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An analysis of modern scientific and practical literature leaves no doubt that among the many problems of a dentist, an important place is occupied by the optimization of wound healing of the oral mucosa in case of injuries. Immunodeficiencies of various origins, elderly and senile age, diabetes mellitus, and severe pathology of the cardiovascular system, hypoxic conditions and other somatic diseases reduce the repair potential of the oral mucosa. In addition, all types of metabolism are disrupted, synthesis and humoral transport are reduced, as well as the secretion of growth factors with saliva and their content in the blood and tissue. The healing process of the oral mucosa is slowed down by bacterial colonization of the wound surface, which can increase the area of inflammation. A common method of treating such patients in a dental clinic is the use of agents aimed at regenerative processes in tissues. Along with the positive qualities of modern drugs (good fixation, comfort when used in the oral cavity, wound healing, bactericidal effect on the damaged mucous membrane, reducing pain, accelerating the processes of epithelialization and scar organization), a noticeable weakening of their action in immunodeficient conditions should be noted. Based on this, it is advisable to use recombinant epidermal growth factor (EGFR) in the treatment of traumatic lesions of the oral mucosa, including those caused by the use of orthopedic structures, surgical interventions, especially those accompanied by conditions involving a decrease in the immune status.

Decreased neurogenesis after brain exposure to ionizing radiation is linked to neurocognitive impairments. Using transgenic mouse models, we previously showed that abrogation of radiation-induced senescence, or apoptosis, can partially rescue neurogenesis in the subventricular and hippocampus regions. Here, we evaluate whether the injection of recombinant epidermal growth factor (rEGF) or mesenchymal stromal cells (MSC) engineered to secrete EGF (MSC-EGF) can preserve neurogenesis. Using doublecortin (Dcx) expression and BrdU incorporation assays, we found that the injection of rEGF into the subventricular zone (SVZ) promotes neurogenesis, despite increasing apoptosis, in the brain of irradiated mice. The effect of rEGF was mostly localized, as Dcx expression was not induced in the hippocampus region and limited in the contralateral SVZ. Surprisingly, the injection of bone marrow-derived MSC alone, or secreting EGF, did not result in increased neurogenesis despite the fact that part of the MSC survived a few weeks after injection. Our results suggest that only a supraphysiological concentration of rEGF can promote neurogenesis, likely through a direct mitogenic effect.

OBJECTIVE To test whether or not topically administered recombinant human epidermal growth factor (rhEGF) accelerates the early healing phase of oral soft tissue wounds. METHODS One day following the creation of palatal defects (n = 6/animal), 14 dogs were allocated to one of the following five groups: spontaneous healing (SH), vehicle ointment (V), vehicle ointment + rhEGF at concentrations of 1 μg/g (EGF1), 10 μg/g (EGF10) or 50 μg/g (EGF50). Topical administration of ointments was repeated twice per day until sacrifice at days 8 and 16. Wound area was clinically monitored. Keratinocytes proliferation (Ki67-immunolabelling), inflammatory response (IR) and areas of collagen (C) and granulation tissue (GT) were histologically measured. Kruskal-Wallis test with Dunnett correction was used for multiple group statistical comparisons. RESULTS Clinically, in comparison with SH, a significantly smaller wound area was observed in groups EGF1 and EGF10 at day 8 (p

Recently, gene-editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) technique has attempted to utilize fibroblasts of livestock animals for somatic cell nuclear transfer. In this study, we establish the procedure for preparing skin fibroblast clones whose genes were edited by the CRISPR/Cas9 technique. After isolating fibroblasts from earlobes of Japanese Black cattle, subsequent collagenase-digestion and extensive wash procedures enabled us to avoid contamination of fungi. Electroporation using NEPA21, rather than lipofection using commercially available liposome reagents, allowed us to perform more efficient transfection of plasmid constructs. Although bovine ear-derived fibroblasts were not able to proliferate in single cell cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum, supplementation with insulin-transferrin-selenium mixture, human recombinant epidermal growth factor, or human recombinant basic fibroblast growth factor promoted proliferation of the cells, even in a single cell culture. Taking advantage of our established protocol, we eventually obtained eight ear-derived fibroblast clones with a recessive mutation in the isoleucyl-tRNA synthetase gene corrected by the CRISPR/Cas9 technique.

OBJECTIVES This study aims to compare the histological healing process and gene expression profile in tendon after the administration of human recombinant epidermal growth factor (hrEGF) and platelet-rich plasma (PRP). MATERIALS AND METHODS A total of 24 mature New Zealand white rabbits (6-month-old; weight 2.5-3.0 kg) were used in the study. Patellar tendons of rabbits were tenotomized and then repaired. Rabbits were separated into three groups and repair areas were injected with 1 mL hrEGF in group 1 (n=9) and 1 mL PRP in group 2 (n=9). No injection was performed in group 3 (controls, n=6). Tissue samples were obtained from the repaired patellar tendons of three rabbits each from groups 1 and 2 and of two rabbits from group 3 at the end of the first, second, and fourth weeks, and these tissues were histologically and genetically assessed. Expression levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, and chemokine receptors (CXCR1, CXCR2) were examined. RESULTS The frequency of neovascularization was detected to be higher in group 1 compared to group 3 at the end of the second and fourth weeks (p=0.018 and p=0.009, respectively). Group 1 was detected to show more increase in terms of the prevalence of tenocytes (p=0.014 and p=0.009, respectively) at the end of the second week, and in terms of collagen intensity at the end of the fourth week (p=0.0018 and p=0.034, respectively) compared to groups 2 and 3. Highest levels of TNF-α, IL-6, and IL-8 were detected in group 1, followed by groups 2 and 3 at all time points. Highest CXCR2 gene expression was detected in group 1. CONCLUSION Compared to PRP, hrEGF caused more increase in healing tissue at neovascularization, tenocyte, fibroblast, collagen, and tissue macrophage levels; and higher levels of TNF-α, IL-6, IL8, and CXCR2. Intralesional hrEGF administration can effectively accelerate tendon healing.

Background: Major complications of diabetes mellitus include cardio vascular disease, chronic kidney disease, diabetic foot ulcers and non-healing wounds. Lack of knowledge and awareness has led to worsening of wounds which can involve deeper tissues and bones also. If treated on time majority of patients can be prevented from undergoing major debilitating surgical procedures such as amputation of toes, foot etc.Methods: In this proposed study, 60 cases of diabetic wounds were studied for healing who were randomly allocated into two groups of 30 patients each. Group A included topical recombinant epidermal growth factor (EGF) application and group B included conventional Betadine dressing for wound healing.Results: Anaemia had a significant role in delaying wound healing in group B patients. Blood sugars had no significant role in delaying wound healing in this study as patients in group A had mean FBS more than group B and still the wound healing was observed better in group A patients. Topical recombinant EGF resulted in faster wound healing than conventional dressings in diabetic wounds. 90% wound healing rates were seen with topical EGF dressings in comparison to 36.67% in conventional betadine dressings. Signs of wound healing i.e., early granulation, decreased discharge, early wound closure were seen faster in group A. Moreover, all patients in group A showed healthy granulation tissue by the end of our study.Conclusions: We concluded that topical recombinant EGF resulted in faster and better wound healing than conventional betadine dressing.

Objective: The aim of this study was to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. Methods: 36 male Sprague Dawley rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: 1- Sham control group where no local augmentation was made, 2- Local Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) augmentation group and 3- Local HA/β-TCP + EGF augmentation group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Results: Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p = 0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p = 0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42–0.59), 0.73 Nmm (0.49–0.88) and 0.91 Nmm (0.66–1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p = 0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p = 0.087). Conclusion: This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates. Our findings may provide insights to further studies, investigating EGF's clinical usage as an alternative fusion enhancer. Keywords: Epidermal growth factor, Spinal fusion, Bone formation, Posterolateral fusion, Rat model

WOS: 000414976900026, PubMed ID: 29151322, Background/aim: Intralesional recombinant epidermal growth factor (EGF) is a new treatment approach for diabetic foot ulcer, approved in 2006. EGF therapy is given as an adjunct to the standard treatment regimen of antibiotics, surgery, and hyperbaric oxygen. EGF accelerates the healing of diabetic foot ulcers and reduces healing time. This single-center study was conducted to evaluate the outcomes of intralesional EGF therapy in patients with diabetic foot ulcers. Materials and methods: We present the data of the follow-up patients treated in our clinics. Fifteen patients with diabetic foot ulcers or infections, who had been followed up and treated in our clinics, were included in this retrospective study. All patients were administered intralesional injections of 75 mu g of EGF after treatment for infection on their diabetic foot ulcers, three times a week on alternate days. The patients were monitored with respect to treatment response and side effects of EGF. Results: Thirteen patients (86.7%) developed new granulation tissue, 10 patients (66.7%) had complete wound closure, and three patients (20%) showed partial wound closure. No serious side effects requiring discontinuation of EGF therapy were observed. A total of twenty-one bacterial agents were isolated in thirteen patients, and no bacterial growth was observed in the tissue cultures of two patients. Pseudomonas aeruginosa was the most common isolated infectious agent in the tissue cultures (n: 6, 28%). Conclusion: Intralesional injection of EGF on top of the standard treatment regimen appears to be a useful adjuvant therapy option in selected patients.

Apocrine sweat glands in bovine skin are involved in thermoregulation. Human, horse, and sheep sweat gland epithelial cells have been isolated and grown in vitro. The present study was conducted to identify a method to isolate bovine sweat glands and culture apocrine bovine sweat gland epithelial cells in vitro. Mechanical shearing, collagenase digestion, centrifugation, and neutral red staining were used to identify and isolate the apocrine glands from skin. Bovine sweat glands in situ and after isolation comprised 2 major cell types consisting of a single layer of cuboidal epithelial cells resting on a layer of myoepithelial cells. In situ, the glands were embedded in a collagen matrix primarily comprising fibroblasts, and some of these cells were also present in the isolated material. The isolated material was transferred to complete medium (keratinocyte serum-free medium, bovine pituitary extract, and human recombinant epidermal growth factor + 2.5% fetal bovine serum) in a T 25 flask (Falcon, Franklin Lakes, NJ) with media film and then incubated at 37°C for 24 h. After sweat glands adhered to the bottom of the flask, an additional 2 mL of complete medium was added and the medium was changed every 3 d. Isolated apocrine sweat glands and bovine sweat gland epithelial cells were immunostained for cytokeratin and fibroblast specific protein, indicating fibroblast-free cultures.

Background: Human recombinant epidermal growth factor has been shown to be effective in corneal healing when applied topically. The purpose of this preliminary study was to observe whether re-epithelization occurred in patients with non-healing corneal defects treated with a bandage contact lenses impregnated with epidermal growth factor. Design: Prospective non-comparative interventional case series study. Epidermal growth factor-impregnated bandage contact lenses (created through passive transfer of epidermal growth factor into hydrogel contact lenses of high water content) were used to passively release epidermal growth factor to the corneal surface of the damaged eye. Participants: Nine clinical patients who presented for tertiary care at the University of British Columbia Eye Care Centre at Vancouver General Hospital. Methods: All patients had clinically significant delayed corneal re-epithelization that had not healed despite standard treatments including conventional bandage contact lenses and topical medications. Causes of delayed re-epithelization varied from corneal injuries (e.g. alkali burns, recurrent corneal erosions) to recent corneal surgery (photorefractive keratectomy, phototherapeutic keratectomy, penetrating keratoplasty). Main Outcome Measures: Closure of wounds. Results: Re-epithelialization was seen in the corneas of seven of the nine patients within 8 days after insertion of the epidermal growth factor-treated bandage contact lens into the damaged eye. The drug delivery system appeared to be most effective in non-inflamed corneas. Conclusions: Preliminary results indicate that bandage contact lenses impregnated with epidermal growth factor may be helpful in promoting re-epithelization in corneas with delayed healing. Efficacy appears to be reduced for vascularized and significantly inflamed corneas.

Agonist and antagonist drugs acting on epidermal growth factor receptor (EGFR) signaling are emerging as a new possibility for pharmaceutical study and clinical manipulation of some skin and corneal disorders. EGFR activation appears to be effective in reducing the time of reepithelialization after corneal wound healing, with potential uses in penetrating keratoplasty, refractive surgery, alkali burns, diabetic keratopathy, keratopathy following chemotherapy, cornea transplantation, and dry eye. Most of the studies show therapeutic advantages of human recombinant epidermal growth factor (hrEGF) eye drops without showing adverse effects. In contrast, EGFR inhibition delays epithelial cell proliferation and stratification during corneal regeneration.The aim of this review is to summarize the most seminal discoveries and recent advances so as to clarify the role of the EGFR system in corneal physiology and pharmacology. Epidermal growth factor eye drops could be a first-choice treatment for promoting regeneration in numerous epithelial defects in the medium to long term.

Fernández‐Montequín JI, Valenzuela‐Silva CM, González Díaz O, Savigne W, Sancho‐Soutelo N, Rivero‐Fernández F, Sánchez‐Penton P, Morejón‐Vega L, Artaza‐Sanz H, García‐Herrera A, González‐Benavides C, Hernández‐Cañete CM, Vázquez‐Proenza A, Berlanga‐Acosta J, López‐Saura PA, for the Cuban Diabetic Foot Study Group. Intra‐lesional injections of recombinant human epidermal growth factor promote granulation and healing in advanced diabetic foot ulcers: multicenter, randomised, placebo‐controlled, double‐blind study. A multicenter, double‐blind, placebo‐controlled trial was carried out to evaluate the intra‐lesional infiltration of recombinant epidermal growth factor (EGF) in Wagner's grade 3 or 4 diabetic foot ulcers (DFUs). Subjects (149) were randomised to receive EGF (75 or 25 µg) or placebo, three times per week for 8 weeks and standard good wound care. The main endpoint was granulation tissue covering ≥ 50% of the ulcer at 2 weeks. It was achieved by 19/48 controls versus 44/53 in the 75 µg group [odds ratio (OR): 7·5; 95% confidence interval (CI): 2·9–18·9] and 34/48 in the 25 µg group (OR: 3·7; 1·6–8·7). Secondary outcome variables such as end‐of‐treatment complete granulation response (28/48 controls, 46/53 with 75 µg and 34/48 with 25 µg EGF), time‐to‐complete response (controls: 5 weeks; both EGF dose groups: 3 weeks), and wound closure after follow‐up (25/48 controls, 40/53 with 75 µg and 25/48 with 25 µg EGF) were also treatment dependent. Multivariate analyses yielded that they were significantly enhanced by 75 µg EGF treatment and neuropathic versus ischemic ulcers. Most adverse events were mild and no drug‐related severe adverse reactions were reported. It was concluded that recombinant human EGF (rhEGF) local injections offer a favourable risk–benefit balance in patients with advanced DFU.

An optimized process for protein encapsulation was applied to formulate epidermal growth factor (rhEGF)-poly-epsilon-caprolactone microspheres. Microparticles mean size was 3.8 microm +/- 0.2 and the encapsulation efficiency was 41.9% +/- 2.6. rhEGF recovery after the encapsulation process was approximately 70% (41.9% inside the microspheres and 30% still active in the external phase). In vitro release experiments in McIlvaine buffered solution showed a rhEGF sustained release over 4 days. Skin absorption studies conducted on full-thickness human skin using the Franz cell method showed that 20% rhEGF was released from the microspheres after 24 h exposure. Microspheres accumulated in the stratum corneum where they may act as a rhEGF reservoir. Therefore, rhEGF-PCL microparticles seemed to be promising systems due to their ability to provide locally a sustained release of rhEGF in skin layers.

Background : Intralesional recombinant epidermal growth factor (EGF) was produced in the Centre for Genetic Engineering and Biotechnology (CIGB), Cuba, in 1988 and licensed in 2006. Because it may accelerate wound healing, it is a potential new treatment option in patients with a diabetic foot wound (whether infected or not) as an adjunct to standard treatment (i.e. debridement, antibiotics). We conducted the initial evaluation of EGF for diabetic foot wounds in Turkey. Methods : We enrolled 17 patients who were hospitalized in various medical centers for a foot ulcer and/or infection and for whom below the knee amputation was suggested to all except one. All patients received 75 μg intralesional EGF three times per week on alternate days. Results : The appearance of new granulation tissue on the wound site (≥75%) was observed in 13 patients (76%), and complete wound closure was observed in 3 patients (18%), yielding a ‘complete recovery’ rate of 94%. The most common side effects were tremor ( n= 10, 59%) and nausea ( n= 6, 35%). In only one case,a serious side effect requiring cessation of EGF treatment was noted. That patient experienced severe hypotension at the 16th application session, and treatment was discontinued. At baseline, a total of 21 causative bacteria were isolated from 15 patients, whereascultures were sterile in two patients. The most frequently isolated species was Pseudomonas aeruginosa . Conclusion : Thus, this preliminary study suggests that EGF seems to be a potential adjunctive treatment option in patients with limb-threatening diabetic foot wounds. Keywords: diabetic foot; intralesional epidermal growth factor; treatment (Published: 11 August 2015) Citation: Diabetic Foot & Ankle 2015, 6: 28419 - http://dx.doi.org/10.3402/dfa.v6.28419

This research addresses the development and in vitro evaluation of lipid nanoparticle (NP)-based dressings to optimize the delivery of human recombinant epidermal growth factor (rhEGF) for the topical treatment of chronic wounds. The systems investigated were rhEGF-loaded solid lipid nanoparticles (rhEGF-SLN) and rhEGF-loaded nanostructured lipid carriers (rhEGF-NLC) formulated in wound dressings comprising either semi-solid hydrogels or fibrin-based solid scaffolds. Following detailed characterisation of the NP, in vitro diffusion cell experiments (coupled with dermatopharmacokinetic measurements), together with confocal microscopic imaging, conducted on both intact skin samples, and those from which the barrier (the stratum corneum) had been removed, revealed that (a) the particles remained essentially superficially located for at least up to 48 h post-application, (b) rhEGF released on the surface of intact skin was unable to penetrate to the deeper, viable layers, and (c) sustained release of growth factor from the NP “drug reservoirs” into barrier-compromised skin was observed. There were no significant differences between the in vitro performance of rhEGF-SLN and rhEGF-NLC, irrespective of the formulation employed. It is concluded that, because of their potentially longer-term stability, the fibrin-based scaffolds may be the most suitable approach to formulate rhEGF-loaded lipid nanoparticles.

Objectives This study was conducted to examine the tissue distribution of human recombinant epidermal growth factor (EGF) after multiple intravenous and subcutaneous injections in mice. Methods Male BALB/c mice were divided into (1) EGF 1 mg/kg intravenous dose, (2) EGF 5 mg/kg intravenous dose, (3) drug-free intravenous control, (4) EGF 1 mg/kg subcutaneous dose, (5) EGF 5 mg/kg subcutaneous dose and (6) drug-free subcutaneous control groups. EGF and drug-free dosing solutions were injected by intravenous and subcutaneous injections once a day for 3 days. EGF concentrations in serum and tissues of kidney, liver, lung, small intestine and tongue were determined by ELISA. Key findings As the intravenous and subcutaneous doses were increased from 1 to 5 mg/kg, serum Cmax and area under the concentration–time curve (AUC) values were increased dose-proportionally. In lung, tongue and small intestine, increases in AUC were dose-proportional after intravenous injections, but greater than dose-proportional after subcutaneous injections. The fold-increases in Cmax and AUC values were lowest in liver and highest in kidney. Conclusion Based on Cmax and AUC data, the systemic exposure achieved by subcutaneous injections was comparable with that achieved by intravenous injections.

Epidermal growth factor (EGF) is a potent stimulant of epithelialisation. However, topical application of EGF to achieve facilitated re-epithelialisation in partial thickness wounds has been controversial. A total of 10 pigs, each with eight 4 x 4 cm partial thickness wounds, were treated twice a day for 10 days to observe the effect of human recombinant EGF in concentrations of 0.1, 1, 5, 10, 25 ug/g, vehicle only and two controls. The control and the vehicle-only wounds each demonstrated 100% healing time (HT100) of 9.31 +/- 1.34 and 8.5 +/- 1.12 while the wounds treated with EGF ointment with concentrations of 0.1 (HT100 = 6.4 +/- 0.71), 1 (HT100 = 5.2 +/- 0.63), 5 (HT100 = 5.8 +/- 0.85), 10 (HT100 = 7.1 +/- 1.45) and 25 ug/g (HT100 = 7.4 + 0.57) demonstrated significant reduction in time to achieve re-epithelialisation. Among the EGF-treated wounds, the wounds treated with EGF concentrations of 1 and 5 ug/g achieved the fastest re-epithelialisation with evidence of substantial increase in basal keratinocyte activity observed through Ki-67 activity. In conclusion, this article demonstrates the efficacy of human recombinant EGF in facilitating re-epithelialisation of partial thickness wounds with the most efficient healing found in EGF concentrations of 1 and 5 ug/g.

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [ 3 H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [ 35 S]sulfate incorporation, and collagen synthesis by [ 3 H]proline incorporation. The presence of specific receptors was confirmed by [ 125 I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5–2.5 ng/ml) stimulated [ 3 H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (≤1%) and was lost at high (≥7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-β and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites ( Kd 0.18 n m ). There were about 4.5 × 10 9 binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions.

The intralesional injection of recombinant human epidermal growth factor (rhEGF) has been recently approved and introduced in several countries for the treatment of advanced diabetic foot ulcers (DFU), based on the results of five exploratory and one confirmatory, phase III clinical trials in 344 subjects. A significant stimulatory effect of this product on the healing process, given by development of granulation tissue and re-epithelization was shown in these trials, as well as a reduction in lesion recurrences during follow-up, and a tendency to a reduction of the risk of amputations, with an acceptable safety profile. However, products not always perform the same way in current medical practice. The present review summarizes the clinical information available from the intralesional use of rhEGF for advanced DFU and shows that in this case the postmarketing experiences in more than 2000 subjects confirm the results of the clinical trials, with 75% probability of complete granulation response, 61% healing, and a 16% absolute and 71% relative reduction of the risk of amputation. The benefit includes ischemic patients. The safety profile in current practice was satisfactory. Serious adverse events are not attributable to the treatment but to the underlying conditions of the patients. No evidence of neoplasia promotion by the growth factor has been found. The benefit-risk ratio of the procedure is favorable.

An enhanced formation of bone, dentin, and collagen fibers in periodontal wounds after application of polypeptide growth factors has recently been reported. However, the complex environment in vivo makes it impossible to determine the specific effects of growth factors on various cells involved in the wound-healing process. We have therefore investigated the mitogenic and morphogenic effects of recombinant epidermal growth factor (rEGF), natural platelet-derived growth factor (nPDGF), and natural fibroblast growth factor (nFGF) on periodontal ligament fibroblast-like cells. A cell line was established from rat PDL tissue. The cell line was characterized according to morphology, growth pattern, cytoskeletal proteins, and growth kinetics. The mitogenic effect of growth factors was assessed by incorporation of [3H]thymidine in the cellular DNA for 4 hours. Differences between groups of observations were assessed by the Student t-test. The morphogenic effects of growth factors were described with respect to growth pattern, cell orientation, and cell and nucleus form after a random photographic recording. The fibroblast-like cell type and the non-transformed phenotype of the cell line have been identified by the presence of parameters considered to be characteristic of a normal fibroblast-like cell line. The morphogenic analysis of both experimental and control cultures showed a monolayer of adherent cells with spindle or stellate morphology, a random alignment and round or elongated nuclei. Incorporation of [3H]-thymidine was increased in a dose-dependent manner by all growth factors. Maximal effect on the DNA synthesis was: rEGF, 131%; nPDGF, 274%; and nFGF, 182%.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of human recombinant epidermal growth factor (EGF) on the growth of MKN-28 human gastric carcinoma, transplanted into nude mice were studied. Modulation of tissue cAMP and EGF receptor levels by EGF was also studied to reveal the mechanism of the growth inhibitory effects of EGF. EGF exhibited a dose-dependent growth inhibitory effect on MKN-28 human gastric carcinoma transplanted into nude mice. Local injection of 2 ng of EGF moderately inhibited the growth of MKN-28 gastric carcinoma, while injections of 20 ng, 200 ng and 2 μg of EGF significantly inhibited tumour growth. EGF decreased tissue cAMP levels in a dose-dependent manner within 24 h after EGF injection. On the other hand, EGF increased the EGF receptor levels up to two or three fold within 24 h after EGF injection. Conversely, the EGF receptor affinity for EGF decreased according to the increase in EGF receptor levels.

We investigated the effects of epidermal growth factor on the survival and growth of caprine preantral follicles. Ovarian fragments were cultured for 1 and 7 days in enriched minimal essential medium with epidermal growth factor (0, 1, 10, 50, 100, or 200 ng/mL). Non-cultured and cultured tissues were processed for histological and ultrastructural studies. Results showed that after 7 days, the epidermal growth factor (1 and 10 ng/mL) maintained the percentage of normal follicles similar to control. An increase in the percentage of primary follicles was observed with 1, 10, and 50 ng/mL of epidermal growth factor compared to enriched minimal essential medium. Ultrastructural studies confirmed follicular integrity after 7 days in epidermal growth factor (1 and 10 ng/mL). In conclusion, the low concentrations of epidermal growth factor maintain caprine follicular viability and promote the transition from primordial to primary follicles.

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.

Recombinant epidermal growth factor (EGF) may be useful to treat severe ulcerative gastrointestinal injury. There is concern, however, that systemic use of this potent mitogen might increase tumour development and/or progression in susceptible subjects. We therefore examined the effect of chronic administration of systemic EGF to multiple intestinal neoplasia (Min) mice, who have a genetic defect in the adenomatous polyposis coli (APC) gene, leading to increased polyp development. Min mice (n=26) and wild-type littermates (n=26) received saline or EGF (223 μg of EGF/kg per day) for 4 weeks using subcutaneous osmotic mini-pumps. Cell proliferation and crypt fission were analysed using microdissection techniques and the number and size of polyps in the small and large intestines were determined. EGF increased wet weight and crypt cell proliferation rate by approx. 20% (all P

Organ cultures of human skin were incubated for 8 days under growth factor-free conditions or exposed to 10 ng ml−1 of human recombinant epidermal growth factor (EGF) during the incubation period. Normal histological features were preserved in the absence of growth factor, while epithelial cells underwent a proliferative response and invaded the underlying stroma in the presence of exogenous EGF. The same concentrations of EGF that induced stromal invasion also resulted in up-regulation of matrix metalloproteinase-9 (MMP-9; 92-kD gelatinase B) in organ culture and keratinocyte monolayer culture, and expression of MMP-1 (interstitial collagenase) in organ culture and fibroblast monolayer culture. When skin organ cultures were exposed to a potent, irreversible EGF–receptor tyrosine kinase (EGF–RTK) antagonist along with EGF, abnormal histological features were reversed, and MMP-9 production was suppressed. In contrast, EGF-RKT antagonism had only a modest inhibitory effect on MMP-1 production. Culture fluid from keratinocytes grown in monolayer culture stimulated fibroblast proliferation and MMP-1 elaboration. Treatment of fibroblasts with the same EGF–RTK antagonist inhibited keratinocyte-induced fibroblast proliferation but had only a modest inhibitory effect (approximately 20% inhibition) on MMP-1 production. In contrast, treatment of dermal fibroblasts with Interleukin-1 Receptor Antagonist had no effect on keratinocyte-induced fibroblast growth but strongly inhibited MMP-1 production (greater than 70% inhibition). These data indicate that stromal invasion by epithelial cells in EGF-treated skin is associated with events occurring in both the epidermis and dermis. The direct effect of the exogenous growth factor appears to be primarily on the epidermis. Dermal events reflect, at least in part, a response to factors elaborated in the epidermis. © 2001 Cancer Research Campaign http://www.bjcancer.com

Our aim was to study the efficacy of oral human recombinant epidermal growth factor (EGF) in the treatment of duodenal ulcers, on the basis of its repairing actions in the gastrointestinal tract.A placebo-controlled, multicenter, randomized, and double-blind study was conducted. Treatment groups were A) placebo solution, B) 10 microg/ml of human recombinant (hr)-EGF, and C) 50 microg/ml of hr-EGF, three times daily during 6 weeks. Patients, 15-65 years old, with a duodenal ulcer4 mm, who gave their written informed consent to participate were eligible. Exclusion criteria were gastric ulcer and more than one duodenal ulcer, ulcer-related complications, and previous treatment with oral EGF or other specific anti-ulcer drugs in the previous 2 weeks. The main outcome variable was ulcer healing, evaluated by endoscopy after the 2nd, 4th, and 6th week.One hundred and three patients were included. The groups were comparable with regard to age, sex, toxic habits, antecedents of ulcerous disease, initial size and depth or the ulcer, initial symptoms, and positivity for Helicobacter pylori. The ulcers were healed in a larger proportion of patients treated with hr-EGF at the highest dose (70.6% in group C versus 40.0% and 35.3% in placebo and low-dose groups, respectively (P = 0.007)). The difference was significant from week 4 on. Groups A and B did not differ. Eighty-eight percent of group C patients were cured or improved versus 57% and 56% in groups A and B, respectively. No adverse reactions were reported.Oral hr-EGF was effective in the treatment of duodenal ulcer at a 50-microg/ml dose every 8 h but not at 10 microg/ml.

We examined the effects of treatment with human recombinant epidermal growth factor (EGF) on the functioning small intestine in the rat. Male Wistar rats, 7-8 weeks old, were treated with EGF administered subcutaneously in doses of 0 (n = 7) or 150 micrograms/kg/day (n = 8) for 4 weeks. The histological composition and mucosal surface area of the perfusion-fixed small intestine was quantified with stereological principles. The length of the gut remained unchanged. The amount of tissue and surface area per length of gut (median (ranges)) were increased from 117 (101-131) mg/cm and 2.6 (2.1-3.5) cm2/cm in the controls to 146 (138-152) mg/cm and 3.5 (2.5-3.8) cm2/cm for the complete small intestine (both comparisons P0.02). The weight increase was due to mucosal growth in all parts of the intestine, whereas the surface area was only increased in proximal and middle parts. It is concluded that EGF treatment in rats increases the mucosal weight and surface area of the functioning small intestine.

1. The availability of recombinant epidermal growth factor provides a potentially exciting development for the treatment of gastrointestinal ulceration. However, because of its potent mitogenic activity, there is a need for strategies which reduce the dose required. Intestinal trefoil factor stimulates mucosal healing without increasing proliferation. Studies were undertaken to examine the biological effects of rat intestinal trefoil factor and/or human epidermal growth factor upon gastrointestinal epithelial cell functions pertinent to mucosal protection, using two wounding models. 2. The study of epithelial restitution in vitro demonstrated a marked synergistic effect on the rate of migration of the wound edge when intestinal trefoil factor was used in combination with epidermal growth factor. There was no increased cellular proliferation due to the addition of intestinal trefoil factor to the cells when given alone, or to the stimulatory effect of cells treated with epidermal growth factor. In the rat model of gastric ulceration, the presence of both epidermal growth factor and intestinal trefoil factor protected against the development of indomethacin-induced gastric lesions. 3. We conclude that combination therapy of epidermal growth factor with intestinal trefoil factor could provide a more potent, safer approach to the treatment of human gastrointestinal ulceration.

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.

OBJECTIVE: To determine whether extracts of unincubated osteoarthritis (OA) and rheumatoid arthritis (RA) synovial tissue contain connective tissue activating peptide-III (CTAP-III) isoforms and prostaglandin E2 (PGE2), and whether such extracts have growth-promoting activity, and to determine whether binary combinations of CTAP-III with other cytokines reported to be present in synovial tissue lead to synergistic, additive, or inhibitory effects on growth. METHODS: Acid-ethanol extracts of human synovium were examined for growth-promoting activity by measuring formation of 14C-glycosaminoglycan (14C-GAG) and 3H-DNA in synovial cell cultures; PGE2 was measured by enzyme immunoassay, and CTAP-III isoforms were identified by Western blotting of extracted proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Growth-promoting activity of CTAP-III and other cytokines was tested in synovial cultures treated with the agonists singly and in binary combination, by measuring changes in synthesis of 14C-GAG and 3H-DNA. RESULTS: Platelet-derived CTAP-III and a cleavage isoform with the electrophoretic mobility of CTAP-III-des 1-15/neutrophil-activating peptide-2 (NAP-2) and PGE2 were found in biologically active extracts of synovial samples from patients with RA and OA. Five growth factors (recombinant epidermal growth factor [rEGF], recombinant interleukin-1 beta [rIL-1 beta], basic fibroblast growth factor [bFGF], PGE1, and PGE2) in binary combination with CTAP-III showed synergism in stimulating GAG synthesis; two (recombinant platelet-derived growth factor type BB [rPDGE-BB] and recombinant transforming growth factor beta [rTGF beta]) had an additive effect. In combination with CTAP-III, rEGF and rPDGF-BB had a synergistic effect in promoting DNA synthesis, rTGF beta and rbFGF had an additive effect, and rIL-1 beta, PGE1, and PGE2 were antagonistic. CONCLUSIONS: The results suggest that, in addition to endogenous factors, CTAP-III and other platelet-derived cytokines may play roles in regulating synovial cell metabolism in RA and OA, and that combinations of growth factors may be more significant than single agents in amplification or suppression of important cell functions.

AIM:To determine whether recombinant human epidermal growth factor (rhEGF) can protect gastric mucosa against ethanol induced injury in rats.METHOD: Fifty-four SD rats weighing 200g-500g each were divided into six groups after fasting for 24 hours.Three groups received different doses of oral rhEGF (30, 60 and 120mgr;gcenter dotkg(-1)center dotd(-1)), one group was given cimetidine,one subcutaneous rhEGF (rhEGF IV) and one received saline as control.RESULTS:Acute gastric dilatation developed in the control and cimetidine groups and bloody gastric juice was found in the control group. The ulcer index was 58 in control group, 53 in rhEGF I, 46 in rhEGF II (P0.01), 11 in rhEGFIII (P0.01), 19 in rhEGF IV (P0.01), and 39 in cimetidine group (P0.05).CONCLUSION: rhEGF protected gastric mucosa against ethanol induced damage. The effect was dose-dependent with blood levels of epidermal growth factor (EGF) at a dosage range of 60mgr;gcenter dotkg(-1)center dotd(-1)-120mgr;gcenter dot kg(-1)center dotd(-1). It was more effective by injection than via oral route at the same dosage.

Athymic mice (C3H/HeN) parasitized by Brugia malayi develop massively dilated lymphatics. The lymphatic endothelial lining is perturbed, and numerous mononuclear and giant cells are closely apposed to the endothelium. The hyperplastic endothelial cells and low opening pressure of the lymphatics suggest abnormal multiplication of these cells may be important in the dilation. We studied the in vitro growth rate of human umbilical vein endothelial cells cultured with adult worms and microfilariae of B. malayi. The tetrazolium salt reduction assays were used to quantify possible direct mitogenic or inhibitory effects. The growth factor-induced proliferation of endothelial cells was significantly suppressed by 44-51% on day 1, 46-81% on day 3, and 45-79% on day 5 in cultures containing adult female worms, which had greater suppressor activity on endothelial cell proliferation than male worms, microfilariae, or soluble adult worm extract. Culture supernatant containing female worm excretory-secretory products significantly inhibited the growth and multiplication of cells, suggesting that adult female worms release antigens or proteins that have inhibitory activity on growth factors necessary for endothelial cell proliferation in vitro. Excess human recombinant epidermal growth factor and bovine brain extract partly reversed the inhibitory activity of worms in culture and restored the endothelial cell proliferation when incubated with worm culture supernatant. Indomethacin and BW 775Hcl failed to restore normal endothelial proliferation in the presence of female worms, suggesting that parasite-derived prostanoids and cyclooxygenase products did not cause the inhibition. Lymph from dilated lymphatics, but not serum from infected mice, increased the proliferation of cells in vitro. Together, these data demonstrate that excretory-secretory products of B. malayi parasites suppress vascular endothelial proliferation in vitro. Furthermore, increases in the number of these cells in vitro in the presence of lymph suggest that parasite-induced host factors may be important in modulating the degree of proliferation.

Ex vivo expansion of hematopoietic stem cell (HSC) is an attractive technology for its potency of a variety of clinical applications. Such a technology has been achieved to some extent with combinations of various cytokines or continuous perfusion cultures. However, much more improvement is required especially for expansion of primitive hematopoietic progenitors. We propose here a novel molecular approach that might have the potential to compensate the current expansion. We designed an adenovirus vector to transiently express human epidermal growth factor receptor (EGFR), which is known to transduce only a mitogenic, but not a differentiation signal to mouse bone marrow cells on human purified CD34+ peripheral blood (PB) cells, and tried to expand these cells with EGF ex vivo. Because we found that exposure of CD34+ PB cells to cytokines induced surface expression of adenovirus-internalization receptor and rendered these cells permissive to adenovirus infection, we infected these cells with the adenovirus vector carrying EGFR gene in the presence of cytokines. Two-color flow cytometric analysis demonstrated that 60.3% ± 22.4% of CD34+ cells expressed the adenovirus-mediated EGFR. Moreover, long-term culture-initiating cell assay showed that adenovirus vector could transduce more primitive progenitors. Subsequently, we tried to expand these cells in suspension culture with EGF for 5 days. Methylcellulose clonal assay showed that EGF induced 5.0- ± 2.4-fold proliferation of the colony-forming unit pool during 5 days of expansion. The simple procedure of efficient adenovirus gene delivery to immature hematopoietic cells proved promising, and this technique was potentially applicable for a novel strategy aiming at ex vivo expansion of hematopoietic progenitors.