Modulatorni efekat makrofaga na osteogeni proces u ektopičnom in vivo modelu i u različitim uslovima in vitro

Abstract

U kliničkoj praksi velike povrede, bolesti ili nasledne malformacije često dovode do stvaranja velikih koštanih defekata, koji ne mogu spontano da zarastu. Popravka ovakvih koštanih defekata predstavlja već dugo vremena izazov za regenerativnu medicinu i tkivno inženjerstvo. Proces zarastanja fraktura prolazi kroz tri faze: inflamacije, obnove i remodeliranja. Dakle, popravka koštanih defekata (fraktura) je okarakterisana inicijalnom inflamatornom reakcijom čiji su glavni akteri makrofagi. U odnosu na obilje literaturnih podataka o ulozi makrofaga u procesima zarastanja mekih tkiva, malo je podataka o njihovoj ulozi u procesima reparacije kosti. Predmet ove doktorske disertacije je ispitivanje modulatornog efekta različito tretiranih makrofaga na ektopični osteogeni proces in vivo, kao i delovanja makrofaga različitog porekla i posle različitih tretmana na in vitro diferencijaciju preostablastnih ćelija. Istraživanje je izvedeno na singenim miševima Balb/c soja, muškog pola, starosti 10-12 nedelja. Implanti su formirani od nosača za makrofage koji su imali i funkciju skafolda, a to su bili mineralni matriks kosti (MMK) i krvni ugrušak. Implanti su se međusobno razlikovali po tretmanu unetih peritonealnih makrofaga (PM). Kontrolni implanti nisu sadržavali makrofage, već samo nosače. Ispitivano je sedam tipova implanata koji su bili formirani od: nosača i rezidentnih (nestimulisanih) PM; nosača i tioglikolatom elicitiranih PM; nosača i PM tretiranih lipopolisaharidom u koncentraciji 5 ng/ml; nosača i PM tretiranih lipopolisaharidom u koncentraciji 500 ng/ml; nosača i PM tretiranih sa 5 x 10-6 M deksametazona; nosača MMK i krvnog ugruška; nosača MMK i fiziološkog rastvora. Pokazano je da osteogeni proces zavisi od načina tretmana makrofaga, pa je tako najizraženiji u implantima sa rezidentnim, kao i deksametazonom tretiranim makrofagima, dok je u implantima sa makrofagima tretiranim lipopolisaharidom u koncentraciji 500 ng/ml osteogeni proces inhibiran...

In clinical practice, major injuries, diseases or congenital malformations often lead to the formation of large bone defects, which can not heal spontaneously. Repair of these bone defects has long been a challenge for regenerative medicine and tissue engineering. The process of fracture healing passes through three phases: inflammation, reparation and remodeling. So repair of bone defects (fractures) is characterized by an initial inflammatory response, whose main participants are macrophages. Compared to the abundance of literature data about the role of macrophages in the healing process of soft tissues, there is lack of information on their role in the process of bone repair. The subject of this doctoral thesis was to investigate the modulatory effect of differently treated macrophages on ectopic osteogenic process, as well as effects of macrophages of different origin and after different treatments on osteogenic differentiation of preosteoblasts in vitro. The study was performed on male Balb/c mice 10-12 weeks of age. Implants were formed from the carriers which served as scaffolds for macrophages and these were bone mineral matrix (BMM) and blood clot. Experimental implants contained differently treated peritoneal macrophages (PM). Control implants did not contain macrophages, but only cell carriers. We examined seven types of implants, which have been formed from: carriers and resident (non-stimulated) PM; carriers and thioglycollate elicited PM; carriers and PM treated with 5 ng/ml of lipopolysaccharide; carriers and PM treated with 500 ng/ml of lipopolysaccharide; carriers and PM treated with 5 x 10-6 M dexamethasone; BMM and blood clot; BMM and saline. The results of in vivo investigation showed that the osteogenic process depends on previous treatment of macrophages, and is the most pronounced in implants which contained resident PM or PM treated with dexamethasone...