Elsevier

Acta Biomaterialia

Volume 60, 15 September 2017, Pages 181-189
Acta Biomaterialia

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

https://doi.org/10.1016/j.actbio.2017.07.017Get rights and content

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.

Introduction

Craniofacial bone tissue engineering currently relies extensively on bone grafting procedures [1], [2], [3]. However, there are numerous well-documented drawbacks related to this therapeutic approach [4], [5], [6], [7]. Mesenchymal stem cells (MSCs) represent an alternative treatment modality in regenerative medicine [4], [5], [6], [7], [8], [9], [10]. MSCs derived from orofacial tissues (e.g., stem cells from human exfoliated deciduous teeth (SHED)) are attractive postnatal stem cells with desirable self-renewal capacity and plasticity and osteogenic properties comparable to bone marrow mesenchymal stem cells (BMMSCs) [10], [11], [12], [13], [14], [15], [16], [17]. SHED are easily found in pediatric patient’s mouth or in tissue waste in pediatric dental clinics. Preclinical and clinical investigations have revealed that SHED are able to generate new bone with great potential for bone repair/regeneration [15], [16]. Moreover, these cells are originated from neural crest that makes them particularly compatible for regeneration and repair of neural crest-derived tissues (e.g., jaw bone) [13], [14], [15], [16], [17], [18], [19]. Recent studies have emphasized, however, that the cell source is not the sole determinant of success in stem cell-mediated regenerative medicine. The host immune system can have an adverse effect on outcomes. For example, proinflammatory T-cells and cytokines inhibit MSC-mediated bone tissue regeneration [20], [21], [22]. These effects can be modulated by local administration of anti-inflammatory agents (e.g. aspirin or indomethacin), which have been shown to improve MSC-mediated bone regeneration [21], [22].

The choice of carrier can also strongly affect the performance of MSCs in tissue engineering applications, in part due to immunomodulatory effects, and also because they offer the opportunity to direct the stem cells fate towards the desired phenotype [22], [23], [24]. However, regulation of the encapsulated MSCs’ is still the main challenge. Hydrogel scaffolds have been largely utilized to study the interaction of biomaterial and MSCs [25], [26], [27], [28] RGD-coupled alginate hydrogel has been used extensively for cell encapsulation in bone tissue regeneration [27], [28], [29], [30], [31], [32], [33], [34], [35]. Alginate hydrogel can delay the penetration of the inflammatory cytokines and T cells, and act as a physical shield to protect the implanted MSCs from the immune cell attack [22]. Therefore, the scaffold material can affect the cross-talk between MSC and host immune cell, control the implanted MSCs fate, toward improved tissue regeneration quality. Furthermore, it is understood that physiomechanical properties including porosity of the biomaterial influence MSC differentiation, but their roles in the MSC-host immune interaction are fairly unknown [36]. There are no studies evaluating the role of porosity and elasticity of the biomaterial in MSC-proinflammatory T cell/cytokine interplay. How this interplay affects the bone regenerative properties of dental-derived MSCs in particular, has received little attention.

Understanding the factors influencing the fate of encapsulating MSCs is of major therapeutic interest [36]. To develop effective MSC-based regenerative therapies it is crucial to have a clear understanding of how the physiomechanical properties including porosity and elasticity of the encapsulating biomaterial affect the cross-talk between the immune cells and MSCs. Hence, the main goal of our study was to clarify the role of mechanical properties and microarchitecture of the hydrogel biomaterial in directing the fate of encapsulated dental-derived MSCs toward osteogenic tissues.

Section snippets

Cell isolation and culture

SHED were used and cultured according to published protocols [15], [16] with required IRB approval. The pulp tissues were separated from exfoliated human primary upper and lower central or lateral incisors (isolated from twelve children aged 6–12) and then processed according to methods in the literature [15], [16]. Mouse Pan T lymphocytes were isolated according to previously published protocols [21], [22] using a magnetic cell sorter (Pan T Cell Isolation Kit II, Miltenyi Biotec, San Diego,

Characterization of the fabricated alginate hydrogel and permeability analysis

In the current study, SHED were isolated and encapsulated in an alginate hydrogel delivery system. Flow cytometric study was completed to evaluate the stemness of the isolated cells. Specific MSC markers including STRO-1 and CD146 were expressed by SHED (Supplementary Fig. 1).

Fig. 1a shows the elastic moduli of the alginate hydrogels, confirming that an increase in the calcium ion concentration increases the elasticity of the hydrogel. In addition, through light microscopy, it was determined

Discussion

Our research group and others have reported that inflammatory T-lymphocytes from the host are capable of prevention of osteo-differentiation of implanted MSCs via IFN-γ-mediated downregulation of osteogenesis and upregulation of TNF-α signaling, leading to cell apoptosis [21], [22]. Additionally, it has been shown that encapsulating cells in alginate hydrogel can retard the infiltration of proinflammatory T cells and cytokines by acting as a physical barrier, and therefore buffer the implanted

Conclusions

Here, we demonstrate that the porosity and elasticity of the encapsulating hydrogel biomaterial play an important role in dental-derived MSC-immune cell interplay and therefore in MSC viability and fate determination. Our findings suggest that the physical properties and microarchitecture of the encapsulating hydrogel biomaterial regulate the permeation of pro-inflammatory cytokines and T-lymphocytes and therefore osteogeneiss of MSCs. These findings might lead to development of an innovative

Acknowledgments

This work was supported by the National Institutes of Health (K08DE023825 to A.M. and K99E025915 to C.C.).

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    These authors contributed equally to this work.

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