Microsoft Word - 13CotrutzActiveMolecules 94 Carmen Elena Cotrutz et al Active molecules in regenerative medicine Active molecules in regenerative medicine Carmen Elena Cotrutz1, T.Petreuş1, Cristina Dascălu2 1Department of Cell and Molecular Biology 2Department of Medical Informatics and Biostatistics University of Medicine and Pharmacy “Gr.T. Popa”, Iasi, Romania Background Biomaterials play central roles in modern strategies in regenerative medicine and tissue engineering as designable biophysical and biochemical environments that direct cellular behavior and function. New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and ECM- bound growth factors, both for therapeutic applications and basic biological studies. The ultimate decision of a cell to differentiate, proliferate, migrate, apoptosis or perform other specific functions is a coordinated response to the molecular interactions with these ECM effectors. The flow of information between cells and their ECM is highly bidirectional, as, for example, observed in processes involving ECM degradation and remodeling. Matrix metalloproteinases (MMP) are Zn and Ca dependent enzymes and represent a class of structural and functional kindred enzymes that are involved in altering the natural compounds of the extracellular matrix [1,2]. MMP are synthesized as zymogens that can be inhibited by 4 classes of natural inhibitors called TIMPs (tissue inhibitor for matrix metalloproteinases) [3]. MMP plays important roles in physiological processes, but their overexpression plays also crucial roles in pathological processes as multiple sclerosis, arthritis, Alzheimer disease and especially in cancer and metastasis [4-8]. Among these biological molecules we have investigated expression of metalloproteinase 8 (MMP-8) and metalloproteinase 9 (MMP-9) in the periimplantar tissues remodelling. Methodology We have performed an indirect immunohistochemical technique using hematoxilin/eosin (HE) staining by peroxidase induced conversion of DAB (3,3-diaminobenzidine), an enzyme conjugate, which reveals the localisation of antibody-bound antigenic sites by means of a coloured reaction. DAB, a chromogenic substrate for peroxidase, stains the antigen- antibody sites in brown, indicating the presence of the metalloproteinases-8 (MMP-8) in the cells surrounding the implant. MMP-8 are usually present in the neutrophils infiltrating the connective tissue or bone in an accute phase of inflamatory process. Data analysis was done using an Olympus BX40 optical microscope with a color CCD camera mounted on. We have also performed molecular docking and molecular dynamics investigations regarding simulations of the catalytic Romanian Neurosurgery (2011) XVIII 1: 94 – 95 95 domain of a known matrix metalloproteinase (MMP), in the absence of the substrate or a known inhibitor, starting from ProteinDataBank published data (ID-1QIB). This study emphasizes the role of the atomic position in this site regarding to further simulations for conceiving a rather modulating inhibitor for these enzymes. Results and conclusions Immunolabelling with MMP-8 is reduced and limited to the perivascular area. MMP-9 has a more diffuse presence, without precise localization and therefore shows no remodeling processes (1st and 2ndtype). After 2 months MMP-8 immunolabelling was more pronounced outside blood vessel walls as a proof of chronic inflammation. MMP-9 was found perivascular (in the wall of small capillaries) more diffuse but also more intense. This suggests a lot of active remodeling here (3rd and 4th type). For 2nd type MMP-9 is present in a diffuse but more intense way suggesting accelerated tissue remodeling. These preliminary phenomenon modulated by the presented molecules are crucial for newly grown bone tissue formation and healing, even allowing implant primary fixation. Molecular modelind and dynamic studies on MMP2 catalytic site showed that Root Mean Square Fluctuation (RMSF) was performed, compared with the NMR starting structure and the hydrophobic solvent accessible surface area (HSAS) of the MMP2 catalytic site. We have observed small fluctuations for the catalytic Zn area (high stability); however we have noticed some fluctuations, especially in some loop regions near the catalytic site of the MMP, in 192-250 region of the MMP2 PDB file. 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