Cartilage tissue engineering on biosynthetic polymer polyHIPE scaffold

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.Cartilage tissue engineering remains a field with possibilities for improvement despite very intense research efforts in recent times. Development of artificial materials and 3-D cellular scaffolds has an important role in facilitation of cartilage regeneration. An interesting approach to scaffold formation presents the so-called emulsion templating technique. The resulting materialtermed polyHIPE (PHP), is a synthetic highly porous polymer prepared by the polymerization of high internal phase emulsions (HIPEs). The main objective of this PhD thesis is to investigate the potential for neocartilage formation within cellular scaffolds prepared from PHP material. The fabrication of 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. The research results suggested that the designed PHP cellular scaffold is suitable for further use in cartilage tissue engineering

New biological approaches in the reconstruction of anterior cruciate ligament (ACL)

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.Background: Anterior cruciate ligament (ACL) rupture is one of the most common knee injuries. The operative reconstruction is usually performed as the standard treatment. The success of the operative technique is between 73-95 %, and 37-75 % of patients return to the preoperative physical activity. The main problem after surgical reconstruction is the long rehabilitation period. Patients are not allowed to perform sports activities for 6 months after surgery. The outcome after ACL reconstruction depends upon the healing of the graft in the bone tunnel and upon the ligamentization of the intraarticular part of the graftthis is a process during which the graft attains the features of a native ACL. The biomechanical factors (anatomical tunnel position, fixation, adequacy of the graft and postoperative rehabilitation) and biological response (revascularization and the number of collagen fibers) seem to play a crucial role after surgery. The biological response can be accelerated with the use of growth factors (GF), periosteum and mesenchymal stem cells (MSCs). Nowadays platelet-rich plasma (PRP) from autologous blood is applied at the graft insertion site to promote healing and ligamentization of the graft. Conclusion: The review focuses on the factors that influence the outcome of ACL reconstruction. A better understanding of the graft\u27s biological response and its modulation is the basis of new approach in the treatment of ACL rupture, which enables the patient and athlete to quicker resume their everyday and sports activities

Polyester type polyHIPE scaffolds with an interconnected porous structure for cartilage regeneration

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