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Growth factor-collagen relationship in wound healing
Abstract
Various growth factors such as epidermal growth factor (EGF) are effective in wound healing. For this reason, it was targeted in our study to observe the impact of systematically given EGF on collagen and protein synthesis in hepatic tissue. Rats to which dorsolateral excisional wound was made were divided into 2 equal groups: 1-Untreated control group, 2-Systemic EGF applied group. A daily dose of EGF (10 ng/mL) was given to the animals intraperitoneally (ip) in the application group. Rats were sacrificed in 1st, 5th, 7th and 14th days of the study. Collagen and protein concentrations in hepatic tissues were measured spectrophotometrically. In consideration of the phases of systemic EGF application and its comparison with the controls, it was determined that EGF stimulates the synthesis of collagen up to 7 days and protein up to 5 days in liver and this stimulation stops in the later days and EGF begins to make pressure on collagen and protein synthesis.
... The first event after the synthesis of procollagen chains on ribosomes is their introduction into the rough endoplasmic reticulum. They underwent a series of post-translational modifications there, which led to the assembly of procollagen molecules [21]. ...
One of the most abundant proteins formed in the human body is collagen. It is responsible for the strength of body tissues in all cellular systems by establishing support networks. Collagen fibers are generally opaque and white, due to this reason they are easily recognized in the tissues. Collagen fibers, which are viscoelastic, have low extensibility and high tensile strength. The isoelectric point is around pH 5.8. Collagen is widely used as it helps blood coagulate, remodeling tissue. Although there are no known side effects, there is some concern about its role in inflammation, group-to-group variability, and possible disease transfection, given that animal-derived (natural) collagen is used in many clinical applications. Not every protein is of equal value, so not every collagen is of equal value. Thanks to its low molecular weight, hydrolyzed collagen is quickly digested by our body and enters our blood circulation. According to study, the most suitable type of collagen is the one in powder or liquid form for collagen peptides that can be easily absorbed and used by our body. It is necessary to rely on the most appropriate analysis technique to evaluate the quality feature. Techniques such as SEC, MS, HPLC and NMR are used to characterize complex peptide mixtures. Classical fibril-forming collagen includes collagen types I, II, III, V, and XI. These collagenes are characterized by their ability to aggregate into highly oriented supramolecular aggregates, forming fibrillar arrays with diameters between 25 and 400 nm. Being a natural protein and its technical characteristics, it is expected that development and consumption of collagen hydrolyzate will increase in the coming years. In this review, physical and chemical properties of collagen, types, pharmacology properties, quantity determination methods are briefly described.
A visible cutaneous scar develops from the excess formation of immature collagen in response to an inflammatory reaction. This study examined the role of epidermal growth factor (EGF) in the formation of cutaneous scars. Twenty Crl:CD-1 (ICR) mice were used and 2 full-thickness skin wounds were made on the dorsum of each mouse. One of the wounds was treated with recombinant human EGF by local application and the other was treated with saline for control until complete healing was achieved. The EGF-treated group's wounds healed faster than the control group's. The width of the scar was smaller by 30% and the area was smaller by 26% in the EGF-treated group. Inflammatory cell numbers were significantly lower in the EGF-treated group. The expression of transforming growth factor (TGF)-beta(1) in the EGF-treated group was increased. It was observed that the amount of collagen in the EGF-treated group was larger than the control group. In the EGF-treated group, the visible external scars were less noticeable than that in the control group. These results suggest that EGF can reduce cutaneous scars by suppressing inflammatory reactions, decreasing expression of TGF-beta(1), and mediating the formation of collagen.
The tooth organ provides a model for discrete patterns of morphogenesis over short periods of developmental time. Studies were designed to test the hypothesis that endogenous epidermal growth factor (EGF) functions to regulate multiple cusp molar tooth morphogenesis during embryonic mouse development. The relative levels of endogenous EGF and EGF receptor (EGFR) transcripts were determined in both enamel organ epithelia and dental ectomesenchyme by reverse transcription-polymerase chain reaction (RT-PCR) assays. EGF and EGFR were localized by immunohistochemistry; both antigenic determinants were demonstrated on the same odontogenic cells in cultured tooth explants. To examine EGF-mediated signal transduction, cap stage mouse molar tooth organs (E16) were cultured in serumless, chemically-defined medium as either (i) controls, or supplemented with (ii) tryphostin (an EGF receptor kinase inhibitor), (iii) tyrphostin plus exogenous EGF, and (iv) exogenous EGF. Antisense oligodeoxynucleotide (ODN) strategy was used to investigate the functions of endogenous EGF employing (i) non-treated control, (ii) sense ODN control, (iii) antisense ODN, (iv) exogenous EGF, (v) sense ODN with exogenous EGF, and (vi) antisense ODN with exogenous EGF. Tyrphostin inhibited DNA synthesis and produced a significant decrease in the volume of the explants. These effects were recovered by addition of exogenous EGF. Antisense ODN inhibition resulted in abnormal cusp formations, decreased DNA synthesis, total DNA, RNA and protein content, and decreased stellate reticulum and tooth explant volumes. The decreased tooth size was not uniform, the most pronounced effect was in the stellate reticulum. This pattern of changes was not seen when antisense ODN treatment was supplemented with exogenous EGF. These results suggest that during cap stage of odontogenesis endogenous EGF acts to stimulate DNA synthesis, which increases the cell number of specific phenotypes within the enamel organ epithelia, and thereby regulates molar tooth morphogenesis.
This work was undertaken to study the effects of various doses of locally applied epidermal growth factor (EGF) on developing granulation tissue in rats. Cylindrical hollow sponge implants were used as an inductive matrix for the growth of granulation tissue. In the test groups, the implants were injected daily with a solution containing 0.2, 1, or 5 micrograms of EGF in 0.1% albumin while the implants of the control group were treated correspondingly with the carrier solution only. Analyses of granulation tissue in the sponge cylinders were carried out 7 days after implantation. A stimulatory, dose-dependent effect of EGF on granulation tissue formation was observed: cellularity increased, as evidenced by the elevated amounts of nucleic acids, and accumulation of collagen and glycosaminoglycans was enhanced.
The effects of epidermal growth factor (EGF) on granulation-tissue formation and collagen-gene expression were studied in experimental sponge-induced granulomas in rats. After daily administration of 5 micrograms of EGF into the sponge, total RNA was extracted from the ingrown granulation tissue at days 4 and 7 and analysed by Northern hybridization for the contents of mRNAs for types I and III procollagens. EGF treatment increased procollagen mRNA, particularly at day 4. To determine whether this elevation was due to increased proliferation of collagen-producing fibroblasts or to activation of collagen-gene expression in these cells, fibroblast cultures were started from granulation tissue and treated with EGF. These experiments confirmed that EGF is a potent mitogen for granuloma fibroblasts in a dose-dependent manner. The effect of EGF treatment on radioactive hydroxyproline production in cultured cells was inhibitory. The decreased rate of collagen synthesis was also indicated by decreased amounts of procollagen mRNAs. The results suggest that the stimulation of wound healing and collagen production by EGF is due to increased fibroblast proliferation, and not to increased expression of type I and III procollagen genes.
Growth factors exert a powerful influence over wound healing. They control the growth, differentiation, and metabolism of cells. Although their influence on wound repair has been studied, there are only a few reports in which growth factors applied topically exerted a positive influence on wound repair. However, there is no doubt that they do control the wound environment. It is likely that their actions will be defined further, and the wound environment will be able to be controlled to achieve complete and durable wound healing in patients.
Traumatic wounds can be labor intensive and expensive to manage. Furthermore, full-thickness wounds often must heal by second intention, which ultimately leads to scarring. The optimal objective of repair is re-establishment of the epithelial cover and recovery of tissue integrity, strength and function. Veterinarians can positively influence wound repair by understanding its mechanisms, which will ensure selection of appropriate wound management techniques. Repair begins the moment a cellular barrier is broken and follows a predictable pattern. The events can be divided into synchronized and interrelated phases including: acute inflammation, cellular proliferation, and finally, matrix synthesis and remodeling. The exact manner in which dermal repair is regulated is not fully understood, but it is increasingly apparent that the process involves complex interactions between many cell types, their mediators (in particular cytokines and growth factors) and the extracellular matrix. This article will review the cellular, physiologic, biochemical and molecular mechanisms involved in the repair process.
The process of wound healing begins immediately following surface lesions or when skin proteins become exposed to radiation, chemical damage or extreme temperatures. Wound repair requires close control of degradative and regenerative processes, involving numerous cell types and complex interactions between multiple biochemical cascades. Growth factors released in the traumatized area promote cell migration into the wound area (chemotaxis), stimulate the growth of epithelial cells and fibroblasts (mitogenesis), initiate the formulation of new blood vessels (angiogenesis), and stimulate matrix formation and remodeling of the affected region. Animal studies have shown that exogenously added growth factors can accelerate the normal healing process. Growth factors have also been used successfully in humans to treat previously incurable wounds. The most intensively studied growth factors are EGF, FGFs, PDGF, TGF-, and TGF-s. Each of these factors is currently the focus of intense commercial development.
Epidermal growth factor (EGF) has been reported to stimulate healing of wounds in skin, cornea, and gastric mucosa. In the present study, we further investigate the effect of endogenous and exogenous EGF in healing of connective tissue wounds using the rat perforated mesentery model. Healing of mesenteric perforations is accomplished by the connective tissue fibroblasts since there are no interfering variables such as interactions of epithelial cells, desiccation, or foreign materials such as sutures or subcutaneous implants. We performed laparotomy in 114 adult male Sprague-Dawley rats and made 20 standardized perforations in the mesentery of each rat with a scalpel. Rats were randomly assigned to five groups. Group I received no treatment after surgery; Group II received intraperitoneal injections of phosphate-buffered saline (PBS) after surgery and then twice daily for the following 3 days; Group III received 10 micrograms of EGF in the PBS injections according to the same regimen as Group II; Group IV had sham exploration of the submandibular salivary glands; and Group V animals had excision of the submandibular glands 3 days before laparotomy to deprive the main source of EGF in rat. On Days 4 through 10 after surgery rats were sacrificed and the percentage of perforations in each rat which were closed was determined. The curves for the time course of wound closure for Groups IV and V were not different indicating that endogenous submandibular EGF does not play a role in healing of mesenteric wounds.(ABSTRACT TRUNCATED AT 250 WORDS)
A great deal of interest has been focused recently on the potential use of synthetic polypeptide growth factors to stimulate healing of chronic wounds. In this pilot double-blind randomized study conducted at a single center, we used human recombinant epidermal growth factor (h-EGF) to treat 44 patients with venous ulceration of the lower extremities. An aqueous solution (10 micrograms/mL) of h-EGF was applied topically to the ulcers twice a day until healing occurred or for a maximum of 10 weeks. Patients were evaluated weekly for measurements of ulcer size and for the formation of granulation tissue suitable for grafting. Nine patients were excluded from efficacy evaluation because of protocol violations. Therefore, 35 patients (17 h-EGF, 18 placebo) were evaluable for efficacy, and 44 patients (22 h-EGF, 22 placebo) were available for safety. The median baseline ulcer size for all patients was 18.5 cm2, and was not significantly different between h-EGF and placebo group (12.9 cm2 versus 19.2 cm2, respectively, P = .27). By study end, six (35%) of h-EGF treated patients and two (11%) in the placebo group had healed completely (P = .10). Another 6 patients (2 of 17 h-EGF, 4 of 18 placebo; P = .50) developed healthy granulation tissue that was suitable for grafting. The median ulcer size reduction was 7% for h-EGF versus 3% for placebo per week (P = .29), and 73% versus 33% at study end (P = .32). No untoward side effects were related to the application of h-EGF. We conclude that topical application of h-EGF, in the dose and manner used in this study, was safe but failed to significantly enhance re-epithelialization of venous ulcers. However, a greater reduction in ulcer size and a larger number of healed ulcers with the use of h-EGF are encouraging results.
The healing of acetic acid-induced gastric and duodenal ulcers was examined together with biochemical indices of growth in gastric and duodenal mucosa in rats with intact or removed salivary glands after treatment with epidermal growth factor (EGF) or somatostatin, or both. After the extirpation of salivary glands, the healing rate of gastric and duodenal ulcerations was delayed and gastric content of immunoreactive EGF was reduced. This was accompanied by a significant decrease in the contents of deoxyribonucleic acid and ribonucleic acid in the gastric and duodenal mucosa. Repeated administration of EGF either subcutaneously or orally accelerated the healing of gastroduodenal ulcers in rats with intact salivary glands and completely reversed the delay in ulcer healing in sialoadenectomized animals. These effects were also accompanied by a significant increase in the growth parameters of gastric and duodenal mucosa. Administration of somatostatin, which prevented the growth-promoting action of subcutaneous EGF, resulted in a significant decrease in the EGF-stimulated healing of gastric and duodenal ulcerations in both intact and sialoadenectomized rats. Our findings suggest that cell proliferation is an important factor in healing of gastric and duodenal ulcerations and that EGF plays an important role in ulcer healing due to its mitogenic action.
This review summarizes briefly some of the historical aspects of the discovery, isolation, and mode of action of epidermal growth factor (EGF). Also considered are some of the key experiments which relate the biochemical mechanisms of EGF action to the mechanism involved in oncogenic transformation by the retroviruses.
The failure of chronic wounds to heal remains a major medical problem. Recent studies have suggested an important role for growth factors in promoting wound healing. We investigated the mitogenic effect of basic fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF), comparing their effects with those of media alone (MEM) in a human skin explant model. A stable organ culture system for maintaining the histologic structure of human epidermis for 10 days in vitro was developed. DNA synthesis was measured on Days 1, 3, and 7 of organ culture using [3H]thymidine ([3H]thy) uptake and expressed as cpm/mg dry weight (mean +/- SEM). FGF, IGF-1, and EGF were each capable of stimulating [3H]thy uptake on Day 1 of culture (2372 +/- 335 FGF, 2226 +/- 193 IGF-1, 4037 +/- 679 EGF vs 1108 +/- 70 MEM, P < 0.05). IGF-1 and EGF also stimulated [3H]thy uptake on Days 3 and 7 of culture. The organ culture system was further employed to observe epidermal outgrowth. Longest keratinocyte outgrowth from the explant periphery (simulating epithelial regeneration from the wound edge) was observed on Day 7. EGF resulted in maximum stimulation of epithelial outgrowth (440 +/- 80 microns), followed by FGF (330 +/- 56 microns), IGF-1 (294 +/- 48 microns), and MEM (189 +/- 50 microns). We postulate, therefore, that FGF, IGF-1, and EGF are important mitogens for wound healing and that EGF in particular is capable of stimulating epithelialization.(ABSTRACT TRUNCATED AT 250 WORDS)
Liposomes, modified into bioadhesive systems by the covalent anchoring of hyaluronic acid (HA) to their surface, were investigated for their abilities to act as site-adherent and sustained-release carriers of drugs for the topical therapy of wounds and burns. Epidermal growth factor (EGF) served as the test drug for growth factors and monolayers of the A431 cell line served as models of the in vivo designated targets. The hyaluronic acid was radiolabeled to carry 3H, through a deacetylation-reacetylation procedure, yielding specific activities in the range of 1 x 10(6) dpm/mg. The HA was bound to the surface of the liposomes, using carbodiimide to crosslink its carboxyl residues to amine residues at the liposomal surface. The latter was provided through the inclusion of phosphatidylethanolamine in the liposome formulation. Varying the initial HA/lipid ratio over the range 1-500 mg HA/mmol lipid, it was found that the increase in bound HA was linear, providing up to 80 mg HA/mmol lipid. The HA-modified, but not the nonmodified, liposomes were found to bind with high affinity (delta G degree in the range of -7 to -9 Kcal/mol) to monolayers of the A431 cell line. The HA-modified liposomes were found to encapsulate EGF at high yields (% encapsulation, > 87) and to act as sustained-release depots, the rate constant for the release of the encapsulated EGF in the range of 1.4 x 10(-3) h-1. Taking all of these findings together, it is concluded that these liposomes are bioadhesive sustained-release carriers of drugs, as desired, meriting further cellular and in vivo studies.
The effect of topically administered epidermal growth factor (EGF) dosage forms was investigated on skin wound healing in mice. Two EGF dosage forms were prepared containing 100 ng mL-1 EGF. The solution dosage form was prepared in 0.9% w/v NaCl. A bioadhesive gel form was prepared in 0.2% Carbopol 940 polymer. The two dosage forms were applied on the skin incision wounds of mice at the rate of 5 microL twice a day for 7 and 15 days. The wound tear strength was tested for skin wound healing at the 7th and 15th days of treatment and compared with controls. The results indicate that the wound tear strength of mice were significantly higher at the 15th day of treatment in the gel-treated group compared with the solution-treated mice and controls (P < 0.001).
The tolerability and efficacy of epidermal growth factor (EGF) in the topical treatment of herpetic corneal ulcers in addition to topical acyclovir have been evaluated in a double-blind, placebo-controlled, randomized study in two groups of patients. The time required for complete reepithelialization of the cornea was recorded, and the data obtained were analyzed statistically. In the EGF group, the reepithelialization was significantly faster than in the control group. Tolerability of EGF was always excellent. These results indicate that EGF is safe and effective in reducing the healing time of herpetic corneal ulcers.
A procedure for the determination of collagen concentration is described. It is simple and sensitive and is based on a modification of Lowry's method. The modification includes an initial heating of collagen samples in alkaline solution and changes in the concentrations of reagents used. It may be used for different collagen types, i.e. I, II, V and XI, from various sources as well as for gelatine. The influence of different buffers, salts, detergents, etc. on the precision of the method was studied. The precision of the measurements was not influenced by pH, the turbidity of the sample and the presence of certain salts, osmolytes and preservatives, routinely used in biochemical, microbiological and immunological experimental protocols. The modification is suitable for routine determinations of collagen concentration in a wide variety of samples.
The healing of wounds caused by accident, assault, welfare and surgical operations has always been a central consideration in surgical practice because any breach in continuity of skin or mucous membrane exposes the deeper tissues to the danger of infections. The understanding of the mechanism of wound healing has increased dramatically during last few years. Today wound healing abnormalities are among the greatest causes of disability and deformity. "I dressed the wound, God healed it" (Ambroise Pare) wound healing involves multiple complicated events. It is the amount and quality of scar tissue and ultimately its remodelling that is of greater importance. The understanding of this process of wound healing and factors affecting it forms the basis of any surgical procedure.
Various growth factors play significant roles during the process of wound healing. A systematic study was carried out to evaluate the effects of topically applied epidermal growth factor (EGF) and platelet-derived growth factors (PDGF-BB, PDGF-AB, PDGF-AA) in various concentrations on wound healing. Various combinations of these growth factors were also studied to find the best combination for wound healing.
Wounds were created on a mouse model. Various concentrations and combinations of EGF and PDGFs were applied topically and the effects on wound healing were monitored visually.
EGF, PDGF-AB and PDGF-BB when applied alone or in combination, healed the wounds in significantly less time and with less scar tissue formation compared to the controls. The best results were obtained with a combination of EGF (10 ng) and PDGF-BB (15 ng). PDGF-AA even at the maximum concentration of 20 ng did not enhance the wound repair process. A combination of PDGF-AA (20 ng) with EGF (10 ng) did not enhance the wound repair process either.
Topically applied EGF, PDGF-AB and PDGF-BB enhanced the wound repair process and had a role in decreasing the formation of scar tissue. PDGF-AA either alone or in combination with EGF did not enhance the wound repair process significantly. It appears that PDGF-AA might be limiting the EGF-mediated wound repair and such observation has not been previously reported in studies on wound healing using topically applied growth factors.
Extracellular matrix components play a key role during the angiogenic process for a correct development of blood vessels: fibroblasts are the main cell type involved in the regulation of ECM protein production. In this study we characterize H. medicinalis fibroblasts and demonstrate that they take part to the regulation of angiogenesis that occurs during wound healing process. Massive proliferation and phenotypic modification are two distinctive markers of fibroblast activation. These cells, that are usually responsible for collagen production and function as an energy reservoir, are recruited during wound healing to form a collagen scaffold through a direct mechanic action and through secretion of specific proteoglycans. In addition we show that the activity of fibroblasts is modulated by EGF, a growth factor involved in wound healing in vertebrates. The formation of bundles of collagen fibrils by fibroblasts is fundamental for the development and migration of new blood vessels in lesioned areas during wound repair: administration of lovastatin in explanted leeches affects fibroblasts, damages collagen "scaffold" and indirectly causes the reduction of neo-capillary formation.
LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the actions of inflammatory mediators, growth factors, and nitric oxide involved in wound healing. 2. Describe the different cellular elements and their function in wound healing. 3. Discuss the three phases of wound healing and the relationships between mediators and cells for each. 4. Discuss the similarities and differences between keloids and hypertrophic scar and the treatment options for each. 5. Discuss the systemic and external factors involved in wound healing. 6. Discuss future wound-healing opportunities. SUMMARY: Understanding wound healing today involves much more than simply stating that there are three phases: inflammation, proliferation, and maturation. Wound healing is a complex series of reactions and interactions among cells and "mediators." Each year, new mediators are discovered and our understanding of inflammatory mediators and cellular interactions grows. This article will attempt to provide a concise overview on wound healing and wound management.