Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells
Introduction
The requirement for new bone to replace or to restore the function of traumatized, damaged, or lost bone is a major clinical and socioeconomic need [1]. Bone tissue engineering has been heralded as an alternative strategy for bone regeneration and multipotent mesenchymal stem cells (MSCs) are a promising option for bone tissue engineering [1], [2], [3], [4], [5].
To generate bone, MSCs should undergo differentiation into the osteogenic lineage. They can be successfully induced to differentiate into osteoblasts by ascorbate-2-phosphate (AsAP), dexamethasone (Dex) and β-glycerophosphate in vitro [6]. These three reagents comprise the routine ‘osteogenic media’ [6], [7], [8], [9]. However, β-glycerophosphate is an in vitro source of phosphate ions necessary for mineralization rather than an inducer of osteogenic differentiation [6], [10], [11], [12], [13]. MSCs cultured in AsAP and Dex supplemented media generated bone tissue in vitro [6], [8] and in vivo [14], [15] but those cultured in the absence of AsAP and Dex generated little bone tissue or differentiated to form other kinds of tissue including cartilage or fibrous tissue both in vitro [6] and in vivo [14], [15], [16].
Osteogenic induction of MSCs by AsAP and Dex has been carried out only in vitro because the method of localizing the delivery of AsAP and Dex for the in vivo tissue engineering has not been established. However, in vitro osteogenic induction alone has several limitations. First, it takes several days or weeks for MSCs to differentiate into osteoblasts in vitro. To fabricate vascularized and innervated bone tissue, in vivo implantation is necessary. Moreover, it may take several weeks or months for vascularization [17] and innervation to be established in vivo. To reduce the total time required to regenerate functional bone tissue, the in vitro differentiation time should be reduced. Second, in vitro differentiation is less physiologic because the in vivo mileu can supply the correct oxygen and CO2 pressure, proper mechanical stimuli, bone tissue specific growth factors, contacts with peer cells and other factors that may be both bone tissue specific and necessary for the osteogenic differentiation. Moreover, osteogenesis of MSCs might be more successfully established if the in vivo mileu were combined with AsAP and Dex. A method of delivering AsAP and Dex via tissue engineering scaffolds would offer a highly efficient way of osteogenic induction of MSCs in vivo.
Tissue-engineering porous scaffolds using poly(D,L-lactide-co-glycolide) (PLGA) have been developed to serve as vehicles for the delivery of drugs or bioactive factors that can direct cellular responses within or around the scaffolds [18], [19], [20], [21], [22], [23], [24], [25], [26]. All the drugs used in these studies have been proteins or DNA. Other kinds of drugs except DNA and protein have not been incorporated into porous tissue engineering scaffolds. DNA is a hydrophilic polymer and most proteins are amphiphilic and labile. AsAP is hydrophilic but not polymeric like DNA and Dex is a hydrophobic molecule. Thus the study of the incorporation and release of AsAP and Dex also becomes a model study on the incorporation and release of hydrophilic and hydrophobic drugs from porous PLGA scaffolds.
In this study, porous PLGA scaffolds loaded with AsAP and Dex were prepared by the solvent casting/particulate leaching method. AsAP and Dex were continuously released from the scaffolds and the osteogenic effect of the released AsAP and Dex on MSCs was confirmed by the increased mineralization of MSC cultures in the scaffolds.
Section snippets
Materials
Ascorbate-2-phosphate (AsAP), dexamethasone (Dex), chloroform, β-glycerophosphate and sodium chloride (NaCl) were purchased from Sigma Chemical Co. (St. Louis, MO, USA), poly(D,L-lactide-co-glycolide) 18:15 (PLGA; inherent viscosity=1.16 dl/g) from Purac (Gorinchem, Netherlands), acetonitrile from Mallinckrodt Baker (Phillipsburg, NJ, USA) and n-octylamine from Aldrich Chemical Co. (Milwaukee, WI, USA).
Fabrication of ascorbate-2-phosphate particles in chloroform
Three kinds of methods were employed to control the size of the AsAP particles, as follows:
The sizes of AsAP particles in chloroform
It was expected that AsAP would be incorporated as particles into the septa or walls of the polymeric scaffolds and that the incorporation efficiency would increase as the size of the AsAP particles decreased; this was confirmed by preliminary studies. Three methods were used to reduce the size of the AsAP particles in chloroform. Method A resulted in significantly smaller AsAP particles than the other methods (Fig. 1). Mild sonication of AsAP particles (method B) resulted in a slight decrease
Conclusion
Porous PLGA scaffolds were prepared that continuously released the osteogenic factors Dex and AsAP continuously for at least a month in vitro. AsAP was incorporated into the scaffold walls as particles and small AsAP particles, which were obtained by the lyophilization of quick-frozen AsAP solutions, showed incorporation efficiencies of over 90%. Water uptake was increased by AsAP incorporation in the scaffolds. An in vitro release study showed that Dex and AsAP showed an initial burst release
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