SOX9 gene plus heparinized TGF-β 3 coated dexamethasone loaded PLGA microspheres for inducement of chondrogenesis of hMSCs

doi:10.1016/j.biomaterials.2012.06.023

Biomaterials

Volume 33, Issue 29, October 2012, Pages 7151-7163

https://doi.org/10.1016/j.biomaterials.2012.06.023 Get rights and content

Abstract

Microparticulated types of scaffolds have been widely applied in stem cell therapy and the tissue engineering field for the regeneration of wound tissues. During application of simple genes or growth factors and cell delivery vehicles, we designed a method that employs dexamethsone loaded PLGA microspheres consisting of polyplexed SOX9 genes plus heparinized TGF-β 3 on the surface of polymeric microspheres prepared using a layer-by-layer (LbL) method. The fabrication of the polyplexed SOX9 genes plus heparinized TGF-β 3 and their subsequent coating onto dexamethsone loaded PLGA microspheres represents a method for functionalization of the polymeric matrix. The use of SOX9 gene plus heparinized TGF-β 3 coated dexamethsone loaded PLGA microspheres was evaluated to determine their potential as both gene carriers and cell delivery vehicle. By adhesion of hMSCs onto SOX9 gene plus heparinized TGF-β 3 coated dexamethsone loaded PLGA microspheres, the chondrogenesis-related specific genes of collagen type II were increased 30 times comparing to control. Also, the specific extracellular matrix of glycosaminoglycan (GAG) production of hMSCs adhered onto SOX9 gene plus heparinized TGF-β 3 coated dexamethasone loaded PLGA microspheres increased more 2.5 times than control group. Not only in vitro culture but in vivo results, the specific genes of COMP, aggrecan, collagen type II, and SOX9 showed much more gene expressions such as 20, 15, 10, 8 times.

Introduction

In stem cell therapy research, many studies have focused on the specific factors related to stem cell differentiation that can be loaded or coated onto carrier molecules using organic or inorganic materials [1], [2], [3], [4]. Of the stem cell differentiation-related factors available, drugs, growth factors, plasmid DNA (pDNA) and small interference RNAs (siRNAs) have been shown to induce cell differentiation in specific cells or tissues [5], [6], [7]. However, it is important to select factors suitable for specific cells or tissues that possess a potent capability for stem cell differentiation [8], [9], [10]. Moreover, the safe delivery of such materials is also vital for the differentiation of stem cells into the desired cell lineage. During the fabrication of the delivery vehicle, several methods are available for functionalizing the vehicle surface to enable targeted delivery of the gene or drug loaded onto the vehicle [11], [12], [13], [14].

The coupling of differentiation factors to particulated carrier vehicles using layer-by-layer (LbL) assembly is known to be a suitable method for their safe delivery to the target tissue. LbL assembly is also useful for fabricating polyplexed structures composed of two oppositely charged surfaces that allows the coating of a three dimensional (3D) surface [15], [16], which effectively increases the coated surface area, so that adhered cells have greater contact with the bioactive material. Immobilization of differentiation factors onto a variety of 3D substrates, including hydrogels or particulated matrix, has been shown to enhance the differentiation process. Of the 3D substrates available, hydrogels and poly(lactide-co-glycolide) (PLGA) microspheres have been used successfully to immobilize diverse bioactive materials, including proteins, peptides, drugs, DNA, siRNAs and small molecules such as drugs [17], [18], [19], [20], [21].

Thus, polymeric microspheres have been constructed to immobilize and stabilize factors for delivery to the required target cell or tissue. Furthermore, previous studies have described a simple and highly efficient method for the complete stabilization of 3D nanostructures by embedding them into a microspherical structure developed for stem cell differentiation [22], [23].

One specific stem cell differentiation factor, pDNA, has been used to target cancer cells for apoptosis, to determine the mechanism underlying the origins of stem cells, or to prepare specific cells for cell therapy. Transfecting pDNAs carrying genes for specific transcription factors into stems cells facilitated their differentiation into specific cell types [24], [25].

One such transcription factor, the SOX9 gene, plays a crucial role in chondrogenesis due to the induction and enhancement of collagen type II gene expression, which is involved in the differentiation of early stage stem cells into chondrocytes. SOX9 also has a potential role in the condensation of prechondrogenic mesenchymal cells and is involved in the maintenance of high expression levels of Col2a1 and aggrecan, which are expressed in fully differentiated chondrocytes [26], [27], [28], [29], [30].

During chondrogenesis, stem cells must be stimulated by the appropriate growth factors. In previous studies, we suggested that the angiogenic growth factors, BMP-2 and BMP-7 (members of the bone morphogenic protein family) and TGF-β1 and TGF-β3 (members of the transforming growth factor family) strongly stimulated human mesenchymal stem cells (hMSCs) to switch into the differentiation pathway leading to the formation of bone and cartilage cells under both in vitro and in vivo conditions [31], [32], [33].

In this study, we have developed a method for high levels of gene adsorption via immobilization to PLGA microspheres. To accomplish this, negatively charged pDNA polyplexed with positively-charged polyethyleneimine (PEI) were complexed to give a positively-charged polyplex, which was incorporated onto PLGA microspheres using the LbL method. The SOX9 gene, a key transcription factor involved in chondrogenesis, was fused to the gene for red fluorescence protein (RFP) and to counteract the negatively charged, RFP-fused SOX9, PEI was employed as a typical cationic material in designing the delivery vehicle for transfection of the genes into hMSCs. Both gene delivery and cell delivery to the target tissue (the subcutis of nude mice) were attempted simultaneously. The hMSCs were cultured or transplanted with the SOX9 gene, PEI polyplexes coated onto PLGA microspheres without detaching the transfected cells unlike conventional methods. After delivery of SOX9, the success of the gene transfection and chondrogenesis of hMSCs adhering to the PLGA microspheres were determined by scanning electron microscope (SEM), confocal laser microscope, Xenogene bioimaging, RT-PCR, real-time qPCR, Western blotting analysis, histology and immunofluorescence.

Section snippets

Preparation of PLGA microspheres

PLGA microspheres were prepared using solvent evaporation in an oil-in-water emulsion. Briefly, PLGA (4 g) was dissolved in 30 ml of dichloromethane, after which a glass syringe and needle (needle gauge: 20G) was used to drop the polymer solution into 300 ml of aqueous solution containing 2% (w/v) of poly(vinyl alcohol) (PVA), while mixing using a magnetic stirrer at 600 rpm. The suspension was then gently stirred for 2–3 h at 35 °C with a magnetic stirrer at 600 rpm to evaporate the

Results

The images of the SOX9 gene plus heparinized TGF-β3-coated, DEX-loaded PLGA microspheres were obtained using an SEM. Fig. 1A shows typical images of DEX-loaded PLGA microspheres without SOX9 gene complexes or heparinized TGF-β3. Fig. 1B and C (a-e) show the total binding sites of the SOX9 gene complex and heparinized TGF-β3-coated, DEX-loaded PLGA microspheres, respectively. Fig. 1D (a-e) shows the total binding sites of the SOX9 gene plus heparinized TGF-β3-coated, DEX-loaded PLGA

Discussion

In stem cell differentiation, many different proteins and growth factors are known to be involved in switching stem cells to differentiate towards specific lineages. The addition of particular drugs or proteins can stimulate stem cells and control cell proliferation and the switching of cell function. Thus, efficient stem cell differentiation depends on the direct delivery of differentiation-related drugs and growth factors into stem cells, such as in the in situ delivery of drugs or growth

Conclusions

To summarize our findings, the use of a polyplexed SOX9 gene plus heparinized TGF-β3-modified, DEX-loaded PLGA microspheres is an efficient method for the induction of chondrogenesis from hMSCs embedded on these scaffolds. This technique facilitated the transfection of SOX9 into the hMSCs adhering to the microsphere surfaces. The transfected hMSCs were then stimulated with the heparinized TGF-β3, which was also attached to the microsphere surfaces. This method has the potential for use as a

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (20120006111, 2012R1A1A3003063, & 2011K000823).

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