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Cooperability
| Project leader |
Vukicevic Slobodan |
| Project co-leader: |
Boro Dropulic |
| Administering organization: |
School of Medicine, University of Zagreb, Salata 11, 10000 Zagreb, Croatia |
| Partner Institution/Company: |
Lentigen Corporation |
| Grant type: |
1B |
| Project title: |
Bone morphogenetic protein-1 isoforms in bone regeneration |
| Project summary: |
Bone morphogenetic proteins (BMPs) are growth and differentiation factors able to induce new bone formation. Originally purified and characterized from bone, they co-localize with BMP-1, a protease related to tolloid (TLD). BMP-1 regulates extracellular matrix maturation, and activates TGF-ß family members. Utilizing plasma from healthy individuals and patients with acute fractures, we detected and characterized the BMP-1-3 (mTld) isoform of the Bmp-1 gene by proteomic techniques, and showed that isolated endogenous BMP-1-3 protein is proteolytically active. We hypothesize that BMP-1-3 is systemically orchestrating the deposition of extracellular matrix and activation of latent growth factors important in bone regeneration. Boro Dropulic at Lentigen Corporation as a recognized scientist will develop necessary tools for this project proposal, including: constructing a BMP-1-3 lentiviral vector, a transduced CHO cell line and master cell bank for BMP-1-3 protein production. BMP-1-3 protein will be validated for its enzymatic activity. The role of BMP-1-3 in bone physiology will then be tested in in vitro and in vivo assays.
At the Laboratory for Mineralized Tissues we will explore the molecular mechanisms of BMP-1-3 using gene and protein markers of bone metabolism in mesenchymal stem cells and osteoblastic lines, and investigate BMP-1-3 signalling through integrin, Wnt- and BMP-related cellular pathways. Also, we will test physical and chemical interaction between BMP-1-3 and other BMPs in a bioassay using a C2C12 myoblast cell line transfected by the BRE-LUC Id gene, as well as in vitro assays testing the rhBMP-1-3 efficacy in processing extracellular matrix and latent growth factors. In vivo the efficacy of rhBMP-1-3 will be tested in a rat femur fracture model and rabbit ulna critical size bone defects, alone or in combination with BMP-6. The efficacy of BMP-1-3 in vivo will be tested in rabbits by using a homologous whole blood modified coagulum as a carrier. This project may lead to development of an improved or novel bone device for stimulating bone repair.
Young investigators from Zagreb School of Medicine will acquire state-of-the-art technology for lentiviral protein expression at our collaborator’s laboratory which will enhance the quality of science at a national level. Finally, this project aims to position itself to the national and international forefront of the control of acute bone fracture, contributing to the wellbeing of patients, Croatian academia and SME to compete for EU and NIH funds and open more business opportunities.
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| Hrvatski sažetak: |
Koštani morfogenetski proteini (BMP) jesu faktori rasta i diferencijacije sposobni potaknuti stvaranje nove kosti. Originalno pročišćeni i karakterizirani iz kosti, oni se lokaliziraju zajedno sa BMP-1 koji je proteaza slična toloidu (Tld). BMP-1 regulira sazrijevanje izvanstaničnog matriksa i aktivira članove TGFß obitelji. Analizirajući plazmu zdravih osoba i bolesnika s akutnim prijelomima otkrili smo i karakterizirali BMP-1-3 (mTld) izoformu Bmp-1 gena pomoću proteomskih tehnika, te pokazali da je izolirani endogeni BMP-1-3 protein proteolitički aktivan. Pretpostavili smo da BMP-1-3 sistemski regulira odlaganje izvanstaničnog matriksa i aktivaciju latentnih faktora rasta važnih za regeneraciju kosti. Boro Dropulić iz Lentigen Corporation, ugledan je znanstvenik koji će razviti pomagala za ovaj projekt, koja uključuju: konstruiranje BMP-1-3 lentiviralnog vektora, transduciranih CHO stanica i master stanične banke za proizvodnju BMP-1-3 proteina. BMP-1-3 će biti vrednovan u eseju procjene enzimatske aktivnosti. Uloga BMP-1-3 u koštanoj fiziologiji bit će potom testirana uporabom in vitro i in vivo eseja.
U Laboratoriju za mineralizirana tkiva proučit ćemo molekularne mehanizme BMP-1-3 uporabom genskih i proteinskih markera koštanog metabolizma u mezenhimalnim matičnim stanicama i osteoblastičnim linijama te istražiti BMP-1-3 signaliziranje putem integrina, Wnt- i BMP-signalnih putova. Isto tako testirat ćemo fizikalne i kemijske interakcije između BMP-1-3 i drugih BMPeva u bioeseju uporabom C2C12 stanične linije mioblasta transfecirane sa BRE-LUC Id genom kao i u in vitro esejima u kojim ćemo testirati učinkovitost BMP-1-3 u procesiranju izvanstaničnog matriksa i latentnih faktora rasta. In vivo učinkovitost BMP-1-3 testirat će se u štakorskom modelu prijeloma bedrene kosti i modelu zarastanja defekta kritične veličine ulne u kunića, u kombinaciji s BMP-6. Učinkovitost BMP-1-3 in vivo testirat će se u kunića upotrebom homolognog ugruška modificiranog uporabom periferne krvi koji će služiti kao nosač za BMP-1-3. Taj projekt ima mogućnost razviti poboljšanu ili novu osteogenu napravu za stimulaciju srastanja kosti.
Mladi istraživači sa zagrebačkog Medicinskog fakulteta steći će nova saznanja iz tehnologije ekspresije proteina pomoću lentiviralne tehnologije u našem suradnom laboratoriju što će povećati kvalitetu znanosti na nacionalnoj razini. Konačno, ovaj projekt ima znanstvenu mogućnost pozicioniranja na nacionalnu i međunarodnu razinu kontrole akutnog prijeloma kosti što će značajno doprinijeti dobrobiti bolesnika, hrvatskoj akademskoj zajednici i gospodarstvu u kompeticiji za EU i NIH fondove te otvoriti više gospodarskih mogućnosti.
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| Amount requested from UKF: |
1.007.500,00 HRK |
| Amount of matching funding: |
300.000,00 HRK |
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