Biodegradable scaffolds for cartilage culture

Primjenom inženjerstva tkiva nastoji se naći biokompatibilne, bioaktivne, mehanički izdržljive i za okoliš pogodne materijale koji će uzrokovati minimalnu neželjenu imunološku reakciju kod pacijenata. Pri tome je potreban multidisciplinaran pristup za razvoj nosača željenih svojstava. Kitozan je izvrstan kandidat zbog svoje slične strukture s komponentama koje grade izvanstaničnu matricu. Lako se oblikuje u različite oblike te ga odlikuje niz poželjnih svojstava poput hidrofilnosti, mukoadhezije, antimikrobnosti, hemostatskog djelovanja i drugih. Cilj ovog rada bio je pripraviti kitozanske okosnice za primjenu u inženjerstvu hrskavičnog tkiva. Nosači kitozana srednje (MC) i visoke (HC) molekulske mase stupnja deacetilacije (SD) >75% i >95% pripremljene su liofilizacijom. Karakterizacija nosača je provedena metodama rendgenske difrakcije (XRD), infracrvene spektroskopije (FTIR) i pretražnog elektronskog mikroskopa (SEM). Nosačima je pobliže određena mikrostruktura (raspodjela veličina pora te poroznost) te fizikalna (bubrenje i razgradnja) i biološka svojstva (imunohistološka analiza uzgojenih hondrocita). Dodatno, površina kitozana modificirana je proteinom fibronektinom (FN) te su kao takvi podvrgnuti biološkim ispitivanja. Promatran je utjecaj različite molekulske mase i SD-a te su rezultati pokazali kako su sva tri nosača visokoporozna uz relativno uski raspon pora optimalan za inženjerstvo hrskavice. Svi uzorci pokazali su znatnu moć bubrenja pri čemu je vrijednost za HC75 najveća. Preko 50% svih nosača izuzev MC95 je bilo razgrađeno nakon razgradnje uz lizozim. Također je dokazan značajan utjecaj fosfatnog pufera (PBS) na otapanje nosača pri fiziološkim uvjetima. Kapacitet adsorpcije nosača je bio povoljniji pri višoj koncentraciji FN-a. Desorpcijom je u prosjeku uklonjeno oko 40% FN-a s uzorka, dok MC95 čini iznimku sa samo 5%. Rezultati histološke i imunohistokemijske analize specifičnih gena ukazuju na stvaranje izvanstanične matrice hrskavice te ekspresiju kolagena tipa II i agrekana karakterističnih za zglobnu hrskavicu.The main purpose of tissue engineering is to find biocompatible, bioactive, mechanically appropriate and eco-friendly materials which will elicit minimal undesired immunologic reaction in patients. For that, a multidisciplinary approach is needed to develop scaffolds with desirable properties. Chitosan is an excellent candidate because of its structural similarity to the components which are building block of the extracellular matrix. It is easily shaped in various forms and has plenty of favorable properties, such as, hydrophilicity, mucoadhesion, antimicrobial properties, haemostatic action etc. The aim of this work was to prepare chitosan scaffolds for articular cartilage engineering. Chitosan scaffolds of medium (MC) and high (HC) molecular weight with deacetylation degrees (SD) >75% and >95% were prepared by the lyophilization method. Scaffolds were characterized using X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The scaffolds’ microstructure was investigated in terms of pore size distribution and porosity, as well as their physical (swelling and degradation) and biological (immunohistochemical analysis of cultured chondrocytes) properties. Additionally, chitosans’ surface was modified by the fibronectin (FN) protein and they were used as such in biological experiments. The effect of varying molecular weight and SD was observed, and the results exhibit high porosity in all three scaffolds with a relatively narrow pore range optimal for cartilage engineering. All samples showed significant swelling capacity with HC75 being the highest. Over 50% of all the scaffolds excluding MC95 were degraded at the end of the degradation experiment. A significant effect of phosphate buffer saline solution (PBS) on scaffolds dissolution has also been observed. The adsorption capacity of scaffolds was more favorable at higher concentration of FN. The desorption experiments showed 40% of desorbed FN from scaffolds with SD >75%, while MC95 showed only 5% of desorbed protein. Histological and immunohistochemical analyses of specific genes indicated the formation of cartilaginous extracellular matrix, as well as the expression of collagen type II and aggrecan characteristic for articular cartilage