Novel muscle-derived extracellular matrix hydrogel promotes angiogenesis and neurogenesis in volumetric muscle loss.

Volumetric muscle loss (VML) represents a clinical challenge due to the limited regenerative capacity of skeletal muscle. Most often, it results in scar tissue formation and loss of function, which cannot be prevented by current therapies. Decellularized extracellular matrix (DEM) has emerged as a native biomaterial for the enhancement of tissue repair. Here, we report the generation and characterization of hydrogels derived from DEM prepared from WT or thrombospondin (TSP)-2 null muscle tissue. TSP2-null hydrogels, when compared to WT, displayed altered architecture, protein composition, and biomechanical properties and allowed enhanced invasion of C2C12 myocytes and chord formation by endothelial cells. They also displayed enhanced cell invasion, innervation, and angiogenesis following subcutaneous implantation. To evaluate their regenerative capacity, WT or TSP2 null hydrogels were used to treat VML injury to tibialis anterior muscles and the latter induced greater recruitment of repair cells, innervation, and blood vessel formation and reduced inflammation. Taken together, these observations indicate that TSP2-null hydrogels enhance angiogenesis and promote muscle repair in a VML model.

Dysregulation of TSP2-Rac1-WAVE2 axis in diabetic cells leads to cytoskeletal disorganization, increased cell stiffness, and dysfunction.

Fibroblasts are a major cell population that perform critical functions in the wound healing process. In response to injury, they proliferate and migrate into the wound space, engaging in extracellular matrix (ECM) production, remodeling, and contraction. However, there is limited knowledge of how fibroblast functions are altered in diabetes. To address this gap, several state-of-the-art microscopy techniques were employed to investigate morphology, migration, ECM production, 2D traction, 3D contraction, and cell stiffness. Analysis of cell-derived matrix (CDM) revealed that diabetic fibroblasts produce thickened and less porous ECM that hindered migration of normal fibroblasts. In addition, diabetic fibroblasts were found to lose spindle-like shape, migrate slower, generate less traction force, exert limited 3D contractility, and have increased cell stiffness. These changes were due, in part, to a decreased level of active Rac1 and a lack of co-localization between F-actin and Waskott-Aldrich syndrome protein family verprolin homologous protein 2 (WAVE2). Interestingly, deletion of thrombospondin-2 (TSP2) in diabetic fibroblasts rescued these phenotypes and restored normal levels of active Rac1 and WAVE2-F-actin co-localization. These results provide a comprehensive view of the extent of diabetic fibroblast dysfunction, highlighting the regulatory role of the TSP2-Rac1-WAVE2-actin axis, and describing a new function of TSP2 in regulating cytoskeleton organization.

Treating 'Septic' With Enhanced Antibiotics and 'Arthritis' by Mitigation of Excessive Inflammation.

Bacterial infection within the synovial joint, commonly known as septic arthritis, remains a clinical challenge as it presents two concurrent therapeutic goals of reducing bacterial burden and preservation of articular cartilage from destructive host inflammation. We hypothesized that mitigation of MRSA-induced inflammatory signaling could diminish destruction of articular cartilage in the setting of septic arthritis when used in conjunction with antibiotics. Herein, we provide evidence which supports a new therapeutic notion that concurrent antimicrobial therapy to address the 'septic' component of the disease with inflammation mitigation to manage the destructive 'arthritis' component. We established a murine model to mimic septic knee arthritis, as well as a variety of other inflammatory joint conditions. This murine septic arthritis model, in conjunction with in vitro and ex-vivo models, was utilized to characterize the inflammatory profile seen in active septic arthritis, as well as post-antibiotic treatment, via transcriptomic and histologic studies. Finally, we provided the clinical rationale for a novel therapeutic strategy combining enhanced antibiotic treatment with rifampin and adjuvant immunomodulation to inhibit post-infectious, excess chondrolysis and osteolysis. We identified that septic arthritis secondary to MRSA infection in our murine model led to increased articular cartilage damage compared to various types of inflammatory arthritis. The activation of the pERK1/2 signaling pathway, which is implicated with the mounting of an immune response and generation of inflammation, was increased in intracellular MRSA-infected synovial tissue and persisted despite antibiotic treatment. Trametinib, an inhibitor of ERK signaling through suppression of MEK1/2, alleviated the inflammation produced by the addition of intra-articular, heat-killed MRSA. Further, when combined with vancomycin and rifampin, mitigation of inflammation by pERK1/2 targeting improved outcomes for MRSA septic arthritis by conferring chondroprotection to articular cartilage and diminishing inflammatory osteolysis within bone. Our results support a new therapeutic notion that cell/biofilm-penetrating antibiotics alongside adjuvant mitigation of excessive intra-articular inflammation accomplish distinct therapeutic goals: reduction of bacterial burden and preservation of articular cartilage integrity.

Foreign body response to synthetic polymer biomaterials and the role of adaptive immunity.

Implanted biomaterials elicit a series of distinct immune and repair-like responses that are collectively known as the foreign body reaction (FBR). These include processes involving innate immune inflammatory cells and wound repair cells that contribute to the encapsulation of biomaterials with a dense collagenous and largely avascular capsule. Numerous studies have shown that the early phase is dominated by macrophages that fuse to form foreign body giant cells that are considered a hallmark of the FBR. With the advent of more precise cell characterization techniques, specific macrophage subsets have been identified and linked to more or less favorable outcomes. Moreover, studies comparing synthetic- and natural-based polymer biomaterials have allowed the identification of macrophage subtypes that distinguish between fibrotic and regenerative responses. More recently, cells associated with adaptive immunity have been shown to participate in the FBR to synthetic polymers. This suggests the existence of cross-talk between innate and adaptive immune cells that depends on the nature of the implants. However, the exact participation of adaptive immune cells, such as T and B cells, remains unclear. In fact, contradictory studies suggest either the independence or dependence of the FBR on these cells. Here, we review the evidence for the involvement of adaptive immunity in the FBR to synthetic polymers with a focus on cellular and molecular components. In addition, we examine the possibility that such biomaterials induce specific antibody responses resulting in the engagement of adaptive immune cells.

Dual therapeutic targeting of intra-articular inflammation and intracellular bacteria enhances chondroprotection in septic arthritis.

Bacterial infections involving joints and vital organs represent a challenging clinical problem because of the two concurrent therapeutic goals of bacterial eradication and tissue preservation. In the case of septic arthritis, permanent destruction of articular cartilage by intense host inflammation is commonly seen even after successful treatment of bacterial infection. Here, we provide scientific evidence of a novel treatment modality that can protect articular cartilage and enhanced eradication of causative bacteria in septic arthritis. Locally delivered cell-penetrating antibiotics such as rifampicin effectively eradicate intracellular reservoirs of methicillin-resistant Staphylococcus aureus within joint cells. Furthermore, mitigation of intra-articular inflammation by targeting the NLRP3 (nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3) inflammasome protects articular cartilage from damage in a murine model of knee septic arthritis. Together, concurrent mitigation of intra-articular inflammation and local adjuvant targeting of intracellular bacteria represents a promising new therapeutic strategy for septic arthritis.

Integrin ß3 targeting biomaterial preferentially promotes secretion of bFGF and viability of iPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) and their secretome have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC's functions. In this study, we investigated the effect of specific ECM ligand-integrin interaction on hiPSC-VSMC's paracrine secretion, cell viability, and morphology. Here, we show precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin ß3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvß3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin ß3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC-VSMC's morphology and cell viability. Finally, the secretome with enhanced bFGF shows potential for future wound healing applications.

Biocompatibility of platinum-based bulk metallic glass in orthopedic applications.

Bulk metallic glasses (BMGs) are a class of amorphous metals that exhibit high strength, ductility paired with wear and corrosion resistance. These properties suggest that they could serve as an alternative to conventional metallic implants that suffer wear and failure. In the present study, we investigated Platinum (Pt)-BMG biocompatibility in bone applications. Specifically, we investigated osteoclast formation on flat and nanopatterned Pt57.5Cu14.7Ni5.3P22.5(atomic percent) as well as titanium (control). Specifically, receptor activator of NF-κB (RANK) ligand-induced murine bone marrow derived mononuclear cell fusion was measured on multiple nanopatterns and was found to be reduced on nanorods (80 and 200 nm in diameter) and was associated with reduced tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase (MMP9) expression. Evaluation of mesenchymal stem cell (MSC) to osteoblast differentiation on nanopatterned Pt-BMG showed significant reduction in comparison to flat, suggesting that further exploration of nanopatterns is required to have simultaneous induction of osteoblasts and inhibition of osteoclasts.Invivo studies were also pursued to evaluate the biocompatibility of Pt-BMG in comparison to titanium. Rods of each material were implanted in the femurs of mice and evaluated by x-ray, mechanical testing, micro-computed tomography (micro-CT), and histological analysis. Overall, Pt-BMG showed similar biocompatibility with titanium suggesting that it has the potential to improve outcomes by further processing at the nanoscale.

Loss of endothelial glucocorticoid receptor promotes angiogenesis via upregulation of Wnt/ß-catenin pathway.

OBJECTIVE: The glucocorticoid receptor (GR) is a member of the nuclear receptor family that controls key biological processes in the cardiovascular system and has recently been shown to modulate Wnt signaling in endothelial cells. Wnt/ß-catenin signaling has been demonstrated to be crucial in the process of angiogenesis. In the current study, we studied whether GR could regulate angiogenesis via the Wnt/ß-catenin pathway. APPROACH AND RESULTSA: Key components of the Wnt/ß-catenin pathway were evaluated using quantitative PCR and Western blot in the presence or absence of GR. Enhanced angiogenesis was found in GR deficiency in vitro and confirmed with cell viability assays, proliferation assays and tube formation assays. Consistent with these in vitro findings, endothelial cell-specific GR loss GR in vivo promoted angiogenesis in both a hind limb ischemia model and sponge implantation assay. Results were further verified in a novel mouse model lacking endothelial LRP5/6, a key receptor in canonical Wnt signaling, and showed substantially suppressed angiogenesis using these same in vitro and in vivo assays. To further investigate the mechanism of GR regulation of Wnt signaling, autophagy flux was investigated in endothelial cells by visualizing auto phagolysosomes as well as by assessing P62 degradation and LC3B conversion. Results indicated that potentiated autophagy flux participated in GR-Wnt regulation. CONCLUSIONS: Lack of endothelial GR triggers autophagy flux, leads to activation of Wnt/ß-catenin signaling and promotes angiogenesis. There may also be a synergistic interaction between autophagy and Wnt/ß-catenin signaling.

Biocompatibility of nanomaterials and their immunological properties.

Nanomaterials (NMs) have revolutionized multiple aspects of medicine by enabling novel sensing, diagnostic, and therapeutic approaches. Advancements in processing and fabrication have also allowed significant expansion in the applications of the major classes of NMs based on polymer, metal/metal oxide, carbon, liposome, or multi-scale macro-nano bulk materials. Concomitantly, concerns regarding the nanotoxicity and overall biocompatibility of NMs have been raised. These involve putative negative effects on both patients and those subjected to occupational exposure during manufacturing. In this review, we describe the current state of testing of NMs including those that are in clinical use, in clinical trials, or under development. We also discuss the cellular and molecular interactions that dictate their toxicity and biocompatibility. Specifically, we focus on the reciprocal interactions between NMs and host proteins, lipids, and sugars and how these induce responses in immune and other cell types leading to topical and/or systemic effects.

Locally delivered adjuvant biofilm-penetrating antibiotics rescue impaired endochondral fracture healing caused by MRSA infection.

Infection is a devastating complication following an open fracture. We investigated whether local rifampin-loaded hydrogel can combat infection and improve healing in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10-12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony-forming units (CFU) of MRSA was inoculated. A collagen-based hydrogel containing low-dose (60 µg) and high-dose (300 µg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (µCT), immunohistochemistry, and histological analyses. All untreated MRSA-infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin-1ß stain were associated with poor callus formation. High-dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low-dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high-dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). µCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin-loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin-loaded hydrogels is required to correlate these findings with clinical efficacy.

An in situ collagen-HA hydrogel system promotes survival and preserves the proangiogenic secretion of hiPSC-derived vascular smooth muscle cells.

Human-induced pluripotent stem cell-derived vascular smooth muscle cells (hiPSC-VSMCs) with proangiogenic properties have huge therapeutic potential. While hiPSC-VSMCs have already been utilized for wound healing using a biomimetic collagen scaffold, an in situ forming hydrogel mimicking the native environment of skin offers the promise of hiPSC-VSMC mediated repair and regeneration. Herein, the impact of a collagen type-I-hyaluronic acid (HA) in situ hydrogel cross-linked using a polyethylene glycol-based cross-linker on hiPSC-VSMCs viability and proangiogenic paracrine secretion was investigated. Our study demonstrated increases in cell viability, maintenance of phenotype and proangiogenic growth factor secretion, and proangiogenic activity in response to the conditioned medium. The optimally cross-linked and functionalized collagen type-I/HA hydrogel system developed in this study shows promise as an in situ hiPSC-VSMC carrier system for wound regeneration.

The role of extracellular matrix in the pathophysiology of diabetic wounds.

Impaired healing leading to the formation of ulcerated wounds is a critical concern in patients with diabetes. Abnormalities in extracellular matrix (ECM) production and remodeling contribute to tissue dysfunction and delayed healing. Specifically, diabetes-induced changes in the expression and/or activity of structural proteins, ECM-modifying enzymes, proteoglycans, and matricellular proteins have been reported. In this review, we provide a summary of the key ECM molecules and associated changes in skin and diabetic wounds. Such information should allow for new insights in the understanding of impaired wound healing and lead to the development of ECM-based therapeutic strategies.

Extracellular matrix-derived biomaterials in engineering cell function.

Extracellular matrix (ECM) derived components are emerging sources for the engineering of biomaterials that are capable of inducing desirable cell-specific responses. This review explores the use of biomaterials derived from naturally occurring ECM proteins and their derivatives in approaches that aim to regulate cell function. Biomaterials addressed are grouped into six categories: purified single ECM proteins, combinations of purified ECM proteins, cell-derived ECM, tissue-derived ECM, diseased and modified ECM, and ECM-polymer coupled biomaterials. Purified ECM proteins serve as a material coating for enhanced cell adhesion and biocompatibility. Cell-derived and tissue-derived ECM, generated by cell isolation and decellularization technologies, can capture the native state of the ECM environment and guide cell migration and alignment patterns as well as stem cell differentiation. We focus primarily on recent advances in the fields of soft tissue, cardiac, and dermal repair, and explore the utilization of ECM proteins as biomaterials to engineer cell responses.

Elevated Thrombospondin 2 Contributes to Delayed Wound Healing in Diabetes.

Impaired wound healing is a major complication of diabetes, and despite the associated risks, treatment strategies for diabetic wounds remain limited. This is due, in part, to an incomplete understanding of the underlying pathological mechanisms, including the effects of hyperglycemia on components of the extracellular matrix (ECM). In the current study, we explored whether the expression of thrombospondin 2 (TSP2), a matricellular protein with a demonstrated role in response to injury, was associated with delayed healing in diabetes. First, we found that TSP2 expression was elevated in diabetic mice and skin from patients with diabetes. Then, to determine the contribution of TSP2 to impaired healing in diabetes, we developed a novel diabetic TSP2-deficient model. Though the TSP2-deficient mice developed obesity and hyperglycemia comparable with diabetic control mice, they exhibited significantly improved healing, characterized by accelerated reepithelialization and increased granulation tissue formation, fibroblast migration, and blood vessel maturation. We further found that hyperglycemia increased TSP2 expression in fibroblasts, the major cellular source of TSP2 in wounds. Mechanistically, high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. Our studies demonstrate that hyperglycemia-induced TSP2 expression contributes to impaired healing in diabetes.

The impact of modulating the blood-brain barrier on the electrophysiological and histological outcomes of intracortical electrodes.

OBJECTIVE: Successful application of chronic intracortical electrodes remains highly variable. The biological mechanisms leading to electrode failure are still being explored. Recent work has shown a correlation between blood-brain barrier (BBB) integrity and long-term recordings. Here we proposed to modulate the BBB healing after intracortical electrode implantation, while evaluating the functional electrophysiology. The CCL2/CCR2 pathway was chosen based on previous work demonstrating the positive histological effects in an intracortical electrode model, as well as in other neurodegenerative models. By disrupting this pathway, recruitment of pro-inflammatory monocytes (a result of a breached BBB) is potentially reduced at the electrode interface. APPROACH: Michigan electrodes were implanted for 2 and 12 weeks in rats, and a CCR2 antagonist (RS 102895) was administered daily to the treatment group. Functional electrodes were used for the 12 week cohort, and weekly electrophysiological recordings were taken. At 2 and 12 weeks, histology was analyzed. MAIN RESULTS: At 12 weeks, the CCR2-antagonist group had significantly higher signal-to-noise ratios (SNRs) than control. CCR2-antagonism at 2 weeks significantly increased the neural population and decreased BBB breach. At 12 weeks, CCR2-antagonism significantly increased number of neurons and BBB + vasculature within 100 µm of the electrode interface. SIGNIFICANCE: This work demonstrates that for intracortical electrodes, disruption of the CCL2/CCR2 pathway improves chronic outcomes in electrophysiology and histology.

Tunable Hydrogels Derived from Genetically Engineered Extracellular Matrix Accelerate Diabetic Wound Healing.

Hydrogels composed of solubilized decellularized extracellular matrix (ECM) are attractive materials because they combine the complexity of native ECM with injectability and ease of use. Nevertheless, these materials are typically only tunable by altering the concentration, which alters the ligand landscape, or by incorporating synthetic components, which can result in an unfavorable host response. Herein, we demonstrate the fabrication of genetically tunable ECM-derived materials, by utilizing wild type (WT) and (thrombospondin-2 knockout) TSP-2 KO decellularized skins to prepare hydrogels. The resulting materials exhibited distinct mechanical properties characterized by rheology and different concentrations of collagens when characterized by quantitative proteomics. Mixtures of the gels achieved intermediate effects between the WT and the KO, permitting tunability of the gel properties. In vivo, the hydrogels exhibited tunable cell invasion with a correlation between the content of TSP-2 KO hydrogel and the extent of cell invasion. Additionally, TSP-2 KO hydrogels significantly improved diabetic wound healing at 10 and 21 days. Furthermore, hydrogels derived from genetically engineered in vitro cell-derived matrix mimicked the trends observed for tissue-derived matrix, providing a platform for faster screening of novel manipulations and easier clinical translation. Overall, we demonstrate that genetic engineering approaches impart tunability to ECM-based hydrogels and can result in materials capable of enhanced regeneration.

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt57.5Cu14.7Ni5.3P22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

Nanopatterned bulk metallic glass-based biomaterials modulate macrophage polarization.

Polarization of macrophages by chemical, topographical and mechanical cues presents a robust strategy for designing immunomodulatory biomaterials. Here, we studied the ability of nanopatterned bulk metallic glasses (BMGs), a new class of metallic biomaterials, to modulate murine macrophage polarization. Cytokine/chemokine analysis of IL-4 or IFNγ/LPS-stimulated macrophages showed that the secretion of TNF-α, IL-1α, IL-12, CCL-2 and CXCL1 was significantly reduced after 24-hour culture on BMGs with 55 nm nanorod arrays (BMG-55). Additionally, under these conditions, macrophages increased phagocytic potential and exhibited decreased cell area with multiple actin protrusions. These in vitro findings suggest that nanopatterning can modulate biochemical cues such as IFNγ/LPS. In vivo evaluation of the subcutaneous host response at 2 weeks demonstrated that the ratio of Arg-1 to iNOS increased in macrophages adjacent to BMG-55 implants, suggesting modulation of polarization. In addition, macrophage fusion and fibrous capsule thickness decreased and the number and size of blood vessels increased, which is consistent with changes in macrophage responses. Our study demonstrates that nanopatterning of BMG implants is a promising technique to selectively polarize macrophages to modulate the immune response, and also presents an effective tool to study mechanisms of macrophage polarization and function. STATEMENT OF SIGNIFICANCE: Implanted biomaterials elicit a complex series of tissue and cellular responses, termed the foreign body response (FBR), that can be influenced by the polarization state of macrophages. Surface topography can influence polarization, which is broadly characterized as either inflammatory or repair-like. The latter has been linked to improved outcomes of the FBR. However, the impact of topography on macrophage polarization is not fully understood, in part, due to a lack of high moduli biomaterials that can be reproducibly processed at the nanoscale. Here, we studied macrophage interactions with nanopatterned bulk metallic glasses (BMGs), a class of metallic alloys with amorphous microstructure and formability like polymers. We show that nanopatterned BMGs modulate macrophage polarization and transiently induce less fibrotic and more angiogenic responses. Overall, we demonstrate nanopatterning of BMG implants as a technique to polarize macrophages and modulate the FBR.