Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using “bead-in-bead” polysaccharide capsules
Introduction
Injuries to articular cartilage, as a consequence of trauma or degenerative diseases such as osteoarthritis fail to spontaneously repair due to the avascular, aneural and alymphatic nature of the tissue. In addition, there is clinical evidence to indicate that full thickness cartilage defects continue to progress and deteriorate [1]. Furthermore, the potential for spontaneous repair is observed to decrease with age as mature cartilage shows a decrease in chondrocyte activity. Current repair techniques have resulted in the formation of fibrous tissue that, typically, is rapidly degraded. With an increasing ageing population and in the absence of clinically viable formation regimes, a tissue engineering approach offers the potential to synthesize functional cartilage constructs for tissue regeneration. The avascular nature of cartilage, coupled with the ability to isolate human chondrocyte populations encapsulated within an appropriate matrix offers a significant, yet achievable, challenge to engineer cartilage at the required site.
In previous studies, the encapsulation of human articular chondrocytes and human bone marrow stromal cells within alginate has resulted in the development of osteoid matrix and osteogenic extracellular matrix with limited chondrogenic extracellular matrix [2]. The regeneration of cartilage remains a clinically relevant target and one potential method in the regeneration of cartilage is gene transfer. Thus, the genetic manipulation of cells able to differentiate into chondrocytes and express an appropriate repertoire of cartilage-specific genes may provide the stimulus to ultimately synthesize functional 3D cartilaginous constructs.
Genetic manipulation of cells allows the study of specific roles of genes and their products, and the regulation of gene expression by transcription factors remains a major mechanism for controlling cellular functions [3]. Furthermore, the development of viable and safe genetic modification strategies is preferable to the direct addition of growth factors to a cell, due to: (i) short half-life of selected growth factors, (ii) the requirement for high doses, and (iii) multiple administration and cost [4]. Gene delivery encompasses either viral (transduction) or non-viral (transfection) approaches. Viral vectors have been shown to induce an inflammatory response whereas non-viral vectors exhibit low immunogenicity and unrestricted plasmid size [5]. Transient transfection provides rapid analysis of genes and limited protein production, whereas stable transfection allows genetic material to be retained following cell division. Non-viral strategies include the use of chemical reagents (lipofection and polyfection), naked plasmid, or physical methods such as microinjection or electroporation. Efficient gene transfer using electroporation strategies and advanced by Amaxa Nucleofector technology allows the direct delivery of DNA into the cell nucleus. The electroporation process uses an electric pulse resulting in cell membrane disruption enabling DNA entry to the cell cytoplasm [6]. The method has been shown to achieve high transfection efficiencies in various primary cells and remains independent of cell division, critical as many primary cells divide slowly or do not divide at all [7].
In the current study we have examined the potential of Sox-9 transfected human bone marrow stromal cells and articular chondrocytes encapsulated within alginate polysaccharide microcapsules to promote chondrogenesis in vitro and in vivo. Sox-9 is a high-mobility-group domain transcription factor that is expressed in chondrocytes, as well as other tissues, and is required for the conversion of condensed mesenchymal cells into chondrocytes. Sox-9 co-operates with Sox-5 and Sox-6 and plays a critical role in the proliferation and function of chondrocytes and has been shown to trans-activate the cartilage-specific collagens type II, IX, and XI as well as the aggrecan core protein gene [3], [8], [9]. When chondrocytes are cultured in monolayer, although an increase in cell number and cell division is observed, a progressive loss of chondrocyte phenotype is also exhibited accompanied by a progressive decrease in Sox-9 expression [10]. The introduction of Sox-9 into cells offers the potential to promote the production of cartilage-specific products and extracellular matrix and, ultimately, the generation of cartilaginous constructs. We have developed a “bead-in-bead” system to provide spatial and temporal separation of cell populations within mineralized alginate/chitosan shells of variable thickness and stability [11] and therefore transfected cell populations can be co-cultured alongside untransfected cell populations to examine the formation of cartilaginous matrix formation within polysaccharide templates.
Section snippets
Materials and methods
All tissue culture and biochemical reagents were obtained from Sigma–Aldrich UK unless indicated otherwise. Anti-type II collagen antibody (rabbit polyclonal, Cat No. 234187) was purchased from Calbiochem (Merck Biosciences, UK). The anti-Sox-9 (rabbit polyclonal, AB 5535) antibody was purchased from Chemicon International (USA). Relevant secondary antibodies and ExtrAvidin Peroxidase were purchased from Sigma–Aldrich (Poole, Dorset, UK) as was the anti-FLAG M2 mouse monoclonal
Sox-9 transfection of human bone marrow stromal cells and articular chondrocytes
Human bone marrow stromal cells and human articular chondrocytes were transfected with GFP and subsequent expression was detected by fluorescence microscopy after 24 h as proof-of-concept and to determine transfection efficacy. High transfection efficiencies were observed for both human bone marrow cells (Fig. 1A) and articular chondrocytes (Fig. 1B) and an overlay of the fluorescent image onto an image of cells taken by light microscopy indicated the relative number of transfected cells in
Discussion
This study has examined the potential of encapsulating Sox-9 transfected cells within alginate polysaccharide templates to establish a chondrogenic phenotype and development of cartilaginous constructs. Alginate has previously been shown to support chondrogenesis as favoring differentiation as well as maintenance of the chondrogenic phenotype both in vitro and in vivo [15], [16], [17]. The differentiation of human bone marrow stromal cells into the chondrogenic lineage depends on a complex
Conclusion
We have investigated a tissue engineering approach utilizing a non-viral gene delivery method coupled with polysaccharide templates to promote cartilaginous construct formation. In summary, the results of the present study demonstrate that localized overexpression of Sox-9 of cells within alginate polysaccharide capsules enhances the development of cartilaginous constructs both in vivo and in vitro. Additionally, the regions of cartilage-like matrix were significantly increased in Sox-9
Acknowledgments
The authors would like to acknowledge Research into Ageing, The Rosetrees Trust and the BBSRC for funding. The monoclonal antibody to type I collagen was a generous gift from Dr Larry Fisher (NIH, Bethesda, MD, USA) and the pcDNA Sox-9 expression construct was a gift from Professor Timothy Hardingham (University of Manchester, UK). The authors are also grateful to the orthopedic surgeons at Southampton General Hospital for their aid in facilitating bone marrow sample and articular chondrocyte
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