Due to well-known disadvantages of the autologous bone graft, many alternatives have been studied for a reliable spinal fusion. Herein, we aimed to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. 36 male SD rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: Sham control group, Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) group and HA/β-TCP + EGF 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. 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). This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates

Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd

Background: The c-ebB-2 gene and its products (also designated HER-2 and c-neu) encode for a 185-kd transmembrane glycoprotein with intracellular tyrosine kinase activity. C-erbB-2 belongs to the epidermal growth factor receptor family, of which there are four known members, and has molecular homology to the epidermal growth factor receptor. It seems that this family is critical in control of growth, differentiation, and mobility of many normal and transformed epithelial cell types. Materials and Methods: We have looked for over expression of c-erbB-2 gene product in 230 cases of soft tissue sarcoma, in order to establish a possible new prognostic marker, and a potentially new treatment option. Results: In all the cases, irrespective of the sarcoma histological type, the immunostaining for erbB-2 was negative. Conclusions: Applications of erbB-2 for prognostication as well as the option of receptor targeting by trastuzumab monoclonal antibodies were aborted

Introduction and Objective. Bone is a tissue which continually regenerates and also having the ability to heal after injuries however, healing of large defects requires intensive surgical treatment. Bioactive glasses are unique materials that can be utilized in both bone and skin regeneration and repair. They are degradable in physiological fluids and have osteoconductive, osteoinductive and osteostimulative properties. Osteoinductive growth factors such as Bone Morphogenetic Proteins (BMP), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF) are well known to stimulate new bone formation and regeneration. Unfortunately, the synthesis of these factors is not cost- effective and, the broad application of growth factors is limited by their poor stability in the scaffolds. Instead, it is wise to incorporate osteoinductive nanomaterials such as graphene nanoplatelets into the structures of synthetic scaffolds. In this study, borate-based 13-93B3 bioactive glass scaffolds were prepared by polymer foam replication method and they were coated with graphene-containing poly (ε-caprolactone) layer to support the bone repair and regeneration. Materials and Methods. Effects of graphene concentration (1, 3, 5, 10 wt%) on the healing of rat segmental femur defects were investigated in vivo using male Sprague–Dawley rats. Fabricated porous bioactive glass scaffolds were coated by graphene- containing polycaprolactone solution using dip coating method. The prepared 0, 1, 3, 5 and 10 wt% graphene nanoparticle-containing PCL-coated composite scaffolds were designated as BG, 1G-P-BG, 3G-P-BG, 5G-P-BG and 10G-P-BG, for each group (n: 4) respectively. Histopathological and immunohistochemical (bone morphogenetic protein, BMP-2; smooth muscle actin, SMA and alkaline phosphatase, ALP) examinations were made after 4 and 8 weeks of implantation. Results. Results showed that after 8-weeks of implantation both cartilage and bone formation were observed in all animal groups. After 4 and 8 weeks of implantation the both osteoblast and osteoclast numbers were significantly higher in the group 4 compared to the control group. Bone formation was significant starting from 1 wt% graphene-coated bioactive glass implanted group and highest amount of bone formation was obtained in group containing 10 wt% graphene (p<0.001). Newly formed vessels expressed this marker and increased vascularization was observed in 8- weeks period compared to the 4-weeks period. In addition, an increase in new vessel formation were observed in graphene-coated scaffold implanted groups compared to the control group. While cartilage tissue was observed in control group, bone formation percentages were significant in graphene-coated scaffold implanted groups. Highest amount of bone formation occurred in group 4 (10 % wt G-C). Conclusions. Additionally, the presence of graphene nanoplatelets enhanced the BMP-2, SMA and ALP levels compared to the bare bioactive glass scaffolds. It was concluded that pristine graphene-coated bioactive glass scaffolds improve osteointegration and bone formation in rat femur defect when compared to bare bioglass scaffolds

Hyperactivation of the epidermal growth factor receptor (EGFR) by gene amplification, mutation as well as overexpression is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR-mediated signals might also contribute to malignant progression and therapy resistance of human sarcomas. Consequently we have investigated if human osteosarcoma cell lines (n=9) express functional EGFR and its useability as therapeutic target. Osteosarcoma cells expressed distinctly differing level of EGFR reaching in some cases high amounts. However, even low expression levels were sufficient to activate both MAPK and PI3K pathways (determined by phosphorylation of ERK1/2 and S6, respectively) following EGF exposure of serum-starved cells. The EGFR-specific inhibitor gefitinib completely blocked EGF-mediated and attenuated serum-induced downstream signal activation. While gefitinib applied as single agent demonstrated only limited growth inhibiting activity in short term experiments (72h drug exposure), it led to reduced colony formation in long term experiments in the majority of cell lines. Importantly, gefitinb sensitized EGFR-expressing osteosarcoma cell lines against chemotherapy with doxorubicin and methotrexate, while it antagonised cisplatin-induced cell death. Summarizing, our data suggest that EGFR-mediated survival signals protect human osteosarcoma cells against the cytotoxic activity of several antineoplastic drugs. Consequently, combination approaches including EGFR inhibitors in addition to chemotherapy should be evaluated for treatment of high grade osteosarcoma patients

Tumorgenesis is often accompanied by transcriptional deregulation of oncogenes, such as the Epidermal Growth Factor Receptor (EGFR). Transcriptional activation of a gene requires the binding of transcription factors (TF) to regulatory DNA elements at specific transcription binding sites (TFBS). A better understanding of these interactions and regulation mechanisms is essential for the development of improved therapeutic applications. ChIP was carried out to prove the existence of four new SP1 binding sites within intron 1 of the egfr gene. Site-directed Mutagenesis was performed on plasmids carrying the regulative sequence of the egfr gene in order to alter these binding sites. Activity of these sites and their influence on the transcriptional regulation were analysed by in vitro transcription and quantification using Ribonuclease Protection Assay (RPA) and qRT PCR. Using ChIP, four novel SP1 binding sites could be confirmed to be active at the egfr gene intron 1 locus. Expression of the egfr gene was found to be highly dependent of these sites. Consequently, their mutation led to a 50% decrease of the transcriptional activity of the egfr gene. The four new SP1 binding sites in the egfr intron 1 have a functional role in the egfr gene regulation, leading to a higher transcription rate. As so far only little is known about egfr gene activation, more TFs and TFBSs have to be analysed in order to gain a comprehensive understanding about the regulation of this important oncogene

Dupuytren’s contracture is characterised by abnormal fibroblast proliferation and extracellular matrix deposition in the palmar fascia. Fibroblast proliferation and matrix deposition in connective tissues are regulated by cytokines. A number of cytokines including transforming growth factor beta (TGFβ), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and epidermal growth factor (EGF) are known to have potent anabolic effects on connective tissue. The aim of this study was to investigate the role played by anabolic cytokines in the pathogenesis of Dupuytren’s disease. Twelve specimens of Dupuytren’s contracture and six control specimens of palmar fascia obtained from patients undergoing carpal tunnel release were cultured using a serumless method under standard conditions for 72 h. Levels of TGFβ-1, bFGF, PDGF and EGF in the medium were estimated using an enzyme linked immunoabsorbent assay technique. Neither Dupuytren’s tissue nor control palmar fascia produced any EGF. The mean (±S.D.)levels of bFGF, PDGF and TGFβ-1 produced by cultured palmar fascia were: 1270 ± 832, 74 ± 24, < 7, and for Dupuytren’s tissue were 722 ± 237, 139 ± 76.6, 645 ± 332, respectively. The levels of PDGF and TGFβ-1 were significantly higher in Dupuytren’s tissue. PDGF is produced in increased amounts by Dupuytren’s tissue. This may contribute to the fibroblast proliferation and increased ECM deposition observed in this condition. TGFβ-1 is not produced by normal palmar fascia but is produced in large amounts by Dupuytren’s tissue. The major physiologic role of TGFβ-1 is to stimulate formation of fibrous tissue. It plays a major role in wound healing and also in pathological conditions where fibrosis is a prominent feature. Inappropriate production of TGFβ-1 in the palmar fascia in Dupuytren’s disease may play a central role in initiating and stimulating the abnormal fibroblast proliferation and collagen synthesis seen in this condition

Introduction: Platelets play a central role in hemostasis and healing processes. Upon their activation, platelet alfa-granules release over 30 cytokines including platelet-derived growth factor (PDGF), transforming growth factor-alfa (TGF-alfa), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF) and also active substances like serotonin, catecholamines, von Willebrand factor, proaccelerin, osteonectin and antimicrobial proteins. By concentrating platelets, platelet-rich plasma (PRP) with higher levels of growth factors might be reached, which could stimulate the healing processes. The activator for PRP is a mixture of thrombin and calcium chloride. After connecting these substances platelet-rich gel (PRG) is formed. Aims: In present study, we investigated in vitro antimicrobial activity of PRG after antibiotic administration. Material and Methods: 30 minutes after iv Amoxillin/ clavulanic acid administration 54 ml of whole blood was collected from each of 10 donors. PRPs were prepared with using GPS system from Biomet. In vitro laboratory susceptibility to PRG was determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Becton Dickinson). Baseline antimicrobial activity was assessed by measuring the zones of inhibition. Agar plates were coated with one of the following strain: Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus ATCC 25923 (MSSA), Klebsiella pneumoniae ATCC 700603 (ESBL), Escherichia coli ATCC 35218 (ESBL), Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 27853. Results: We tested 10 samples of PRG. Zones of inhibition produced by PRG ranged between 6 – 23 mm in diameter. PRG inhibited the growth of Staphylococcus aureus. PG also was active against Escherichia coli, Enterococcus faecalis. No activity against Klebsiella pneumoniae and Pseudomonas aeruginosa was detected. Conclusions: Our previous study showed PRG no activity against Enterococcus faecalis without antibiotic administration. In this investigation we observed PRG strong activity against this bacteria after iv Amoxicillin-clavulanic acid administration. In infections during antibiotic treatment, PRG antimicrobial properties are enhanced by antibiotics that are concentrated in plasma