Usporedba dinamike nastajanja ektopične kosti primjenom koštanog morfogenetskog proteina 2 na nosaču kolagenske spužvice i koštanog morfogenetskog proteina 6 u autolognom krvnom ugrušku

doctoral thesis

• Title (english): Comparison of the dynamics of ectopic bone formation using bone morphogenetic protein 2 on a collagen sponge carrier and bone morphogenetic protein 6 in an autologous blood coagulum
• Author: Natalia Ivanjko
• Mentor: Slobodan Vukičević (mentor)
• Committee member: Marko Pećina (predsjednik povjerenstva)
• Committee member: Domagoj Delimar (član povjerenstva)
• Committee member: Dražen Matičić (član povjerenstva)
• Committee member: Tomislav Đapić (član povjerenstva)
• Committee member: Sven Seiwerth (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2022-12-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Clinical Medical Sciences; Clinical Immunology
• Universal decimal classification (UDC): 616 - Pathology. Clinical medicine

Abstract

U cilju razvoja boljih terapeutskih rješenja za regeneraciju kostiju, razvijen je novi osteoinduktivni sustav koji se sastoji od rhBMP6 i nosača u formi autolognog krvnog ugruška, koji je novo terapeutsko rješenje za regeneraciju kosti koje se trenutno testira u kliničkim ispitivanjima. U sklopu ovog rada istražena je biologija i vremenski tijek ektopičnog stvaranja koštanog tkiva pod utjecajem rhBMP6 u autolognom krvnom ugrušku (ABC) kao nosaču koji je uspoređen sa zlatnim standardom za cijeljenje kostiju, rhBMP2 na nosaču kolagenske spužvice (ACS), pri čemu su oba osteoinduktivna sustava ispitana na modelu potkožne ugradnje implantata u aksilarnu regiju štakora. Također, u seriji in vitro pokusa dinamike otpuštanja pokazano je kako apsorbirajuća kolagenska spužvica nema zadovoljavajući profil otpuštanja. U cilju ispitivanja staničnih populacija koje sudjeluju u ranoj osteogenezi (24h i 72h nakon potkožne ugradnje) koja je inducirana spomenutim osteoinduktivnim sustavima analiziran je udio limfoidnih, mijeloidnih i mezenhimalnih staničnih populacija. Adipoprogenitori su u najvećem broju bili prisutni u rhBMP2/ACS implantatima, dok su matične stanice u najvećem broju prisutne u rhBMP6/ABC implantatima. Nadalje, superiornost rhBMP6/ABC u odnosu na rhBMP2/ACS dokazana je u eseju potkožne ugradnje, gdje je u svim promatranim vremenskim točkama najveću količinu novostvorene kosti inducirao rhBMP6/ABC u dozi od 20 μg. Primijećeno je da se kolagenska spužvica kao nosač na ektopičnom mjestu sporije razgrađuje u odnosu na autologni krvni ugrušak. Posljedično, proces osifikacije je odgođen prilikom primjene rhBMP2/ACS zbog guste kolagenske mreže koja usporava ulazak progenitornih stanica, pa je samim time proces osifikacije odgođen u odnosu na krvni ugrušak. U cilju ispitivanja efikasnosti korištenih rekombinantnih proteina, rhBMP2 i rhBMP6, njihov osteoinduktivni potencijal ispitan je na jednakom nosaču koji se sastoji od autolognog krvnog ugruška i bifazične keramike u rangu veličine čestica od 500 do 1700 μm u tri različite doze (5, 20, 50 μg), pri čemu se rhBMP6 pokazao superiornijim u odnosu na rhBMP2 u dozi od 50 μg. Potencijalni razlog tomu je već dokazana superiornost rhBMP6 na inhibiciju nogginom. Osim toga, dodatkom biokompatibilne keramike u svojstvu matriksa otpornog na kompresiju poboljšavaju se biomehanička svojstva implantata, te je proces osifikacije osteoinduktivnih implantata s dodatkom keramičkih čestica drugačiji u odnosu na krvni ugrušak sam.

Abstract (english)

In order to develop a better therapeutic solutions for bone regeneration, a new osteoinductive device has been developed consisting of rhBMP6 and autologous blood coagulum as a carrier, which is currently being tested in clinical trials as a new therapeutic solution for bone repair and regeneration. Part of this research was focused on the investigation of the biology and time course of ectopic bone formation induced by rhBMP6 in autologous blood coagulum (ABC) and compared with the gold standard for bone healing, rhBMP2 on absorbable collagen sponge (ACS), where both osteoinductive devices were evaluated in the rat subcutaneous assay. Also, in a series of in vitro experiments release dynamics of both osteoinductive devices was tested showing that absorbable collagen sponge does not have a satisfactory release profile. In order to examine the cell populations involved in early osteogenesis (24h and 72h after subcutaneous implantation) induced by the aforementioned osteoinductive devices, the number of lymphoid, myeloid and mesenchymal cell populations were analyzed. Adipoprogenitors were most abundant in rhBMP2/ACS implants, while mesenchimal stem cells were predominately present in rhBMP6/ABC implants. Furthermore, the superiority of rhBMP6/ABC over rhBMP2/ACS was demonstrated in a rat subcutaneous assay, where at all observed time points, the largest amount of newly formed bone was induced by rhBMP6 / ABC at a dose of 20 μg. It has been observed that the absorbable collagen sponge as a carrier at the ectopic site degrades more slowly compared to the autologous blood coagulum. Consequently, the ossification process is delayed when rhBMP2/ACS is used due to the dense collagen meshwork that slows down the entry of progenitor cells, and thus the ossification process is delayed in comparison to the blood coagulum. In order to test the efficacy of the recombinant proteins used, rhBMP2 and rhBMP6, their osteoinductive potential was tested on an equal scaffold consisting of autologous blood coagulum and biphasic ceramics in the particle size range of 500 to 1700 μm in three different doses (5, 20, 50 μg), where rhBMP6 was superior to rhBMP2 at a dose of 50 μg. A potential reason for this is already proven resistance of rhBMP6 to noggin inhibition. Also, the addition of biocompatible ceramics as a compression-resistant matrix improves the biomechanical properties of implants, and the process of ossification of osteoinductive implants with the addition of ceramic particles is different compared to the blood coagulum itself.

• Keywords: Osteogeneza; Rekombinantni proteini; Matične stanice; Biologija
• Keywords (english): Osteogenesis; Recombinant Proteins; Stem Cells; Biology
• Language: croatian
• URN:NBN: urn:nbn:hr:105:109998
• Project: Number: KK.01.1.1.01.0008; Title: Reproduktivna i regenerativna medicina - istraživanje novih platformi i potencijala; Leader: Ana Katušić Bojanac; Jurisdiction: eu; Funder: MZO
• Study programme: Title: Biomedicine and Health Sciences; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
• Type of resource: Text
• Extent: 125 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-01-30 12:26:28