Title Priprava visokoporoznoga biorazgradljivoga i biokompatibilnoga materijala na temelju supstituiranoga hidroksiapatita Title (english) Preparation of highly porous biodegradable and biocompatible material based on substituted hydroxyapatite Author Leonard Bauer Mentor Marica Ivanković (mentor)Committee member Krešimir Košutić (predsjednik povjerenstva)Committee member Anamarija Rogina (član povjerenstva)Committee member Lidija Ćurković (član povjerenstva)Granter University of Zagreb Defense date and country 2021-04-08, Croatia Scientific / art field, TECHNICAL SCIENCES Universal decimal classificationUDC ) 66 - Chemical technology. Chemical and related industries Abstract Koštana oštećenja koja ne mogu zacijeliti normalnim metabolizmom koštanog tkiva u medicini se uobičajeno tretiraju metalnim implantatima koji okolnom tkivu pružaju mehaničku potporu prilikom regeneracije. Nedostatci uobičajenih implantata su mogućnost imunološke reakcije na strano tkivo i potreba za operativnim procesom uklanjanja inertnog implantata nakon završetka regeneracije koštanog oštećenja. Inženjerstvo koštanog tkiva posvećuje veliku pozornost razvoju koštanih nadomjestaka temeljenih na biomaterijalima koji zbog sličnih svojstava s prirodnim tkivom aktivno sudjeluju u metabolizmu koštanog tkiva. Idealan nosač treba biti biokompatibilan i bioaktivan, što znači da ga tijelo ne prepoznaje kao strani materijal i da stvara fizikalno-kemijske veze koje potiču cijeljenje kosti s čvrstim vezanjem za koštano tkivo. Također, bitno je da se nosač biološkim procesom remodeliranja zdrave kosti razgrađuje na netoksične komponente, dok cijelo vrijeme služi kao mehanička i strukturna potpora oštećenom tkivu. Kalcijevi fosfati (CaP) zbog sličnosti s anorganskom komponentom koštanog tkiva najistraživaniji su biomaterijali za primjenu koštanih nadomjestaka. Biomimetičkim pristupom, korištenjem skeleta vodenih beskralježnjaka kao biogenog izvora, moguće je sintetizirati koštani nadomjestak koji je biokemijski, mineraloški i strukturno vrlo sličan ljudskoj kosti. Uz Ca2+, PO43- i OH- ione, koštani kalcij-fosfatni materijal sadrži brojne ione koji imaju velik utjecaj na svojstva koštanog tkiva. Korištenjem polimernih biorazgradljivih materijala moguće je utjecati na svojstva CaP-a i dobiti kompozitni nosač koji oponaša anorgansko-organski kompozitni materijal prirodnog koštanog tkiva. U sklopu istraživanja ovog doktorskog rada hidrotermalnom reakcijom pri 200 °C nakon 48 sati sintetizirani su magnezijem i stroncijem supstituirani CaP nosači. Korištenjem kalcij-karbonatne strukture sipine kosti, amonij dihidrogenfosfata (NH4H2PO4), stroncij nitrata (Sr(NO3)2), magnezij klorid heksahidrata (MgCl2×6H2O) ili magnezij perklorata (Mg(ClO4)2) kao reaktanata dobiveni su dvofazni trodimenzionalni visokoporozni nosači hidroksiapatita (HAp) i vitlokita (WH). Kompozitni nosači pripremljeni su vakuumskom impregnacijom poli(ε-kaprolaktona) na površinu CaP matrice. Utjecaj udjela Mg2+ i Sr2+ iona na sastav i morfološka svojstva pripravljenih nosača ispitan je rendgenskom difrakcijom praha, infracrvenom spektroskopijom s Fourierovim transformacijama, termogravimetrijskom analizom i elektronskim pretražnim mikroskopom (SEM) s energijski razlučujućom rendgenskom analizom. Dodatak Mg2+ utječe na formiranje nove kristalne faze, WH. Strukturna utočnjavanja provedena Rietveldovom metodom ukazuju da se Mg2+ i Sr2+ preferirano ugrađuju u strukturu WH. SEM mikrografi pripravljenih uzoraka pokazuju da je međusobno povezana visokoporozna struktura sipine kosti u potpunosti očuvana nakon hidrotermalne sinteze. Mg2+ ioni smanjuju dimenziju površinskih mikrosfera karakterističnih za hidroksiapatit. Rezultati mehaničke analize primjenom tlačnog opterećenja pokazuju da WH ima pozitivan utjecaj na mehanička svojstva CaP nosača. Površinska tanka prevlaka poli(ε-kaprolaktona) ravnomjerno prekriva površinu nosača i poboljšava mehanička svojstva kompozitnih nosača za jedan red veličine u odnosu na CaP nosače. Ispitivanjem stanične vijabilnosti dokazano je da supstituirani CaP nosači nisu citotoksični. Provedene su stanične kulture ljudskim mezenhimskim matičnim stanicama (engl. human mesenchymal stem cells, hMSC) in vitro na CaP i kompozitnim nosačima u osteogenom mediju u trajanju od 21 dana. Imunohistokemijsko bojenje pokazuje da Mg-CaP nosači s masenim omjerom HAp : WH 90 : 10 i 70 : 30 pokazuju veću ekspresiju kolagena tipa I i osteokalcina nego čisti HAp nosač. Nastanak kalcijevih depozita potvrđen je bojenjem s alizarinskim crvenilom. Pozitivan utjecaj Mg2+ iona na diferencijaciju matičnih stanica na poroznom 3D CaP nosaču potvrđen je i kvantifikacijom ekspresije gena svojstvenih za osteogenu aktivnost (alkalne fosfataze, koštanog sijaloproteina i proteina matrice dentina) korištenjem kvantitativne lančane reakcije polimeraze u stvarnom vremenu. U odnosu na kompozitni nosač supstituiran Mg2+ ionima, kompozitni nosač kosupstituiran Mg2+ i Sr2+ ionima pokazao je veću ekspresiju kolagena tipa I i pozitivan utjecaj stroncija na rani stupanj osteogene diferencijacije matičnih stanica.
Abstract (english) Bone defects that cannot heal with normal bone tissue metabolism in medicine are usually treated with metal implants that provide mechanical support to nearby tissue during the regeneration process. Drawbacks of conventional implants are the possibility of an immunologic reaction to foreign tissue and need for a postoperative process where the inert implant is removed after complete bone regeneration. Bone tissue engineering dedicates great attention to the development of bone scaffolds based on biomaterials that actively participate in bone tissue metabolism due to properties similar to natural tissue. The ideal scaffold should be biocompatible and bioactive, which means that the organism does not recognise it as foreign material and it forms physicochemical bonds that promote bone healing with tight attachment to bone tissue. Additionally, it is essential that the scaffold is degradable with biological remodelling process to nontoxic components, while providing mechanical and structural support to damaged tissue the whole time. Calcium phosphates (CaP), due to their similarity to the inorganic component of bone tissues, are the most researched biomaterial for bone substitute applications. With a biomimetic approach, use of aqueous invertebrates’ skeletons as a biogenic source, it is possible to synthesise bone substitutes that biochemically, mineralogically and structurally resemble the human bone. Besides Ca2+, PO43- and OH- ions, bone calcium-phosphate materials consist of numerous ions that have a great effect on the bone tissue properties. With biodegradable polymer materials, it is possible to affect the CaP properties and synthesise a composite scaffold that resembles inorganic-organic composite material of natural human tissue. Within the research of this doctoral thesis, magnesium and strontium substituted CaP scaffolds are synthesised by a hydrothermal reaction at 200 °C after 48 h. Using the calcium-carbonate structure of the cuttlefish bone, ammonium dihydrogen phosphate (NH4H2PO4), strontium nitrate (Sr(NO3)2), magnesium chloride hexahydrate (MgCl2×6H2O) or magnesium perchlorate (Mg(ClO4)2) as reagents, biphasic three-dimensional highly porous scaffolds based on hydroxyapatite (HAp) and whitlockite (WH) are obtained. Composite scaffolds are prepared by a vacuum impregnation of poly(ε-caprolactone) to the surface of the CaP matrix. The effect of Mg2+ and Sr2+ ions content on the compositional and morphological properties of scaffolds was studied by means of X-ray powder diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis and scanning electron microscopy (SEM) with energy-dispersive X-ray analysis. The addition of Mg2+ affects the formation of new crystalline phase, WH. Structural refinements performed by the Rietveld method indicate that Mg2+ and Sr2+ were preferentially incorporated into the WH phase structure. SEM micrographs of prepared samples showed that the interconnected highly porous structure of the cuttlefish bone was completely maintained after the hydrothermal synthesis. Mg2+ ions reduce the dimension of surface microspheres characteristic for hydroxyapatite. Mechanical analysis results of compression tests showed a positive impact of the WH on the mechanical properties of CaP scaffolds. The thin surface layer of poly(ε-caprolactone) covers the scaffold surface evenly, enters the structure of spherical clusters and, in comparison to CaP scaffolds, enhances the mechanical properties of composite scaffolds by one order of magnitude. Cell viability tests proved that substituted CaP scaffolds are not cytotoxic. In vitro cell cultures with human mesenchymal stem cells (hMSC) are conducted on CaP and composite scaffolds in osteogenic medium for 21 days. Immunohistochemical staining showed that Mg-CaP scaffolds with the HAp : WH wt. ratio of 90 : 10 and 70 : 30 exhibited higher expression of collagen type I and osteocalcin than the pure HAp scaffold. Calcium deposition was confirmed by Alizarin Red staining. The positive effect of Mg2+ ions on the differentiation of stem cells on porous 3D scaffolds was also confirmed by quantification of gene expression inherent to osteogenic activity (alkaline phosphatase, bone sialoprotein and dental matrix protein) with real-time quantitative polymerase chain reaction analysis. In comparison to composite scaffold substituted with Mg2+, composite scaffold cosubstituted with Mg2+ and Sr2+ showed higher expression of collagen I and the positive effect of strontium on the early stage of stem cells differentiation.
Keywords
hidroksiapatit
magnezij
matične stanice
poli(ε-kaprolakton)
sipina kost
stroncij
supstitucija
visokoporozni nosač
vitlokit
Keywords (english)
hydroxyapatite
magnesium
stem cells
poly(ε-caprolactone)
cuttlefish bone
strontium
substitution
highly porous scaffold
whitlockite
Language croatian URN:NBN urn:nbn:hr:149:548897 Promotion 2021 Project Number: IP-2014-09-3752 Study programme Title: Chemical Engineering and Applied Chemistry - Doctoral course Type of resource Text Extent VII, 104 str. ; 30 cm File origin Born digital Access conditions Open access Terms of use Created on 2023-06-02 11:34:30
