1. Tkivni inženiring hrustančnega tkiva na biosintetičnem polimernem polyHIPE nosilcuJakob Naranđa, 2017, doktorsko delo/naloga Opis: Tkivni inženiring hrustančnega tkiva še vedno nudi številne možnosti za izboljšavo, navkljub intenzivnim raziskovalnim naporom v zadnjem času. Razvoj umetnih materialov in 3-D celičnih nosilcev ima pomembno vlogo pri regeneraciji hrustančnega tkiva. Zanimiv pristop pri izdelavi celičnih nosilcev predstavlja izgradnja s pomočjo emulzij. Nastali material, imenovan polyHIPE (PHP), je sintetični visoko porozen polimer, ki ga pripravimo s polimerizacijo visokega deleža notranje faze emulzij (HIPEs – high internal phase emulsions).
Glavni cilj te doktorske disertacije je raziskati možnosti za tvorbo hrustančnega tkiva znotraj celičnih nosilcev pripravljenih iz PHP materiala. Proizvodnjo PHP nosilcev smo posebej prilagodili tkivnemu inženiringu hrustanca, tako da smo pripravili porozne (85 %) strukture s primarno velikostjo por v območju 50–170 m. Pokazali smo, da je PHP material biokompatibilen s človeškimi sklepnimi hondrociti, kar smo ovrednotili s pomočjo testa za preživetje celic (Live/Dead kit) in histološko analizo. Opazovali smo hondrocite z okroglimi jedri, ki so bili organizirani v večceličnih plasteh na površini PHP nosilca in so rastli približno 300 m v notranjost nosilca. Kopičenje kolagena tipa 2 smo dokazali s pomočjo imunohistokemije, molekularna analiza je pokazala izražanje hrustančno specifičnih genov z ugodnim razmerjem kolagena tipa 2 in tipa 1. Dodatno so bili PHP vzorci biološko razgradljivi, njihove osnovne mehanske lastnosti pa primerljive z nativnim sklepnim hrustancem.
Izsledki raziskave dokazujejo, da je zasnovan PHP celični nosilec primeren za nadaljnjo uporabo v tkivnem inženiringu hrustančnega tkiva.
Ključne besede: humani sklepni hrustanec, tkivni inženiring, biokompatibilni celični nosilec, sintetični polimer, polyHIPE, diferenciacija hondrocitov
Objavljeno v DKUM: 17.10.2017; Ogledov: 2333; Prenosov: 290
 Celotno besedilo (24,38 MB) |
2. Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)Jakob Naranđa, Lidija Gradišnik, Mario Gorenjak, Matjaž Vogrin, Uroš Maver, 2017, izvirni znanstveni članek Opis: BACKGROUND: Cartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.
METHODS: Isolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.
RESULTS: Cartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.
DISCUSSION: In this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.
Ključne besede: aggrecan, collagen 2, gene expression, human articular chondrocytes, isolation protocol, phenotype preservation, TKA, total knee arthroplasty
Objavljeno v DKUM: 02.08.2017; Ogledov: 1730; Prenosov: 661
Celotno besedilo (42,50 MB)
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3. Polyester type polyHIPE scaffolds with an interconnected porous structure for cartilage regenerationJakob Naranđa, Maja Sušec, Uroš Maver, Lidija Gradišnik, Mario Gorenjak, Andreja Vukasović, Alan Ivković, Marjan Rupnik, Matjaž Vogrin, Peter Krajnc, 2016, izvirni znanstveni članek Opis: Development of artificial materials for the facilitation of cartilage regeneration remains an important challenge in orthopedic practice. Our study investigates the potential for neocartilage formation within a synthetic polyester scaffold based on the polymerization of high internal phase emulsions. The fabrication of polyHIPE polymer (PHP) was specifically tailored to produce a highly porous (85%) structure with the primary pore size in the range of 50–170 μm for cartilage tissue engineering. The resulting PHP scaffold was proven biocompatible with human articular chondrocytes and viable cells were observed within the materials as evaluated using the Live/Dead assay and histological analysis. Chondrocytes with round nuclei were organized into multicellular layers on the PHP surface and were observed to grow approximately 300 μm into the scaffold interior. The accumulation of collagen type 2 was detected using immunohistochemistry and chondrogenic specific genes were expressed with favorable collagen type 2 to 1 ratio. In addition, PHP samples are biodegradable and their baseline mechanical properties are similar to those of native cartilage, which enhance chondrocyte cell growth and proliferation.
Ključne besede: polyester, polymerization, polyHIPE
Objavljeno v DKUM: 23.06.2017; Ogledov: 1985; Prenosov: 416
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4. Tkivno inženirstvo pri zdravljenju hrustančnih poškodbJakob Naranđa, Matjaž Vogrin, 2013, pregledni znanstveni članek Ključne besede: hrustanec, regeneracija, hondrogeneza, tkivno inženirstvo, celični nosilci, rastni faktorji, zarodne celice, bioreaktorji
Objavljeno v DKUM: 30.12.2015; Ogledov: 2094; Prenosov: 100
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5. Novi biološki pristopi pri rekonstrukciji sprednje križne vezi (ACL)Jakob Naranđa, Matjaž Vogrin, 2010, pregledni znanstveni članek Opis: Izhodišča: Ruptura sprednje križne vezi (ACL) je ena pogostejših poškodb kolena. Pri zdravljenju igra ključno vlogo uspešna rekonstrukcija ACL z vstavitvijo presadka. Uspešnost operativnega postopka je 73-95 %, vrnitev na predoperatvno stopnjo dejavnosti pa 37-75 %. Glavna težava po rekonstrukciji je zelo dolga rehabilitacija. Bolniki ne smejo polno obremeniti kolena najmanj 6 mesecev po poškodbi. Uspešnost rekonstrukcije ACL je odvisna od vraščanja presadka v kostnem tunelu in od ligamentizacije, t.j. procesa, pri katerem pridobi lastnosti nativnega ACL. Pri tem igrajo pomembno vlogo biomehanski dejavniki (pravilni položaj kostnih tunelov -anatomska lega presadka, pričvrstitev in ustreznost presadka ter pooperativna rehabilitacija) in biološki odziv presadka po rekonstrukciji (stopnja revaskularizacije in nastajanje kolagenskih vlaken), ki ga lahko pospešimo z dodajanjem rastnih faktorjev (RF), periosta in uporabo mezenhimskih zarodnih celic. Ti pristopi so novost v rekonstrukciji ligamentnih struktur, saj posegajo v biologijo celjenja med kostjo in presadkom. Danes uporabljamo tehniko za pridobivanje s trombociti bogate plazme (PRP) iz avtologne krvi, ki jo damo na mesto vstavitve presadka in tako pospešimo vraščanje in proces ligamentizacije presadka.
Zaključki: Pregledni članek obravnava dejavnike, ki vplivajo na uspešnost zdravljenja po rekonstrukciji ACL. Poznavanje in modulacija biološkega odziva presadka je nov pristop k zdravljenju pretrgane ACL, bolnikom in športnikom pa omogoča hitrejšo vrnitev k vsakodnevnim in drugim telesnim dejavnostim.
Ključne besede: sprednja križna vez (ACL), rekonstrukcija, vraščanje presadka, ligamentizacija, rastni faktorji (RF)
Objavljeno v DKUM: 10.07.2015; Ogledov: 4119; Prenosov: 299
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