Artikel
Stem cell loaded functionalized fibrin-based gels for tissue regeneration
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Veröffentlicht: | 21. Oktober 2010 |
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Gliederung
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Objective: Instant cell-based therapy denotes a promising, clinically relevant alternative to classical two-step tissue engineering strategies including cell expansion prior to implantation [Kasten et al. 2008]. Multipotent mesenchymal stromal cells (MSCs) can be easily isolated and culture expanded from bone marrow aspirates and provide an excellent source of progenitor cells [Stiehler et al. 2006]. In this context, practicable approaches require immediate progenitor cell immobilization in a three-dimensional, biocompatible matrix supporting cellular proliferation and differentiation. Fibrin is a physiologically occuring protein that polymerizes during haemostasis. The aim of this study was to establish a protocol for three-dimensional fibrin-based immobilization and osteogenic stimulation of MSCs. Cell-loaded gels were evaluated for cell viability and osteogenic differentiation. Furthermore the release of bone morphogenetic protein-4 (BMP-4) from fibrin gels was investigated.
Methods: For preparation of gels Tissucol™ fibrin sealant kit (FC) or platelet-rich plasma (PRP) in combination with or without tricalciumphosphat (TCP) granules were used at different fibrin concentrations. BMP-4 release from the gels was measured using BMP-4 ELISA kit. Human immortalized single-cell derived MSCs [Böcker et al. 2008] were added to the fibrin or PRP solution and cultured for 7 days with and without osteogenic supplements. Cell viability was assessed by use of live/dead staining. Lactate dehydrogenase (LDH) activity was determined to investigate cellular proliferation. For evaluation of osteogenic differentiation cell-specific alkaline phosphatase (ALP) activity was quantified.
Results and conclusions: Gel viscosity was optimized by modification of fibrin concentrations. The amount of fibrinogen concentration used was positively correlated to the rate of rhBMP-4 release from the gels. MSCs showed sufficient viability in all types of fibrinogen and PRP-based gels, respectively and no differences concerning cell viability could be observed between FC- and PRP-gels. Cells were able to proliferate and differentiate towards osteogenic lineage as detected by cell-specific ALP-activity. Thereby highest cell numbers and cell-specific ALP activity could be observed for scaffolds made of TCP granules and PRP (p<0.05).
We conclude that fibrin gels can serve as a naturally occuring matrix for the immobilization of stem cells enabling cell proliferation and differentiation at the defect site. We will further pursue this approach for therapy of musculoskeletal defects.