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Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.ORCID iD: 0000-0002-1922-128X
KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
Institutionen for Odontologi, Karolinska Institute.
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2012 (English)In: Biomedical Materials, ISSN 1748-6041, Vol. 7, no 3, 035011- p.Article in journal (Refereed) Published
Abstract [en]

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin.

Place, publisher, year, edition, pages
2012. Vol. 7, no 3, 035011- p.
Keyword [en]
Marrow Stromal Cells, Osteoblast-Like Cells, Osteogenic Differentiation, In-Vitro, Bone Regeneration, Ligand-Binding, Collagen-I, Scaffolds, Laminin, Expression
National Category
Biomaterials Science Polymer Chemistry Cell Biology
URN: urn:nbn:se:kth:diva-47596DOI: 10.1088/1748-6041/7/3/035011ISI: 000303667600011ScopusID: 2-s2.0-84871506788OAI: diva2:455714
EU, European Research Council
QC 20120531. Updated from manuscript to article in journal.Available from: 2011-11-11 Created: 2011-11-11 Last updated: 2012-06-07Bibliographically 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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