔糖尿病溃疡模型,设空白对照组和实验组(对照组创面采用常规包扎治疗处理,实验组创面则采用VAC处理),观察和比较两组动物的创面肉眼观、愈合时间,在致伤前、致伤后3 d、7 d、14 d取创面软组织,检测和比较两组动物的创面组织含水量、血流量以及血浆ET-1和NO含量。结果:与对照组比较,实验组动物的创面肿胀及分泌物得到明显控制,创面坏死组织的清除与肉芽组织的生长明显加快,平均愈合时间明显缩短(P0.05);致伤后3 d、7 d和14 d,创面组织含水量与血浆ET-1含量明显下降(P0.05),创面组织血流量与血浆NO含量明显增加(P0.05)。结论:VAC对兔糖尿病溃疡创面组织的愈合可起到积极的促进作用,这可能与其增加血浆NO含量及降低ET-1的含量有关,其具体机制尚有待于进一步的研究。     

Effect of Alternanthera brasiliana (L) Kuntze on healing of dermal burn wound

Wound healing activity of methanol extract of Alternanthera brasiliana [5% (w/w) ointment] was evaluated in experimental burn wound model in rats. Healing potential was assessed by the rate of wound contraction, estimation of anti-oxidants like catalase, superoxide dismutase, reduced glutathione, protein, vitamin C and hydroxyproline, along with histopathological examination on 8th day post wounding. The statistical data indicated that there was significant increase in wound contraction along with augmented level of antioxidants in granulation tissues in A. brasiliana treated group. Histopathological assessment of the granulation tissue revealed formation of epidermis with keratin layer and deposition of collagen fibers after treatment with the plant extract.     

MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13(-/-)) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13(-/-) mice. Granulation tissue in Mmp13(-/-) mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13(-/-) mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13(-/-) mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13(-/-) granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13(-/-) mice compared to WT mice. Mmp13(-/-) mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis.     

Morphological and cytological characteristics of the burn healing process by various treatment methods

Morphological and cytological methods were used to study the healing of a burn wound in local isolation wards (an open treatment method) and under ointment bandages (a closed treatment method). Biopsy specimens and imprints from the burn wound surface were taken from 40 patients. During treatment in an abacterial medium, burn wound was rapidly decontaminated, which was a consequence of the appearance in wound exudate of segmented neutrophils with marked neutrophilic granularity. Granulation tissue was formed in the wound cavity at the 5th-7th day after the treatment commencement. During treatment under bandages wound decontamination was extremely slow. For a long time wound exudate shows the predominance of dystrophically changed neutrophils, while granulation tissue in the wound cavity if formed only at the 21st-24th day after the treatment commencement.     

Electron microscopic immunochemical localization of actin in fibroblasts in healing skin and palate wounds of beagle dog

Wound contraction in soft tissue has been attributed to the activity of contractile fibroblasts containing actin microfilaments. Immunochemical staining at the electron microscopic level was used to demonstrate the presence of such cells in healing wounds from skin and oral mucosa. Biopsies of granulation tissue from 10 and 16 day old excision wounds in beagle palate mucoperiosteum and skin were fixed and 10 micrometer sections were treated with antiactin serum, peroxidase-anti peroxidase (PAP) and then incubated to reveal the localization of actin. Controls were prepared using non-immune serum or preabsorbed immune serum. Thin sections examined with the electron microscope revealed the presence of PAP particles associated with microfilament bundles beneath the plasma membrane and in processes of fibroblasts. Reaction was also associated with micropinocytotic vesicles at the cell surface. More reactive cells were seen in 16 day than in 10 day old wounds and there were greater numbers of these cells in skin than in oral mucoperiosteum. The results indicate that actin containing cells with the ultrastructural characteristics of contractile fibroblasts (myofibroblasts) are present in the granulation tissue of healing skin and oral mucosal wounds. Such cells may be responsible for the wound contraction observed clinically in the healing palatal mucoperiosteum.     

Differential stimulation of collagenase and chemotactic activity in fibroblasts derived from rat wound repair tissue and human skin by growth factors

Epidermal growth factor and cartilage-derived basic fibroblast growth factor (EGF and CD-bFGF) are mitogens shown to increase the rate of wound repair in animal models. In addition to being a mitogen for granulation tissue, CD-bFGF stimulates the recruitment of cells to the wound site. CD-bFGF and a closely-related chondrosarcoma-derived fibroblast growth factor stimulated chemotaxis of granulation tissue cells in vitro, each factor having a maximum activity at a concentration of 55 pM. Epidermal growth factor was also a potent chemoattractant for rat granulation tissue fibroblasts; however, maximum activity was obtained at 1.7 nM. Cells from all stages of wound repair were chemotactically responsive to these factors, but there was some attenuation of the response to bFGF in cells derived from fully-organized day 28 granulation tissue. Collagenase-catalyzed restructuring of collagen, an additional significant feature of wound repair, is probably critical to cell movement in an extracellular matrix. Cells derived from organizing (6-day old) sponge granulation tissue secreted latent collagenase constitutively in vitro. In the presence of serum, the production of collagenase was stimulated three-four fold by 1.8 nM bFGF derived either from cartilage or chondrosarcoma. When serum was present, as at a wound site, collagenase production was not enhanced by the addition of EGF. Cells from fully organized, day 21 sponge granulation tissue did not secrete latent collagenase constitutively and could not be stimulated to do so by the addition of EGF, bFGF, or phorbol ester. Human skin fibroblast collagenase production was also stimulated by bFGF and was refractory to EGF. While both classes of growth factor have the ability to promote wound healing, the varying responses they elicit in cell populations from the wound site emphasize the different pathways of cellular activation.     

Efficacy of Butea monosperma on dermal wound healing in rats

Wound healing occurs as a fundamental response to tissue injury. Several natural products have been shown to accelerate the healing process. The present investigation was undertaken to determine the efficacy of topical administration of an alcoholic bark extract of Butea monosperma (B. monosperma) on cutaneous wound healing in rats. Full-thickness excision wounds were made on the back of rat and B. monosperma extract was administered topically. The granulation tissue formed on days 4, 8, 12 and 16 (post-wound) was used to estimate total collagen, hexosamine, protein, DNA and uronic acid. The extract increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, total protein and total collagen content of granulation tissues. The extract treated wounds were found to heal much faster as indicated by improved rates of epithelialization and wound contraction, also confirmed by histopathological examinations. Also, the tensile strength of drug-treated wounds was increased significantly. In addition, we show that B. monosperma possesses antioxidant properties, by its ability to reduce lipid peroxidation. The results clearly substantiate the beneficial effects of the topical application of B. monosperma in the acceleration of wound healing.     

Vascular changes in maturing granulation tissue

Granulation tissue was examined in rats on the 20th and 40th day after wounding of the back. It was shown that by both the 20th and the 40th day the vessels with impaired typical structure could be revealed together with the normal ones in the granulation tissue. Specific rearrangement was observed in the structure of these vessels, consisting in the impairment of their wall integrity, separation of the constituent cells and their free position among the other cells and fibrous structures. This process is especially marked by the 40th day. 3H-thymidine was actively incorporated by some fibroblasts and rather often by the cells of both normal and degenerating vessels. The authors suggest the existence of an earlier unknown phenomenon of transformation of capillary vessels, common for both normal dermal and reparative processes after injury. The essence of the phenomenon consists in the fact that small dermal vessels get permanently disintegrated and are included into the composition of cellular elements of the interstitial tissue, and form again, providing for the physiological regeneration of dermal cells and fibrous structures.     

Sustained-release adrenomedullin ointment accelerates wound healing of pressure ulcers

Pressure ulcers are one of the most common complications in elderly, incontinent or paralyzed patients. For the healing of pressure ulcers, the development of granulation tissue and reepithelialization is required. Adrenomedullin (AM), an endogenous vasodilator peptide, is reported to stimulate the proliferation and migration of various cells including endothelial cells, fibroblasts and keratinocytes. Therefore, we hypothesized that AM might accelerate the healing process of pressure ulcers in which these cells were involved. We developed a sustained-release ointment containing human recombinant AM, and applied it in a mouse model of pressure ulcer twice a day for 14 days. Human AM was efficiently absorbed in wound area, but its blood concentration was negligible. AM ointment significantly reduced the wound area on day 5 to 7 after injury. In addition, AM ointment accelerated the formation of granulation tissue and angiogenesis as well as lymphangiogenesis after 7 days of treatment. Immunological analysis revealed that Ki-67-positive proliferating cells in granulation tissue expressed AM receptors. In summary, sustained-release AM significantly improved wound healing of pressure ulcers through acceleration of granulation and induction of angiogenesis and lymphangiogenesis. Therefore, sustained-release AM ointment may be a novel therapeutic agent for pressure ulcers.     

Quantitative characteristics of changes in the cellular composition and vascularization of aseptic wounds in rat skin, healing without treatment and during stimulation of repair processes by exogenous collagen

The changes in cellular composition and vascularization of aseptic wounds on the rat skin were assessed quantitatively using the ocular net without treatment and during stimulation of repair processes by exogenous collagen. An intensive increase in the number of macrophages, endotheliocytes and fibroblasts was observed in wounds without treatment by the fifth day, with maximum vascularization of the granulation tissue occurring by the seventh day. During stimulation of repair processes by collagen the macrophage reaction, proliferation of endotheliocytes and fibroblasts and vascularization of wounds were activated earlier, while the stereotype relationships of the cellular components remained unchanged. The intercellular relationships of the wound healing process are discussed.     

The effect of nerve growth factor on the early responses during the process of wound healing

In this study, we investigated the role of nerve growth factor (NGF)-incorporated collagen on wound healing in rats. Full-thickness excision wounds were made on the back of female rats weighing about 150-160 g. Topical application of NGF-incorporated collagen, at a concentration of 1 microg/1.2 mg collagen/cm(2), once a day, for 10 days resulted in complete healing of wounds on the 15th day. The concentrations of collagen, hexosamine and uronic acid in the granulation tissue were determined. The NGF-incorporated collagen-treated rats required shorter duration for the healing with an increased rate of wound contraction. Histological and electron microscopical evaluations were also performed, which reveal the activation of fibroblasts and endoplasmic reticulum and therefore increased level of collagen synthesis due to NGF application. These results clearly indicate that the topical application of NGF-incorporated collagen enhanced the rate of healing of excision wounds.     

Curcumin improves wound healing by modulating collagen and decreasing reactive oxygen species

Wound healing consists of an orderly progression of events that re-establish the integrity of the damaged tissue. Several natural products have been shown to accelerate the healing process. The present investigation was undertaken to determine the role of curcumin on changes in collagen characteristics and antioxidant property during cutaneous wound healing in rats. Full-thickness excision wounds were made on the back of rat and curcumin was administered topically. The wound tissues removed on 4th, 8th and 12th day (post-wound) were used to analyse biochemical and pathological changes. Curcumin increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, total protein and type III collagen content of wound tissues. Curcumin treated wounds were found to heal much faster as indicated by improved rates of epithelialisation, wound contraction and increased tensile strength which were also confirmed by histopathological examinations. Curcumin treatment was shown to decrease the levels of lipid peroxides (LPs), while the levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), activities were significantly increased exhibiting the antioxidant properties of curcumin in accelerating wound healing. Better maturation and cross linking of collagen were observed in the curcumin treated rats, by increased stability of acid-soluble collagen, aldehyde content, shrinkage temperature and tensile strength. The results clearly substantiate the beneficial effects of the topical application of curcumin in the acceleration of wound healing and its antioxidant effect. Both the authors have contributed equally towards this paper.     

Impaired cutaneous wound healing after sensory denervation in developing rats: effects on cell proliferation and apoptosis

The role of sensory nociceptor nerves in cutaneous wound healing was investigated following full-thickness 4-mm diameter dorsal cutaneous excision wounding of rats on postnatal day 12. In rats with intact innervation, wounds at 3 days contained large numbers of TUNEL- and BRDU-labeled nuclei, consistent with inflammatory cell death and granulation cell proliferation. Wound area and volume decreased through 11 days in concert with a transient appearance of alpha-smooth muscle actin-immunoreactive myofibroblasts, declining rates of cell division, and increased occurrence of apoptotic cells. Sensory denervation by capsaicin injections on postnatal days 2 and 9 reduced calcitonin gene-related peptide-immunoreactive wound innervation persistently by up to 43%. This was associated with increased wound surface area and volume, and delays in scab loss and re-epithelialization. Relative to control wounds, granulation tissue showed increased myofibroblast content at 5-7 days. Capsaicin-treated rats had more BRDU-labeled cells, including myofibroblasts, through day 7. Numbers of TUNEL apoptotic cells per unit area of tissue section were reduced by denervation in both early and late stages of healing. We conclude that partial loss of sensory innervation impairs cutaneous wound healing in developing rats, as manifested by delayed re-epithelialization and failure of the wound area to decrease normally through at least 21 days. This is associated with an abnormally enlarged wound tissue volume resulting from increased granulation cell proliferation without proportionate increases in apoptosis. These findings suggest that nociceptor innervation plays a critical role in wound healing by regulating wound cellularity.     

Characteristics of changes in lipids of granulation fibrous tissue of rats subjected to different doses and modes of melatonin administration

Melatonin influences the time-course of changes of lipids in granulation fibrous tissue in rats. Its effect depends on a dose, modes of administration (intraperitonial, subcutaneous or local) and duration of treatment. Intraperitonial administration of a single dose of melatonin (4 mg/kg) did not influence lipid content in the granulation fibrous tissue, while repeated injections of this hormone limited the increase in contents of lipids and phospholipids on the 5th and 8th days of regeneration. Long-term subcutaneous injections of melatonin caused distinct changes of lipids: at the dose of 0.3 mg/kg it prevented, and at the dose of 4 mg/kg it promoted the increase of lipid content in the granulation fibrous tissue on the fifth day of this study. Local application of melatonin solution (1.5 mg/ml) in early periods of regeneration caused insignificant changes of total lipids and total phospholipids in the granulation fibrous tissue. However, the higher concentration (15 mg/ml) of melatonin caused the decrease of total lipids due to reduced content of cholesterol and triglycerides and the increase of total phospholipids and some of their fractions.     

Appearance of tenascin in healing skin of the mouse: possible involvement in seaming of wounded tissues

Distribution of the extracellular matrix glycoprotein tenascin during wound healing in mouse skin was studied immunohistochemically. Within 24 hours after wounding, and preceding the formation of granulation tissue, tenascin appeared in the basement membranes beneath epidermis and hair follicles adjacent to the wound edges and in the wounded edges of cutaneous muscle layer. Granulation tissue began to form in the wound space at about 1-2 days and was immediately covered by epidermis. Tenascin first appeared in the periphery of the granulation tissue beneath healing epidermis and around the wounded edges of cutaneous muscle layer. Then the tenascin-positive area extended into the inner region of granulation tissue. At about 5-7 days, all of the granulation tissue was intensely stained with anti-tenascin serum. Tenascin immunoreactivity decreased as granulation tissue was replaced with reconstructed dermal tissue at 7-14 days. In most cases, tenascin staining persisted longest in the dermis beneath the healing epidermis and at the juncture of healing edges of cutaneous muscle layer. It disappeared at about 10-14 days after wounding. These findings suggest that tenascin may play an important role in the seaming of wounded tissues.     

VAC 技术治疗兔耳缺血性创面的实验研究

目的:观察间歇和持续负压下缺血创面不同处理与愈合的关系。方法:实验前1天,用脱毛剂(Nair,美国)对兔耳背脱毛。动物用1%戊巴比妥钠耳缘静脉注射麻醉(30 mg/kg体重),固定于手术台。75%乙醇消毒双侧耳背皮肤。距耳根3-3.5cm处分离、结扎兔耳中央神经血管束。在耳背中部形成直径2.5cm全层皮肤缺损创面(保留软骨膜)[1]。止血后置动物于特制木盒内。42只大白兔共84个创面,随机分为-50mmHg-75mmHg和-100mmHg 3大组,分别施以间歇负压(运行2分钟,停1分钟)和持续负压组。实验分别运用-50mmHg,-75mmHg,-100mmHg三个不同负压值进行连续、间歇治疗兔耳缺血性创面,观察伤后1,3,7,10,14,20d创面愈合情况,取伤后7d组织标本进行Western blot、HE染色,观察VEGF(vascular endothelial growth factor)的表达及创面上皮的再生和肉芽组织生长情况[1]。以及各时间点细胞凋亡的检测。结果:-50mmHg(纱布+海绵)间歇负压引流技术治疗兔耳缺血性创面的愈合最快,-75mmHg治疗组次之,-100mmHg治疗组创面愈合最慢。在同一时间点上,-50mmHg治疗组与-75mmHg,-100mmHg治疗组和空白对照组之间相比,能够更快地促进创面VEGF的表达和肉芽组织的再生,毛细血管增多。封闭负压治疗能够降低创面组织细胞的凋亡的发生。结论:(1)封闭负压治疗能够促进缺血创面的肉芽组织再生及VEGF的表达,减少创面组织细胞的凋亡的发生;(2)-50mmHg间歇封闭负压治疗效果最好。     

Effect of Ocimum sanctum Linn. on normal and dexamethasone suppressed wound healing

Ethanolic extract of leaves of O. sanctum was investigated for normal wound healing and dexamethasone depressed healing using incision, excision and dead space wound models in albino rats. The extract of O. sanctum significantly increased the wound breaking strength in incision wound model. The extract treated wounds were found to epithelialize faster and the rate of wound contraction was significantly increased as compared to control wounds. Significant increase in wet and dry granulation tissue weight, granulation tissue breaking strength and hydroxyproline content in dead space wound model was observed. The extract significantly decreased the antihealing activities of dexamethasone in all the wound models. The results indicated that the leaf extract promotes wound healing significantly and able to overcome the wound healing suppressing action of dexamethasone. Histological examination of granulation tissue to determine the pattern of lay-down for collagen confirmed the results.     

Influence of sea buckthorn (Hippophae rhamnoides L.) flavone on dermal wound healing in rats

The present investigation was undertaken to determine the efficacy of topical administration of flavone of sea buckthorn (Hippophae rhamnoides L.) on cutaneous wound healing in rats. Four full-thickness excision wounds were created on the back of rat and 1.0% w/v flavone prepared in propylene glycol was applied topically. Control animals received the vehicle alone in an identical manner. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, hydroxyproline, hexosamine, antioxidants estimation and histopathology of the granulation tissue. The sea buckthorn flavone promoted the wound healing activity as indicated by improved rate of wound contraction, decreased time taken for epithelialization (16.3 days versus 24.8 days in controls) and significant increase in hydroxyproline (26.0%) and hexosamine (30.0%) content. These findings were also confirmed by histopathological examinations. In addition, it was observed that sea buckthorn flavone possesses potent antioxidant properties as evidenced by significant increase in reduced glutathione (55.0%), vitamin C (70.0%) and catalase (20.0%) activities in wound granulation tissue. The flavone treatment also resulted in significant decrease in lipid peroxide levels (39.0%). The results suggest that the sea buckthorn flavone promotes wound healing activity.     

创伤合并海水浸泡后愈合过程的病理学观察

目的观察创伤合并海水浸泡后对伤口愈合时间的影响及愈合过程中的病理学改变。方法以大鼠为实验动物,建立背部双侧圆形创伤模型,创后随机分为对照组和实验组,每组50只,实验组创后置海水中浸泡30min。观察2组创面的愈合生长状况。观察伤口局部愈合过程中的病理学改变,应用免疫组化方法观察愈合过程中伤口修复细胞增殖指数及微血管密度变化。结果对照组平均愈合时间为（12.0±1.0）d,而实验组的平均愈合时间为（14.3±0.8）d;两组在肉芽组织形成时间上以及修复细胞增殖能力上存在差异。结论创伤合并海水浸泡可导致伤口愈合时间延迟,加重创伤局部的炎症反应、延缓肉芽组织形成时间。