Hydrogel elasticity and microarchitecture regulate dental-derived mesenchymal stem cell-host immune system cross-talk

Abstract

The host immune system (T-lymphocytes and their pro-inflammatory cytokines) has been shown to compromise bone regeneration ability of mesenchymal stem cells (MSCs). We have recently shown that hydrogel, used as an encapsulating biomaterial affects the cross-talk among host immune cells and MSCs. However, the role of hydrogel elasticity and porosity in regulation of cross-talk between dental-derived MSCs and immune cells is unclear. In this study, we demonstrate that the modulus of elasticity and porosity of the scaffold influence T-lymphocyte-dental MSC interplay by regulating the penetration of inflammatory T cells and their cytokines. Moreover, we demonstrated that alginate hydrogels with different elasticity and microporous structure can regulate the viability and determine the fate of the encapsulated MSCs through modulation of NF-kB pathway. Our in vivo data show that alginate hydrogels with smaller pores and higher elasticity could prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascades, leading to higher amounts of ectopic bone regeneration. Additionally, dental-derived MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. Taken together, our findings demonstrate that the mechanical characteristics and microarchitecture of the microenvironment encapsulating MSCs, in addition to presence of T-lymphocytes and their pro-inflammatory cytokines, affect the fate of encapsulated dental-derived MSCs.

Statement of significance: In this study, we demonstrate that alginate hydrogel regulates the viability and the fate of the encapsulated dental-derived MSCs through modulation of NF-kB pathway. Alginate hydrogels with smaller pores and higher elasticity prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascade, leading to higher amounts of ectopic bone regeneration. MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. These findings confirm that the fate of encapsulated MSCs are affected by the stiffness and microarchitecture of the encapsulating hydrogel biomaterial, as well as presence of T-lymphocytes/pro-inflammatory cytokines providing evidence concerning material science, stem cell biology, the molecular mechanism of dental-derived MSC-associated therapies, and the potential clinical therapeutic impact of MSCs.

Keywords: Alginate hydrogel; Bone tissue engineering; Elasticity; Host immune system; Porosity.

Figure 1. Characterization of the developed hydrogel biomaterials

(a) The modulus of elasticity of the fabricated alginate hydrogels in the presence of two different concentrations of calcium ions (100 and 200 mM, showing increased elasticity in presence of a higher concentration of calcium. (b) Light microscopic images of the fabricated microspheres. (c) SEM micrographs of prepared hydrogels at the two different calcium ion concentrations. (d) The calculated average pore size for the two different hydrogels based on SEM images. In vitro trypsin inhibitor (e), BSA (f), and IgG (g) release profile from alginate hydrogels with different elastic moduli/porosity showing decreased release amounts from alginate hydrogel with greater stiffness. (h, i) Qualitative and quantitative characterization of the permeability of fabricated alginate microspheres to TNF-α using immunofluorescence staining, showing reduced infiltration of TNF-α in alginate hydrogel with greater elasticity and lower porosity. Scale bar: c, 30 μm; *P<0.05.

Figure 2. Biomaterial physical properties and microarchitecture affect SHED-immune system interplay in vitro

(a) Live/dead staining of encapsulated SHED MSCs in fabricated hydrogels after two weeks of culturing in regular media (scale bar = 200 mm). (b) Viability of the encapsulated MSCs: percentage of live MSCs in alginate microspheres with different elasticity/porosity. (c) Apoptosis of SHED in the presence of activated Pan-T lymphocytes after three days of co-culturing, as confirmed by FACS analysis using Annexin V-FITC apoptosis detection kit. The protective properties of encapsulating biomaterials and the role of their porosity/elasticity were demonstrated by the reduced apoptosis of encapsulated SHED in the presence of activated Pan-T lymphocytes after three days of co-culturing, as confirmed by FACS analysis using an Annexin V-FITC apoptosis detection kit. (d) Western blot analysis showed upregulation of the NF-kB pathway in SHED in the presence of IFN-γ (50 ng/ml) and TNF-α (5 ng/ml), showing the harmful effects of pro-inflammatory cytokines on MSC survival. Encapsulating SHED in alginate hydrogel with higher elasticity (lower porosity) down-regulated NF-kB pathway activity relative to that of SHED encapsulated in more porous alginate hydrogel. *P<0.05, ***P<0.001.

Figure 3. Down-regulation of SHED osteogenesis in the presence of pro-inflammatory cytokines

(a) Alizarin red staining showed reduced mineralized nodule formation for SHED cultured in osteogenic media in the presence of IFN-γ 50 (ng m/l)- TNF-α (5 ng m/L) combination. Mineralization area percentage was defined as the area of stained mineralization divided by the total area. (b) The protective role of the biomaterial was confirmed by osteogenic differentiation of encapsulated SHED in alginate with different degrees of elasticity/porosity in the presence of IFN-γ and TNF-α for 4 weeks and staining with Xylenol orange fluoroprobe. (c) Mineralization area percentage was defined as the area of stained mineralization divided by the total area of the field of view of the image in d. (d) Western blot analysis showed that pro-inflammatory cytokine-treated SHED expressed decreased levels of osteogenic marker. Moreover, Western blot analysis confirmed that the physiomechanical properties of the encapsulating hydrogel biomaterial regulated the expression of osteogenic marker levels (RUNX2) in the presence of IFN-γ and TNF-α. NS= not significant, *P<0.05, **P<0.01.

Figure 4. Biomaterial-T lymphocyte cross-talk regulates SHED-mediated bone regeneration

(a) Micro-CT images of SHED encapsulated in alginate hydrogels with different physiomechanical properties, retrieved 8 weeks after subcutaneous implantation in immunocompromised mice. Pan T lymphocytes were isolated and injected into the mice via the tail vein immediately prior to the surgical procedures. (b) Histological analysis (H&E staining) of retrieved specimens after 8 weeks of subcutaneous implantation. (c) Semi-quantitative analysis via micro-CT images (a) showing the bone volume fraction (BV/TV). (d) Semi-quantitative analysis of the amount of bone regeneration from histological slide presented in panel b. B: regenerated bone, #: connective tissue. NS= not significant, *P<0.05, **P<0.01.

Figure 5. Post-implantation characterization of implanted MSCs

To further analyze the role of the physiomechanical properties of the encapsulating biomaterial on MSC-immune system cross-talk, the encapsulated SHED cells were subcutaneously implanted in wild type mice and SHED apoptosis was evaluated 1 week post-implantation. Encapsulated SHED in alginate hydrogel implanted in nude mice were used as the control. (a) Immunohistochemical staining with antibodies against anti-NF-kB p65 and anti- Cox-2 antibodies of specimens retrieved after one week of implantation showing higher expression levels of (upper panel) Cox-2 and (lower panel) NF-kB p65 (red arrows) in encapsulated SHED in alginate hydrogel with lower elasticity and greater porosity. (b) Semi-quantitative analysis of the percentage of positive surface area in panel a. (c) Fluorescent immuno-staining with antibodies against Caspase-3 (upper panel) and Caspase-8 (lower panel) after 1 week of implantation revealing significantly higher expression levels of Caspase-3 and Caspase-8 in encapsulated SHED in alginate hydrogel with lower elasticity and greater porosity. (d) Semi-quantitative analysis of the number of positive cells in panel c. NS= not significant, *P<0.05, **P<0.01.