Elsevier

Biomaterials

Volume 30, Issue 6, February 2009, Pages 1089-1097
Biomaterials

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

https://doi.org/10.1016/j.biomaterials.2008.10.047Get rights and content

Abstract

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α5β1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α5β1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α5β1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α5β1 versus αvβ3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

Introduction

Biomaterial matrices are being explored to guide stem cell differentiation phenomena, for purposes both in vitro and in vivo [1]. Many efforts are focused on providing a biofunctional surface for cell adhesion through addition of natural extracellular matrix (ECM) proteins such as fibronectin (FN) or cell-adhesive ligand motifs derived from ECM. FN is a core ECM component of many tissues, where it regulates a variety of cell activities predominantly through direct interactions with cell surface integrin receptors. FN is critically important in vertebrate development [2], since it mediates a wide variety of cellular interactions and plays important roles in cell adhesion, migration, growth and differentiation [3]. The capacity of FN to bind up to 20 distinct integrins provides its influence on multiple tissues and cell types. Moreover, physiological molecular unfolding and refolding of the more than 15 FN type III repeats has been proposed as a part of FN's control mechanism for integrin-specific binding [4]. Thus, this capacity to bind multiple integrins represents a design challenge when delivering FN to instruct specific cell behaviors. Small protein fragments corresponding to functional FN domains that contain integrin-specific binding sites should be used instead of the full-length protein, which displays low level of specificity, or small FN-derived peptides such as RGD that notoriously display poor integrin specificity.

The integrin α5β1 is an important FN-specific integrin that can be found in different adhesion structures [5], and has been implicated in the control of differentiation of various cell types, such as precursor cell osteogenic differentiation (OD [6], [7], [8], [9]), while its effect on human mesenchymal stem cells (MSCs) OD is still unknown [10]. Functionally, α5β1 interaction requires both the traditional integrin-binding sequence (RGD) located in the 10th type III repeat (FN III10) as well as the “synergy sequence” (PHSRN) in the adjacent 9th type III repeat (FN III9), whereas most of other RGD dependant integrins such as αvβ3 do not require PHSRN [11]. Interestingly, type III repeats are stabilized only by hydrogen bonding and van der Waals forces [12]. Recent simulations of physiologically relevant force-mediated unfolding of the FN III9–10 structure predict the existence of a stable intermediate structural state prior to complete unfolding of the 10th type III repeat [13]. In this stable intermediate, the PHSRN-to-RGD distance has been shown to be too large for both sites to synergistically bind the same receptor [14] suggesting that synergy-dependent binding of α5β1 can be turned off by simply stretching FN III9–10 into this intermediate state. This mechano-sensitive regulation of α5β1 binding is further supported by the fact that the degrees of conformational stability of FN III9 modulate integrin accessibility to the RGD motif [15]. One intriguing example of this effect is the stabilization of the FN III9 domain attributed to a single human to mouse (Leu1408 to Pro) mutation that enhances both conformational stability of FN III9–10 and affinity for α5β1 [16]. Because α5β1 binding requires this critical and sensitive domain conformation, it is clear that α5β1 engagement cannot be efficiently accomplished with simple or tandem peptides [17] and can possibly be tuned through molecular modifications which alter conformational stability.

In this study, we investigate how engineered integrin-specific ECM fragments can influence MSC behavior in both 2D and 3D environments, determining the effects of the ligation of integrin α5β1 to various degrees. We determined the integrin specificity of full-length FN compared to recombinant FN III9–10 domains, specifically FN III9–10, the structurally stabilized FN III9–10 (Leu1408 to Pro) and FN III10, and their capacity to direct MSC behavior in the context of OD. Cell attachment and spreading, proliferation, and differentiation responses on 2D surfaces and in a clinically relevant 3D fibrin matrix system [18] are reported.

Section snippets

Cell culture

MSCs from human bone marrow purified according to the protocol of Pierre Charbord laboratory (Université François-Rabelais, Tours, France) were purchased from Biopredic Int. (Rennes, France). Cells were expanded until passage 3 in Minimum Essential Medium Alpha (MEM-α) supplemented with 10% bovine growth serum (BGS, HyClone, Logan, UT, USA), 100 U/mL penicillin, 0.1 mg/mL streptomycin and 2 mm l-glutamine (expansion medium). Cells were utilized at passage 4.

MSC integrin expression during OD

MSCs were cultured for 3, 7 and 14

Surface expressions of integrins during MSC OD

Flow cytometry was used to detect surface expression of integrins during the initial steps of differentiation of MSCs in an osteoinductive media [21]. In the presence or absence of osteoinductive media, greater than 90% of the cells were positive for the integrin subunits β1, α5, α2 and αv, whereas αvβ3 integrin was weakly expressed (20% positive cells). Following 14 days in osteogenic media, no significant phenotypic changes in the population were observed (data not shown). However, the mean

Discussion

Our interest in full-length FN and FN fragments arises from a need to better direct stem cell fate in a physiological context, i.e. within a 3D microenvironment capable of displaying adhesion ligands and matrix-binding growth factors so as to mimic the ECM. Such engineered microenvironments may be useful in basic biological studies as well as in tissue engineering, both with transplanted MSCs as well as in influencing the behavior of endogenous infiltrating MSCs. To date, most efforts in this

Conclusion

This study investigates the effect of the degree of α5β1 engagement in the context of MSC OD within 2D and 3D environments. Our data indicate that the integrin α5β1 plays an important role in the OD process of MSCs within both environments but can be mitigated and controlled by the engagement of other integrins. Furthermore, this work extends our basic understanding of the mechanisms controlling integrin specificity to FN and FN fragments (i.e. FN domain stability) and provides new tools for

Acknowledgements

The authors sincerely thank Dr. Michael Smith (ETH, Zurich, Switzerland) for discussions regarding FN. This work was partially supported by the European Commission Framework Project 6 Program GENOSTEM consortium (M.M.M., J.A.H.), FY2008 Researcher Exchange Program between JSPS and SNSF (M.M.), NIH/NCI CA86997 (S.A.R.), and NSF EEC-9731643 (T.H.B).

References (26)

Cited by (0)

View full text