Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors

The 12th-14th type III repeats of fibronectin function as a highly promiscuous growth factor-binding domain

It has recently been shown that some growth factors (GFs) have strong interactions with nonproteoglycan extracellular matrix proteins. Relevant here, the 12th-14th type three repeats of fibronectin (FN III12-14) have been shown to bind insulin-like growth factor binding-protein-3, fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF)-A with high affinity. Since FN III12-14 is known to bind GFs from different families, we hypothesized that this domain could be highly promiscuous in its GF-binding capacity. We used biochemical approaches and surface plasmon resonance to investigate such interactions with recombinant FN III12-14. We found that FN III12-14 binds most of the GFs from the platelet-derived growth factor (PDGF)/VEGF and FGF families and some GFs from the transforming growth factor-β and neurotrophin families, with K(D) values in the nanomolar range, without inhibiting GF activity. Overall, 25 new binding interactions were identified. In a clinically relevant fibrin matrix, a fibrin-binding variant of FN III12-14 was highly effective as a GF delivery system. For instance, in matrices functionalized with FN III12-14, PDGF-BB-induced sprouting of human smooth muscle cell spheroids was greatly enhanced. We show that FN III12-14 is a highly promiscuous ligand for GFs and also holds great potential in clinical healing applications

Immune Regulation of Tissue Repair and Regeneration via miRNAs—New Therapeutic Target

The importance of immunity in tissue repair and regeneration is now evident. Thus, promoting tissue healing through immune modulation is a growing and promising field. Targeting microRNAs (miRNAs) is an appealing option since they regulate immunity through post-transcriptional gene fine-tuning in immune cells. Indeed, miRNAs are involved in inflammation as well as in its resolution by controlling immune cell phenotypes and functions. In this review, we first discuss the immunoregulatory role of miRNAs during the restoration of tissue homeostasis after injury, focusing mainly on neutrophils, macrophages and T lymphocytes. As tissue examples, we present the immunoregulatory function of miRNAs during the repair and regeneration of the heart, skeletal muscles, skin and liver. Secondly, we discuss recent technological advances for designing therapeutic strategies which target miRNAs. Specifically, we highlight the possible use of miRNAs and anti-miRNAs for promoting tissue regeneration via modulation of the immune system

Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem cell-driven tissue regeneration

Tissue injury and the healing response lead to the release of endogenous danger signals including Toll-like receptor (TLR) and interleukin-1 receptor, type 1 (IL-1R1) ligands, which modulate the immune microenvironment. Because TLRs and IL-1R1 have been shown to influence the repair process of various tissues, we explored their role during bone regeneration, seeking to design regenerative strategies integrating a control of their signalling. Here we show that IL-1R1/MyD88 signalling negatively regulates bone regeneration, in the mouse. Furthermore, IL-1β which is released at the bone injury site, inhibits the regenerative capacities of mesenchymal stem cells (MSCs). Mechanistically, IL-1R1/MyD88 signalling impairs MSC proliferation, migration and differentiation by inhibiting the Akt/GSK-3β/β-catenin pathway. Lastly, as a proof of concept, we engineer a MSC delivery system integrating inhibitors of IL-1R1/MyD88 signalling. Using this strategy, we considerably improve MSC-based bone regeneration in the mouse, demonstrating that this approach may be useful in regenerative medicine applications

Evaluation of injectable constructs for bone repair with a subperiosteal cranial model in the rat

While testing regenerative medicine strategies, the use of animal models that match the research questions and that are related to clinical translation is crucial. During the initial stage of evaluating new strategies for bone repair, the main goal is to state whether the strategies efficiently induce the formation of new bone tissue at an orthotopic site. Here, we present a subperiosteal model in rat calavria that allow the evaluation of a broad range of approaches including bone augmentation, replacement and regeneration. Easy and fast to perform, the model is minimally invasive and no defect are created. The procedure enables to evaluate the outcomes quantitatively using micro-computed tomography and qualitatively by histology and immunohistochemistry. For establishing the model, we used bone morphogenetic protein-2 as an osteoinductive factor and hyaluronic acid hydrogel as injectable biomaterial. We showed that this subperiosteal cranial model offers a minimally invasive and promising solution for a rapid evaluation of bone tissue engineering strategies, even for investigator with limited experience in orthopedic surgery. We believe that this approach could be a powerful platform for orthopedic research and regenerative medicine

Regulatory T-Cells: Potential Regulator of Tissue Repair and Regeneration

The identification of stem cells and growth factors as well as the development of biomaterials hold great promise for regenerative medicine applications. However, the therapeutic efficacy of regenerative therapies can be greatly influenced by the host immune system, which plays a pivotal role during tissue repair and regeneration. Therefore, understanding how the immune system modulates tissue healing is critical to design efficient regenerative strategies. While the innate immune system is well known to be involved in the tissue healing process, the adaptive immune system has recently emerged as a key player. T-cells, in particular, regulatory T-cells (Treg), have been shown to promote repair and regeneration of various organ systems. In this review, we discuss the mechanisms by which Treg participate in the repair and regeneration of skeletal and heart muscle, skin, lung, bone, and the central nervous system

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

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 alpha(5)beta(1) and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of alpha(5)beta(1) integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that alpha(5)beta(1) has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for alpha(5)beta(1) versus alpha(v)beta(3) 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