Optimization of extracellular matrix production from human induced pluripotent stem cell-derived fibroblasts for scaffold fabrication for application in wound healing.

Extracellular matrix is a key component of all tissues, including skin and it plays a crucial role in the complex events of wound healing. These events are impaired in chronic wounds, with chronic inflammation and infection often present in these non-healing wounds. Many tissue engineering approaches for wound healing provide a scaffold to mimic the native matrix. Fibroblasts derived from iPS cells (iPSF) represent a novel source of matrix rich in pro-regenerative components, which can be used for scaffold fabrication to improve wound healing. However, in vitro production of matrix by cells for scaffold fabrication requires long cell culturing times which increases cost. The aim of this work is to optimize the iPSF matrix production by boosting matrix deposition, without affecting its composition. A good candidate technique to achieve this goal is macromolecular crowding, which is known to promote conversion of procollagen into mature collagen and its accumulation. We tested two molecular crowders, Ficoll and Carrageenan-in combination with ascorbic acid-over a prolonged period of time. Ficoll in combination with ascorbic acid notably increased collagen deposition and matrix dry weight compared to ascorbic acid alone, and did not affect matrix composition as measured by RT-PCR. Interestingly, Carrageenan did not affect collagen quantity, but it significantly increased glycosaminoglycan deposition. Finally, we successfully fabricated scaffolds from harvested matrix and confirmed their ability for cell growth and viability. This work lays the foundation for development of a time and cost effective protocol for novel iPSF ECM production for tissue engineering scaffolds.

Substrate Stiffness Modulates the Crosstalk Between Mesenchymal Stem Cells and Macrophages.

Upon implantation of a biomaterial, mesenchymal stem cells (MSCs) and macrophages contribute to the wound healing response and the regeneration cascade. Although biomaterial properties are known to direct MSC differentiation and macrophage polarization, the role of biomaterial cues, specifically stiffness, in directing the crosstalk between the two cell types is still poorly understood. This study aimed to elucidate the role of substrate stiffness in modulating the immunomodulatory properties of MSCs and to shed light on their complex interactions with macrophages when presented with diverse biomaterial stiffness cues, a situation analogous to the implant environment where multiple cell types interact with an implanted biomaterial to determine regenerative outcomes. We show that MSCs do not play an immunomodulatory role in the absence of an inflammatory stimulus. Using collagen-coated polyacrylamide gels of varying stiffness values, we demonstrate that the immunomodulatory capability of MSCs in the presence of an inflammatory stimulus is not dependent on the stiffness of the underlying substrate. Moreover, using paracrine and direct contact culture models, we show that a bidirectional crosstalk between MSCs and macrophages is necessary for promoting anti-inflammatory responses and positive immunomodulation, which is dependent on the stiffness of the underlying substrate. We finally show that direct cell-cell contact is not essential for this effect, with paracrine interactions promoting immunomodulatory interactions between MSCs and macrophages. Together, these results demonstrate that biophysical cues such as stiffness that are presented by biomaterials can be tuned to promote positive interactions between MSCs and macrophages which can in turn direct the downstream regenerative response.

Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing.

Impaired wound healing of diabetic foot ulcers has been linked to high MMP-9 levels at the wound site. Strategies aimed at the simultaneous downregulation of the MMP-9 level in situ and the regeneration of impaired tissue are critical for improved diabetic foot ulcer (DFU) healing. To fulfil this aim, collagen/GAG (Col/GAG) scaffolds activated by MMP-9-targeting siRNA (siMMP-9) were developed in this study. The siMMP-9 complexes were successfully formed by mixing the RALA cell penetrating peptide with siMMP-9. The complexes formulated at N:P ratios of 6 to 15 had a diameter around 100 nm and a positive zeta potential about 40 mV, making them ideal for cellular uptake. In 2 dimensional (2D) culture of human fibroblasts, the cellular uptake of the complexes surpassed 60% and corresponded to a 60% reduction in MMP-9 gene expression in low glucose culture. In high glucose culture, which induces over-expression of MMP-9 and therefore serves as an in vitro model mimicking conditions in DFU, the MMP-9 gene could be downregulated by around 90%. In the 3D culture of fibroblasts, the siMMP-9 activated Col/GAG scaffolds displayed excellent cytocompatibility and ~60% and 40% MMP-9 gene downregulation in low and high glucose culture, respectively. When the siMMP-9 complexes were applied to THP-1 macrophages, the primary cell type producing MMP-9 in DFU, MMP-9 gene expression was significantly reduced by 70% and 50% for M0 and M1 subsets, in 2D culture. In the scaffolds, the MMP-9 gene and protein level of M1 macrophages decreased by around 50% and 30% respectively. Taken together, this study demonstrates that the RALA-siMMP-9 activated Col/GAG scaffolds possess high potential as a promising regenerative platform for improved DFU healing.

Scaffolds Functionalized with Matrix from Induced Pluripotent Stem Cell Fibroblasts for Diabetic Wound Healing.

Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product-a cell-free collagen-glycosaminoglycan (GAG) scaffold-demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic-like state. Recent findings describe how iPSCs-derived fibroblasts ("post-iPSF") are proangiogenic, produce more ECM than their somatic precursors ("pre-iPSF"), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar-free). ECM production is 45% higher from post-iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze-dried scaffold using ECM grown by post-iPSF cells (Post-iPSF Coll) is developed and tested vs precursors ECM-activated scaffolds (Pre-iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM-derived scaffolds have more diverse ECM and more robust immune responses to cues. Post-iPSF-Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin-1-receptor antagonist (IL-1ra) vs. pre-iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.

The Use of Genipin as an Effective, Biocompatible, Anti-Inflammatory Cross-Linking Method for Nerve Guidance Conduits.

A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs.

Hydroxyapatite Particle Shape and Size Influence MSC Osteogenesis by Directing the Macrophage Phenotype in Collagen-Hydroxyapatite Scaffolds.

The field of bone tissue engineering has seen the advancement of a variety of biomaterials with a diverse range of material properties. Biomaterial properties such as particle shape and size, stiffness, and pore size all influence the osteogenic capacity of biomaterials, typically evaluated in vitro by analyzing their potential to promote osteogenesis in mesenchymal stem cells (MSCs). There is now accumulating evidence highlighting the role of macrophages in driving bone regeneration responses. In this study, we evaluated the osteogenic capacity of collagen scaffolds functionalized with hydroxyapatite particles of varying shapes (needle vs spherical) and sizes (5 µm vs 100 µm) using an in vitro culture system of MSCs alone and in coculture with macrophages. We show that macrophage response to HA particles was elevated in the presence of a scaffold with 5 µm needle-shaped particles (Coll N5), with an increase in the expression and secretion of both pro-inflammatory (TNFα, IL6, and MIP1α) and anti-inflammatory (IL10 and IL1Ra) factors. When MSCs alone were cultured on the scaffolds, we show that scaffolds with HA particles were highly osteogenic, with superior osteogenesis observed in scaffolds with large 30 µm spherical particles (Coll S30) compared to small 5 µm needle-shaped particles (Coll N5). A coculture of MSCs with macrophages increased osteogenesis in all groups, with the most dramatic increase on Coll N5 scaffolds, leading to an elimination of the differences observed during monoculture. Through gene expression analysis, we showed that this correlated with an enhanced pro-osteogenic macrophage phenotype on Coll N5 scaffolds. These results highlight the potential of modulating material properties such as particle shape and size to develop osteoimmunomodulatory materials that direct osteogenic responses by influencing macrophage response.

Material stiffness influences the polarization state, function and migration mode of macrophages.

Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states (Anderson et al., 2008; Brown and Badylak, 2013). It is known that chemical signals such as bacterial endotoxins and cytokines (IL4) can direct macrophage polarization (Mantovani et al., 2004); however, recent evidence implicates biophysical cues in this process (McWhorter et al., 2015; Patel et al., 2012). Here we report that THP-1 derived macrophages cultured on collagen-coated polyacrylamide gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. Through gene expression and protein secretion analysis, we show that stiff polyacrylamide gels (323 kPa) prime macrophages towards a pro-inflammatory phenotype with impaired phagocytosis in macrophages, while soft (11 kPa) and medium (88 kPa) stiffness gels prime cells towards an anti-inflammatory, highly phagocytic phenotype. Furthermore, we show that stiffness dictates the migration mode of macrophages; on soft and medium stiffness gels, cells display Rho-A kinase (ROCK)-dependent, podosome-independent fast amoeboid migration and on stiff gels they adopt a ROCK-independent, podosome-dependent slow mesenchymal migration mode. We also provide a mechanistic insight into this process by showing that the anti-inflammatory property of macrophages on soft and medium gels is ROCK-dependent and independent of the ligand presented to them. Together, our results demonstrate that macrophages adapt their polarization, function and migration mode in response to the stiffness of the underlying substrate and suggest that biomaterial stiffness is capable of directing macrophage behaviour independent of the biochemical cues being presented to them. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation. STATEMENT OF SIGNIFICANCE: Biomaterial implantation is followed by an inflammatory cascade dominated by macrophages, which determine implant acceptance or rejection through pro- and anti-inflammatory polarization states. It is known that chemical signals can direct macrophage polarization; however, recent evidence implicates biophysical cues in this process. Here we report that macrophages cultured on gels of varying stiffness adapt their polarization state, functional roles and migration mode according to the stiffness of the underlying substrate. The results from this study establish an important role for substrate stiffness in directing macrophage behaviour, and will lead to the design of immuno-informed biomaterials that are capable of modulating the macrophage response after implantation.

Macrophage Polarization in Response to Collagen Scaffold Stiffness Is Dependent on Cross-Linking Agent Used To Modulate the Stiffness.

Macrophages are the first responders to biomaterial implantation and determine the success or failure of an implant through their polarization into proinflammatory (M1) and anti-inflammatory (M2) states. It is known that material properties such as stiffness can influence this response, with these properties typically modulated using a cross-linking agent. However, the cellular response comparing different cross-linking agents is often not analyzed. In this study, collagen scaffolds were cross-linked with one physical (DHT) and two chemical cross-linking methods (EDAC and genipin) in order to independently modulate the stiffness of scaffolds. The physical and structural properties of the scaffolds were thoroughly characterized to ensure that macrophage behaviors toward scaffold stiffness and cross-linking agent employed could be evaluated independent of each other. Through gene expression and protein secretion analysis of THP1 cultures, we demonstrate that the macrophage response to collagen scaffold stiffness is dependent on the cross-linking agent used. Macrophages respond similarly to scaffolds of increasing stiffness generated using the same cross-linking agent. However, when exposed to scaffolds of similar bulk modulus and degradation characteristics cross-linked using different cross-linkers, the cells responded to the cross-linking agent used rather than to the bulk modulus of the scaffolds. Moreover, while genipin cross-linking suppressed both proinflammatory and anti-inflammatory responses from macrophages, EDAC cross-linking promoted a robust proinflammatory and anti-inflammatory response to M1 and M2 factors, respectively. The results demonstrate the potential of using tailored individual cross-linking treatments depending on the clinical indication. Taken together, the results from this study highlight the importance of understanding the macrophage response to both chemical and physical properties of scaffolds in order to promote positive remodeling outcomes after biomaterial implantation.

Functionalising Collagen-Based Scaffolds With Platelet-Rich Plasma for Enhanced Skin Wound Healing Potential.

Porous collagen-glycosaminoglycan (collagen-GAG) scaffolds have shown promising clinical results for wound healing; however, these scaffolds do not replace the dermal and epidermal layer simultaneously and rely on local endogenous signaling to direct healing. Functionalizing collagen-GAG scaffolds with signaling factors, and/or additional matrix molecules, could help overcome these challenges. An ideal candidate for this is platelet-rich plasma (PRP) as it is a natural reservoir of growth factors, can be activated to form a fibrin gel, and is available intraoperatively. We tested the factors released from PRP (PRPr) and found that at specific concentrations, PRPr enhanced cell proliferation and migration and induced angiogenesis to a greater extent than fetal bovine serum (FBS) controls. This motivated us to develop a strategy to successfully incorporate PRP homogeneously within the pores of the collagen-GAG scaffolds. The composite scaffold released key growth factors for wound healing (FGF, TGFß) and vascularization (VEGF, PDGF) for up to 14 days. In addition, the composite scaffold had enhanced mechanical properties (when compared to PRP gel alone), while providing a continuous upper surface of extracellular matrix (ECM) for keratinocyte seeding. The levels of the factors released from the composite scaffold were sufficient to sustain proliferation of key cells involved in wound healing, including human endothelial cells, mesenchymal stromal cells, fibroblasts, and keratinocytes; even in the absence of FBS supplementation. In functional in vitro and in vivo vascularization assays, our composite scaffold demonstrated increased angiogenic and vascularization potential, which is known to lead to enhanced wound healing. Upon pro-inflammatory induction, macrophages released lower levels of the pro-inflammatory marker MIP-1α when treated with PRPr; and released higher levels of the anti-inflammatory marker IL1-ra upon both pro- and anti-inflammatory induction when treated with the composite scaffold. Finally, our composite scaffold supported a co-culture system of human fibroblasts and keratinocytes that resulted in an epidermal-like layer, with keratinocytes constrained to the surface of the scaffold; by contrast, keratinocytes were observed infiltrating the PRP-free scaffold. This novel composite scaffold has the potential for rapid translation to the clinic by isolating PRP from a patient intraoperatively and combining it with regulatory approved scaffolds to enhance wound repair.

The shape and size of hydroxyapatite particles dictate inflammatory responses following implantation.

The extent of regeneration following biomaterial implantation is dependent on the microenvironment surrounding the implant. Since implant composition can have a profound effect on inflammation, it is essential to understand this process as a non-resolving inflammatory response can lead to fibrous encapsulation and insufficient integration. Incorporation of particulates into implants confers structural and functional benefits, thus optimizing particulate characteristics to enhance immune mediated efficacy is important. We investigated the relationship between the nature of hydroxyapatite (HA) particles and the innate immune response, focusing on how particle size (0.1 µm, 5 µm, 20 µm, 100 µm) and morphology (needle-shaped/spherical; smooth/rough surface) modulates inflammatory responses. We observed a shape and size-dependent activation of the NLRP3 inflammasome and IL-1ß secretion; while needle-shaped and smaller HA particles significantly enhanced cytokine secretion, larger particles did not. Moreover, HA particle characteristics profoundly influenced patterns of innate immune cell recruitment and cytokine production following injection. While small, needle-shaped particles induced a strong inflammatory response, this was not observed with smooth, spherical particles of comparable size or with larger particles. These findings indicate that hydroxyapatite particle characteristics dictate immune cell recruitment and the ensuing inflammatory response, providing an opportunity to tailor HA particle characteristics to regulate immune responses induced after biomaterial implantation.

Decellularized grafts with axially aligned channels for peripheral nerve regeneration.

At least 2 million people worldwide suffer annually from peripheral nerve injuries (PNI), with estimated costs of $7 billion incurred due to paralysis alone. The current "gold" standard for treatment of PNI is the autograft, which poses disadvantages such as high fiscal cost, possible loss of sensation at donor site and the requirement of two surgeries. Allografts are viable alternatives; however, intensive immunosuppressive treatments are often necessary to prevent host rejection. For this reason, significant efforts have been made to remove cellular material from allografts. These decellularized nerve grafts perform better than other clinically available grafts but not as well as autografts; therefore, current research on these grafts includes the incorporation of additional components such as growth factors and cells to provide chemical guidance to regenerating axons. However, effective cellular and axonal penetration is not achieved due to the small pore size (5-10µm) of the decellularized grafts. The overall objective of this study was to induce axially aligned channels in decellularized nerve grafts to facilitate enhanced cell penetration. The specific aims of this study were to optimize a decellularization method to enhance cellular removal, to induce axially aligned pore formation in decellularized grafts through a novel unidirectional freeze drying method, to study the bulk mechanical properties of these modified decellularized grafts and to assess cell penetration into these grafts. To this end we modified an existing decellularization protocol to improve cellular removal while preserving matrix structure in rat sciatic nerve sections. Standard freeze drying and unidirectional freeze drying were employed to impart the necessary pore architecture, and our results suggest that unidirectional freezing is a pertinent modification to the freeze drying process to obtain axially aligned channels. These highly porous scaffolds obtained using unidirectional freeze-drying possessed similar tensile properties to native nerve tissue and exhibited enhanced cellular penetration after 14 days of culture when compared to non-freeze dried and standard freeze-dried scaffolds. The results of this study not only highlight the importance of aligned pores of diameters ~20-60µm on cellular infiltration, but also presents unidirectional freeze drying as a viable technique for producing this required architecture in decellularized nerves. To the best of our knowledge, this study represents the first attempt to manipulate the physical structure of decellularized nerves to enhance cell penetration which may serve as a basis for future peripheral nerve regenerative strategies using decellularized allografts.

Bioengineering tools to elucidate and control the fate of transplanted stem cells.

For the last decade, stem cell therapies have demonstrated enormous potential for solving some of the most tragic illnesses, diseases and tissue defects worldwide. Currently, more than 1300 clinical trials use stem cell therapy to solve a spectrum of cardiovascular, neurodegenerative and autoimmune diseases (http://www.clinicaltrials.gov, Jan 2014, search term: stem cell therapy; only currently recruiting and completed studies are included in the search). However, the efficacy of stem cell transplantation in patients has not been well established, and recent clinical trials have produced mixed results. We attribute this lack of efficacy in part to an incomplete understanding of the fate of stem cells following transplantation and the lack of control over cell fate, especially cell-homing and therapeutic functions. In the present review, we present two of our recently developed technologies that aim to address the above-mentioned bottlenecks in stem cell therapy specifically in the areas of MSCs (mesenchymal stem cells): (i) aptamer-based cell-surface sensors to study cellular microenvironments, and (ii) mRNA engineering technology to enhance the homing and immunomodulatory efficacy of transplanted stem cells. The first engineering strategy aims to elucidate the basic cellular signalling that occurs in the microenvironment of transplanted stem cells in real time. The second technique involves a simple mRNA transfection that improves the homing and anti-inflammatory capability of MSCs. Although we have specifically applied these engineering techniques to MSCs, these strategies can be incorporated for almost any cell type to determine and control the fate of transplanted stem cells.

Engineered cell homing.

One of the greatest challenges in cell therapy is to minimally invasively deliver a large quantity of viable cells to a tissue of interest with high engraftment efficiency. Low and inefficient homing of systemically delivered mesenchymal stem cells (MSCs), for example, is thought to be a major limitation of existing MSC-based therapeutic approaches, caused predominantly by inadequate expression of cell surface adhesion receptors. Using a platform approach that preserves the MSC phenotype and does not require genetic manipulation, we modified the surface of MSCs with a nanometer-scale polymer construct containing sialyl Lewis(x) (sLe(x)) that is found on the surface of leukocytes and mediates cell rolling within inflamed tissue. The sLe(x) engineered MSCs exhibited a robust rolling response on inflamed endothelium in vivo and homed to inflamed tissue with higher efficiency compared with native MSCs. The modular approach described herein offers a simple method to potentially target any cell type to specific tissues via the circulation.