Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors

Abstract

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4 weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p < 0.05). Altogether, our findings confirmed that GMSCs encapsulated in RGD-modified alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.

Keywords: Dental mesenchymal stem cells; Muscle regeneration; RGD-coupled alginate hydrogel; Tissue engineering.

FIGURE 1. Development of RGD-coupled alginate hydrogel microenvironment containing myogenic cocktail for encapsulation of MSCs

(a) Characterization and comparison of the cellular morphology of GMSCs and hBMMSCs before and during myogenic differentiation. (b) Evaluation and quantification of the percentage of cells that express stem cell surface markers (passage 4) through flow cytometric analysis. (c) Bright field image of translucent alginate microspheres showing their retained spherical shape with a uniform cell distribution (average microsphere diameter 650 micrometer). (d) SEM image of the alginate hydrogel-MSC construct showing encapsulated MSCs within porous alginate hydrogel microspheres after two weeks of culturing in regular media. NS=not significant.

FIGURE 2. MSC viability and release profile characterization of the alginate microencapsulation system

(a, b) Live/dead staining of the encapsulated MSCs in alginate microspheres after one day and two weeks of culturing (scale bar = 200 mm). Viability of the encapsulated MSCs was measured as a percentage of live cells in either RGD-coupled alginate or non-RGD coupled alginate microspheres after two weeks of culturing in regular media. (c) 3-(4,5-Di- methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay of metabolic activity of encapsulated MSCs. No significant difference was observed between GMSCs and hBMMSCs at each time interval. Also, the myogenic cocktail had no adverse effects of the metabolic activity of the encapsulated MSCs. (d) Characterization of the in vitro release profile of different components of the myogenic differentiation cocktail form the alginate hydrogel microspheres showing the multiple growth factor delivery capacity of the alginate microencapsulation system. Sustained release of FSK, MeBIO, and b-FGF was observed from alginate microsphere for up to 2 weeks. *p < 0.05.

FIGURE 3. In vitro myogenic differentiation of GMSC

(a) Immunofluorescence staining against MF20 (Myosin Heavy Chain), Myf5 (Myogenic factor 5), and MyoD (myogenic differentiation protein) antibodies after four weeks of in vitro culturing in myogenic differentiation. Both GMSCs and hBMMSCs positively immunostained with antibodies against MF20, Myf5, and MyoD. Results confirmed that both hBMMSCs and GMSCs were positively stained for myogenic markers (white arrows), while the negative control (−), cell-free alginate hydrogel microspheres failed to express any of these myogenic markers. (b) Analysis of the percentage of cells positive for anti-MF20, Myf5, and MyoD antibodies, showing higher expression levels of myogenic markers in GMSCs in comparison to hBMMSCs (positive control) and negative control groups. *p < 0.05.

FIGURE 4. Molecular analysis of myogenic differentiation of MSCs in vitro

(a) RT-PCR analysis demonstrate significantly greater expression level (in fold changes) of MyoG, Myf5, and MyoD genes for encapsulated GMSCs after 4 weeks of culturing in myogenic differentiation media in vitro in comparison to hBMMSCs. The obtained data were normalized by the Ct of the housekeeping gene GAPDH and expressed relative to the expression level for the same gene at day 1. (b) The expression level of MyoG and MyoD for encapsulated hBMMSCs and GMSCs in alginate hydrogel in comparison to scaffold-free MSC cultures after two weeks of myogenic differentiation in vitro containing the myogenic cocktail. Data confirmed the important role of the microenvironment and the presence of an encapsulating scaffolds, as the encapsulated MSC expressed greater levels (p<0.05) of expressions for examined myogenic genes in comparison to scaffold free MSC groups. *P <0.05, **P<0.01.

FIGURE 5. Fate determination and myogenic differentiation of MSCs encapsulated in RGD-coupled alginate hydrogel microspheres

(a) Immunofluorescence detection of MyoD protein localized to the GMSCs encapsulated in alginate microspheres with different modulus of elasticity after 4 weeks of culturing in myogenic differentiation media (counterstained with DAPI). (b) Semi-quantitative analysis of the percentage of cells positive for anti-MyoD antibodies via immunofluorescence staining images in panel a. (c) MyoD gene expression evaluation via RT-PCR for GMSCs encapsulated in RGD-coupled alginate hydrogel with different elastic moduli after 2 weeks of culturing in myogenic differentiation media. (d) Western blot analysis presented changes in the expression levels of regulators of myogenesis of GMSCs. The expression level of MyoD gene is elevated in the encapsulated GMSCs in RGD containing alginate microspheres with intermediate modulus of elasticity, while GMSCs encapsulated in alginate hydrogels with higher or lower elastic modulus showed decreased levels of MyoD gene expression conforming the important role of the mechanical properties of the matrix in fate determination of the encapsulated MSCs. *P <0.05, **P<0.01.

FIGURE 6. In vivo myogenesis of encapsulated GMSCs after subcutaneous transplantation

(a) GMSCs or hBMMSCs as the control group encapsulated in alginate hydrogel containing myogenic cocktail were subcutaneously transplantation into immunocompromised mice and myogenic tissue formation was analyzed after 8 weeks. Histological evaluation by hematoxylin and eosine staining (top panel) confirmed partial islands of muscle regeneration with typical myogenic morphology. Extensive positive staining in immunofluorescence analysis against MF20 (middle panel) and MyoD (lower panel) antibodies further confirmed myogenic differentiation of GMSC. The negative control (−) was cell-free alginate hydrogel scaffold failed to exhibit any positive staining or myogenic tissue regeneration. (b) Semi-quantitative analysis of the percentage of cells positive for anti-MF20 and MyoD antibodies via immunofluorescence staining images. *p < 0.05. Alg= unresorbed alginate hydrogel.

FIGURE 7. The origin of the implanted MSCs and their contribution to vascularization in vivo

(a) The human origin of the engrafted GMSCs and hBMMSCs was confirmed by immunohistochemical staining with an antibody specific for human mitochondria (white arrows) (upper panel). Endothelial cell marker was identified by immunohistochemistry using anti- CD31 antibody (middle panel). Histological evaluation by hematoxylin and eosine staining (top panel) (yellow arrows) (lower panel). (b) Semi quantitative analysis of microvessel density based on panel c data. *p < 0.05.