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Osteogenic Differentiation by Rat Bone Marrow Stromal Cells on Customized Biodegradable Polymer Scaffolds
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.ORCID iD: 0000-0002-1922-128X
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2010 (English)In: Journal of bioactive and compatible polymers (Print), ISSN 0883-9115, E-ISSN 1530-8030, Vol. 25, no 2, 207-223 p.Article in journal (Refereed) Published
Abstract [en]

In this report, poly(L-lactide-co-epsilon-caprolactone), poly(LLA-co-CL) and poly(L-lactide-co-1,5-dioxepan-2-one), poly(LLA-co-DXO) were evaluated and compared for potential use in bone tissue engineering constructs together with bone marrow stromal cells (BMSC). The copolymers were tailored to reduce the level of harmful tin residuals in the scaffolding. BMSC isolated from Sprague-Dawley rats were seeded onto the scaffolds and cultured in vitro for up to 21 days. Cell spreading and proliferation was analyzed after 72 h by scanning electron microscopy and thiazolyl blue tetrazolium bromide (MTT) conversion assay. Osteogenic differentiation of BMSC was evaluated by real-time PCR after 14 and 21 days of culture. Hydrophilicity was significantly different between poly(LLA-co-CL) and poly(LLA-co-DXO) with the latter being more hydrophilic. After 72 h, both scaffolds supported increased cell proliferation and the mRNA expression of osteocalcin and osteopontin was significantly increased after 21 days. Further investigation of these constructs, with lower levels of tin residuals, are being pursued.

Place, publisher, year, edition, pages
2010. Vol. 25, no 2, 207-223 p.
Keyword [en]
biopolymers, bone marrow stromal cells, degradable scaffold, tissue, engineering, cell proliferation, poly(L-lactide-co-epsilon-caprolactone), poly(L-lactide-co-1, 5-dioxepan-2-one), low tin residuals, mesenchymal stem-cells, of-the-art, in-vitro, aliphatic polyesters, porous scaffolds, acid) foams, osteoblast, poly(l-lactide), degradation, copolymers
National Category
Industrial Biotechnology Materials Engineering
URN: urn:nbn:se:kth:diva-19210DOI: 10.1177/0883911509358812ISI: 000274532200006ScopusID: 2-s2.0-77749245896OAI: diva2:337257
QC 20110131Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-11-11Bibliographically approved
In thesis
1. Polyester scaffold: Material design and cell-protein-material interaction
Open this publication in new window or tab >>Polyester scaffold: Material design and cell-protein-material interaction
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Tissue engineering has emerged as a valid approach for the regeneration and restoration of bone defects. The concept of bone tissue engineering includes degradable scaffolds, osteogenic cells and osteoinductive growth factors either alone or in any combination of these three. The scaffold bulk material and its design, in particular, are essential for reaching clinically relevant treatments. It is essential that the scaffold is biocompatible and acts as a temporary extra-cellular matrix with a porous 3-dimensional structure, supporting adhesion, proliferation and differentiation of osteogenic cells. Yet another criterion of the scaffold is that is must have sufficient mechanical stability to maintain structural integrity and protect the cells with a gradual transfer of mechanical load to the developing tissue. At the same time, the scaffolds needs to be bioresorbable with a controllable degradation rate depending on its application and the rate of tissue regrowth.

In this thesis, aliphatic polyester scaffolds have been modified and shown to be suitable for bone tissue engineering applications. In addition, a new microfluidic device for live imaging of cell behavior within porous 3-dimensional scaffolds has been developed.

          Highly porous and degradable aliphatic polyester scaffolds with varying pore sizes and interconnected pores were fabricated. The polyesters assayed were random co-polyesters poly(L-lactide-co-ε-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO] and the homopolymer poly(L-lactide) [poly(LLA)]. The inherently different polymers yielded scaffolds with a wide range of properties with respect to surface chemistry, thermal properties, mechanical stability and degradation rate.

The polyester scaffolds were shown to support the increased proliferation of bone marrow-derived stromal cells (BMSC) as well as enhanced osteogenic differentiation, with increased levels of osteocalcin gene expression, which emphasized their potential to act as cells carriers in bone tissue engineering. The potential of poly(LLA-co-CL) scaffolds and common biomedical polyesters in bone tissue engineering was further enhanced by surface functionalization. This involved two different methods of immobilization of bone morphogenetic protein-2 (BMP-2), a potent bone-growth-inducing factor, to the assayed polyesters. The first method used BMP-2 immobilized to heparin functionalized polyesters, while the second method covalently bonded BMP-2 to grafted linker groups on polyesters. Both immobilization techniques retain the bioactivity of BMP-2, and growth-factor-modified polyesters showed an increasing expression of osteogenic genes and production of osteocalcin in osteoblasts-like cells as well as increased proliferation in the mouse cell line, C3H10T1/2.

The rate of degradation of electron-beam-sterilized polyester scaffolds and the subsequent loss of mechanical stability were strongly dependent on the chemical, physical and macroscopic architecture of the samples. The degradation rate and loss of mechanical integrity were much greater in porous scaffolds with hydrophilic co-monomers. By incorporating hydrophobic co-monomers with a limited ability to crystalize instead of hydrophilic co-monomers, the mechanical stability was retained for a longer time during the degradation process.

The polyester supported spreading and flattened the morphology of both BMSC and osteoblast-like cells. The early cell adhesion to synthetic surfaces is mainly governed by the proteins adsorbed from its surrounding fluids. Early adhesion of BMSC to blood-plasma-coated polyesters was limited, despite the ability of the polyesters to adsorb adhesive proteins and expression of appropriate integrins on BMSC. However, adhesion to a purified adhesive matrix protein on the polyesters did occur, suggesting that pretreatment of polyester scaffolds with adhesive proteins or peptides is a feasible way to enhance the efficiency of cell loading into polyester scaffolds. 

                       Polyester scaffolds were combined with microfluidics and soft lithography to develop a new method for high-resolution imaging of live cells within porous scaffolds. The microfluidic device was used to frequently follow live cell proliferation and differentiation on the same spatial location within 3-dimansional porous scaffolds over a period of more than four weeks. This device is attractive for the evaluation of cells and materials intended for tissue engineering.

We conclude that degradable aliphatic co-polyester scaffolds carefully designed with respect to macroscopic structure, bulk material and surface chemistry are able to meet the specific requirements of various bone tissue engineering applications. In addition, microfluidic devices permit reoccurring high resolution imaging of live cells within porous scaffolds and have a potential as a method of evaluating tissue engineering constructs.

Abstract [sv]

Konsten att med kroppens egna celler som utgångspunkt återskapa förlorad eller skadad vävnad, s.k. tissue engineering, har på senare tid blivit allt mer accepterad som ett alternativ  till dagens kliniska metoder. Inom ben-tissue engineering ingår oftast förutom celler, en nedbrytbar matris och lämpliga tillväxtfaktorer. För att kunna användas kliniskt ställs mycket höga krav på materialegenskaperna hos matrisen t.ex. dess utformning. Materialet ska vara biokompatibelt och matrisen ska i möjligaste mån efterlikna det naturliga proteinnätverk som finns mellan cellerna i benvävnaden. Eftertraktade egenskaper innefattar en 3-dimensionell porös struktur med förutsättnigar för adhesion, proliferering och differentiering av benceller.  Matrisens mekaniska egenskaper och nedbrytningshastighet är starkt beroende av varandra och bör anpassas efter den aktuella applikationen och vävnadens naturliga egenskaper.

Den här avhandlingen beskriver utvecklandet av matriser baserade på alifatiska polyestrar med flera eftertraktade egenskaper inom ben-tissue egineering. Dessutom har alifatiska polyestrar används för att utveckla ett nytt mikrofluidikssystem för kontinuerlig visuell utvärdering av levande celler i porösa 3-dimensionella matriser.

De nedbrytbara alifatiska polyestrarna poly(L-laktid-co-ε-kaprolakton) [poly(LLA-co-CL)], poly(L-laktid-co-1,5-dioxepan-2-on) [poly(LLA-co-DXO)] och poly(L-laktid) [poly(LLA)] användes för att tillverka matriser med hög grad av porositet, väl sammanbundna porer och varierande porstorlekar. De olika egenskaperna hos monomererna resulterade i matriser med mångsidiga egenskaper inom nedbrytningshastighet, mekanisk stabilitet, vätnings- och termiska-egenskaper.

De framställda matriserna av alifatiska polyestrar visade sig stimulera proliferering och differentiering mot osteoblaster hos stamceller isolerade från benmärg (BMSC), vilket underströk potentialen hos dessa matriser för ben-tissue engineering. Vidare så förhöjdes potentialen hos poly(LLA-co-CL) matriser och andra kliniskt använda polyestrar genom att ytmodifiera materialen med den beninducerade tillväxtfaktorn, bone morphogenetic protein-2 (BMP-2). För detta ändamål utvecklades två separata immobiliseringsmetoder. I den ena metoden immobiliserades BMP-2 till heparinkopplade ytor och i den andra bands BMP-2 kovalent direkt till yt-ympade kopplingskedjor. I båda metoderna kunde BMP-2 immobiliseras med bibehållen biologisk aktivitet. Immobiliserat BMP-2 stimulerades i respektive metod till förhöjd proliferering hos embryonala C3H10T1/2 celler och ökat uttryck av gener starkt förknippade med mogna benceller samt proteinet osteocalcin i bencells-liknande celler.

Nedbrytningsbeteendet och de mekaniska egenskaperna hos de elektronstråle-steriliserade polyestermatriser visade sig kraftigt beroende av materialens kemiska, fysiska och makroskopiska utfromning. Genom att inkorporera mer hydrofila monomerer i materialen påskyndades nedbrytningen och därmed förlusten av provens mekaniska stabilitet. Att istället sampolymerisera två hydrofoba monomerer med begränsade möjlighet till kristallisation, gav material med förlängd mekanisk stabilitet under nedbrytningen.

BMSC och bencells-liknande celler visade normal utbredning och morfologi vid ahdesion till polyestrarna. Den initiala celladhesionen till syntetiska ytor sker vanligtvis genom ytadsorberade protein från omgivande biologiska vätskor. BMSCs initiala celladhesion till blodplasmabelagda polyestrar var starkt begränsade, oavsett polyestrarnas förmåga att adsorbera adhesiva protein ur blodplasma och att motsvarande proteinreceptorer återfanns på cellytan hos BMSC. Genom att istället adsorbera upprenade adhesiva protein till polyestrarna ökade adhesionen av BMSC till ytorna. Detta påvisade möjligheten att öka effektivitet av celladhesionen till polyestrar genom att på förhand belägga dem med adhesiva protein.

Polyestrar kombinerades även med mikrofluidikteknik och mjuk litografi för att utveckla ett nytt system för högupplöst visuell observation av levande celler i porösa matriser. Systemet kunde användas till att kontinuerligt följa cell-proliferering och –differentiering på exakt samma plats i 3-dimensionella matriser under mer än 4 veckor. Det framtagna systemet kan med fördel användas till utvärdering av både material och celler som ska kan komma att användas inom tissue engineering.

Slutsatsen blir att nedbrytbara matriser baserade på alifatiska polyester som grundligt utformas i avseende på makroskopisk struktur, bulkmaterial och ytkemi kan möta kraven för olika applikationer inom ben-tissue engineering. Dessutom kan alifatiska polyestrar kombineras med mikrofluidiskt system för att tillåta kontinuerligt högupplös visualisering av levande celler i porösa matriser med stor potential som utvärderingsmetod inom tissue engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 104 p.
Trita-CHE-Report, ISSN 1654-1081 ; 2011:51
National Category
Polymer Technologies
urn:nbn:se:kth:diva-46166 (URN)978-91-7501-113-4 (ISBN)
Public defence
2011-11-25, F3, Lindstedsvägen 26, KTH, Stockholm, 10:00 (English)
QC 20111111Available from: 2011-11-11 Created: 2011-11-02 Last updated: 2011-11-11Bibliographically approved

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