Silver-releasing bioactive glass nanoparticles for infected tissue regeneration.

Bacterial infections represent a formidable challenge, often leaving behind significant bone defects post-debridement and necessitating prolonged antibiotic treatments. The rise of antibiotic-resistant bacterial strains further complicates infection management. Bioactive glass nanoparticles have been presented as a promising substitute for bone defects and as carriers for therapeutic agents against microorganisms. Achieving consistent incorporation of ions into BGNs has proven challenging and restricted to a maximum ion concentration, especially when reducing the particle size. This study presents a notable achievement in the synthesis of 10 nm-sized Ag-doped bioactive glass nanoparticles (Ag-BGNs) using a modified yet straightforward Stöber method. The successful incorporation of essential elements, including P, Ca, Al, and Ag, into the glass structure at the intended concentrations (i.e., CaO wt% above 20 %) was confirmed by EDS, signifying a significant advancement in nanoscale biomaterial engineering. While exhibiting a spherical morphology and moderate dispersity, these nanoparticles tend to form submicron-sized aggregates outside of a solution state. The antibacterial effectiveness against MRSA was established across various experimental conditions, with Ag-BGNs effectively sterilizing planktonic bacteria without the need for antibiotics. Remarkably, when combined with oxacillin or fosfomycin, Ag-BGNs demonstrated a potent synergistic effect, restoring antibacterial capabilities against MRSA strains resistant to these antibiotics when used alone. Ag-BGNs exhibited potential in promoting human mesenchymal stromal cell proliferation, inducing the upregulation of osteoblast gene markers, and significantly contributing to bone regeneration in mice. This innovative synthesis protocol holds substantial promise for the development of biomaterials dedicated to the regeneration of infected tissue.

The composition and mechanical properties of porcine placental ECM from three different breeds.

Biologic scaffolds are extensively used in various clinical applications such as musculotendinous reconstruction, hernia repair or wound healing. Biologic scaffolds used in these applications vary in species, breed and tissue of origin, and other variables that affect their properties. Decellularization and sterilization processes also determine the characteristics of these scaffolds. The goal of the present study is to compare the composition and mechanical properties of decellularized porcine placental scaffolds from three different porcine breeds: Landrace, York and Duroc. Placental extracellular matrix (ECM) scaffolds from the three porcine breeds preserved the amnion/chorion ECM structure and the basement membrane markers laminin and collagen type IV. ECM placental scaffolds showed similar contents of collagen, elastin and lipids, and minimal differences in glycosaminoglycans content. Mechanical properties from the three breeds ECM placental scaffolds were also similar and stable for 24 months. While this study serves as preliminary characterization of porcine ECM scaffolds, future studies will determine their compatibility and suitability for tissue engineering applications.

Transcriptomic Regulation of Macrophages by Matrix-Bound Nanovesicle-Associated Interleukin-33.

The innate immune response, particularly the phenotype of responding macrophages, has significant clinical implications in the remodeling outcome following implantation of biomaterials and engineered tissues. In general, facilitation of an anti-inflammatory (M2-like) phenotype is associated with tissue repair and favorable outcomes, whereas pro-inflammatory (M1-like) activation can contribute to chronic inflammation and a classic foreign body response. Biologic scaffolds composed of extracellular matrix (ECM) and, more recently, matrix-bound nanovesicles (MBV) embedded within the ECM are known to direct macrophages toward an anti-inflammatory phenotype and stimulate a constructive remodeling outcome. The mechanisms of MBV-mediated macrophage activation are not fully understood, but interleukin-33 (IL-33) within the MBV appears critical for M2-like activation. Previous work has shown that IL-33 is encapsulated within the lumen of MBV and stimulates phenotypical changes in macrophages independent of its canonical surface receptor stimulation-2 (ST2). In the present study, we used next-generation RNA sequencing to determine the gene signature of macrophages following exposure to MBV with and without intraluminal IL-33. MBV-associated IL-33 instructed an anti-inflammatory phenotype in both wild-type and st2-/- macrophages by upregulating M2-like and downregulating M1-like genes. The repertoire of genes regulated by ST2-independent IL-33 signaling were broadly related to the inflammatory response and crosstalk between cells of both the innate and adaptive immune systems. These results signify the importance of the MBV intraluminal protein IL-33 in stimulating a pro-remodeling M2-like phenotype in macrophages and provides guidance for the designing of next-generation biomaterials and tissue engineering strategies. Impact statement The phenotype of responding macrophages is predictive of the downstream remodeling response to an implanted biomaterial. The clinical impact of macrophage phenotype has motivated studies to investigate the factors that regulate macrophage activation. Matrix-bound nanovesicles (MBV) embedded within the extracellular matrix direct macrophages toward an anti-inflammatory (M2)-like phenotype that is indicative of a favorable remodeling response. Although the mechanisms of MBV-mediated macrophage activation are not fully understood, the intraluminal protein interleukin-33 (IL-33) is clearly a contributing signaling molecule. The present study identifies those genes regulated by MBV-associated IL-33 that promote a pro-remodeling M2-like macrophage activation state and can guide future therapies in regenerative medicine.

Tissue response, macrophage phenotype, and intrinsic calcification induced by cardiovascular biomaterials: Can clinical regenerative potential be predicted in a rat subcutaneous implant model?

The host immune response to an implanted biomaterial, particularly the phenotype of infiltrating macrophages, is a key determinant of biocompatibility and downstream remodeling outcome. The present study used a subcutaneous rat model to compare the tissue response, including macrophage phenotype, remodeling potential, and calcification propensity of a biologic scaffold composed of glutaraldehyde-fixed bovine pericardium (GF-BP), the standard of care for heart valve replacement, with those of an electrospun polycarbonate-based supramolecular polymer scaffold (ePC-UPy), urinary bladder extracellular matrix (UBM-ECM), and a polypropylene mesh (PP). The ePC-UPy and UBM-ECM materials induced infiltration of mononuclear cells throughout the thickness of the scaffold within 2 days and neovascularization at 14 days. GF-BP and PP elicited a balance of pro-inflammatory (M1-like) and anti-inflammatory (M2-like) macrophages, while UBM-ECM and ePC-UPy supported a dominant M2-like macrophage phenotype at all timepoints. Relative to GF-BP, ePC-UPy was markedly less susceptible to calcification for the 180 day duration of the study. UBM-ECM induced an archetypical constructive remodeling response dominated by M2-like macrophages and the PP caused a typical foreign body reaction dominated by M1-like macrophages. The results of this study highlight the divergent macrophage and host remodeling response to biomaterials with distinct physical and chemical properties and suggest that the rat subcutaneous implantation model can be used to predict in vivo biocompatibility and regenerative potential for clinical application of cardiovascular biomaterials.

The effect of normal, metaplastic, and neoplastic esophageal extracellular matrix upon macrophage activation.

INTRODUCTION: Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. METHODS: Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). RESULTS: Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. CONCLUSION: A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.

Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole (n = 6) or omeprazole alone (n = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α+ cell infiltrate compared to control animals. The metaplastic tissue in control animals (n = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.

Lipidomics and RNA sequencing reveal a novel subpopulation of nanovesicle within extracellular matrix biomaterials.

Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

Extracellular Matrix-Based Biomaterials and Their Influence Upon Cell Behavior.

Biologic scaffold materials composed of allogeneic or xenogeneic extracellular matrix (ECM) are commonly used for the repair and remodeling of injured tissue. The clinical outcomes associated with implantation of ECM-based materials range from unacceptable to excellent. The variable clinical results are largely due to differences in the preparation of the material, including characteristics of the source tissue, the method and efficacy of decellularization, and post-decellularization processing steps. The mechanisms by which ECM scaffolds promote constructive tissue remodeling include mechanical support, degradation and release of bioactive molecules, recruitment and differentiation of endogenous stem/progenitor cells, and modulation of the immune response toward an anti-inflammatory phenotype. The methods of ECM preparation and the impact of these methods on the quality of the final product are described herein. Examples of favorable cellular responses of immune and stem cells associated with constructive tissue remodeling of ECM bioscaffolds are described.

Extracellular Matrix Bioscaffolds for Building Gastrointestinal Tissue.

Regenerative medicine is a rapidly advancing field that uses principles of tissue engineering, developmental biology, stem cell biology, immunology, and bioengineering to reconstruct diseased or damaged tissues. Biologic scaffolds composed of extracellular matrix have shown great promise as an inductive substrate to facilitate the constructive remodeling of gastrointestinal (GI) tissue damaged by neoplasia, inflammatory bowel disease, and congenital or acquired defects. The present review summarizes the preparation and use of extracellular matrix scaffolds for bioengineering of the GI tract, identifies significant advances made in regenerative medicine for the reconstruction of functional GI tissue, and describes an emerging therapeutic approach.

Macrophage phenotype in response to ECM bioscaffolds.

Macrophage presence and phenotype are critical determinants of the healing response following injury. Downregulation of the pro-inflammatory macrophage phenotype has been associated with the therapeutic use of bioscaffolds composed of extracellular matrix (ECM), but phenotypic characterization of macrophages has typically been limited to small number of non-specific cell surface markers or expressed proteins. The present study determined the response of both primary murine bone marrow derived macrophages (BMDM) and a transformed human mononuclear cell line (THP-1 cells) to degradation products of two different, commonly used ECM bioscaffolds; urinary bladder matrix (UBM-ECM) and small intestinal submucosa (SIS-ECM). Quantified cell responses included gene expression, protein expression, commonly used cell surface markers, and functional assays. Results showed that the phenotype elicited by ECM exposure (MECM) is distinct from both the classically activated IFNγ+LPS phenotype and the alternatively activated IL-4 phenotype. Furthermore, the BMDM and THP-1 macrophages responded differently to identical stimuli, and UBM-ECM and SIS-ECM bioscaffolds induced similar, yet distinct phenotypic profiles. The results of this study not only characterized an MECM phenotype that has anti-inflammatory traits but also showed the risks and challenges of making conclusions about the role of macrophage mediated events without consideration of the source of macrophages and the limitations of individual cell markers.

Extracellular matrix hydrogels from decellularized tissues: Structure and function.

Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed. STATEMENT OF SIGNIFICANCE: More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel.

Immunomodulation and Mobilization of Progenitor Cells by Extracellular Matrix Bioscaffolds for Volumetric Muscle Loss Treatment.

Acellular bioscaffolds composed of extracellular matrix (ECM) have been effectively used to promote functional tissue remodeling in both preclinical and clinical studies of volumetric muscle loss, but the mechanisms that contribute to such outcomes are not fully understood. Thirty-two C57bl/6 mice were divided into eight groups of four animals each. A critical-sized defect was created in the quadriceps muscle and was repaired with a small intestinal submucosa ECM bioscaffold or left untreated. Animals were sacrificed at 3, 7, 14, or 56 days after surgery. The spatiotemporal cellular response in both treated and untreated groups was characterized by immunolabeling methods. Early time points showed a robust M2-like macrophage phenotype following ECM treatment in contrast to the predominant M1-like macrophage phenotype present in the untreated group. ECM implantation promoted perivascular stem cell mobilization, increased presence of neurogenic progenitor cells, and was associated with myotube formation. These cell types were present not only at the periphery of the defect near uninjured muscle, but also in the center of the ECM-filled defect. ECM bioscaffolds modify the default response to skeletal muscle injury, and provide a microenvironment conducive to a constructive healing response.

Diffusion-Reaction Models of Genipin Incorporation into Fibrin Networks.

Genipin is a naturally derived small molecule that crosslinks compounds containing primary amines including many natural biopolymers. A diffusion-reaction model to predict the rates of delivery and incorporation of genipin into fibrin networks is presented. Genipin crosslink formation within fibrin hydrogels is a multi-step process that requires genipin diffusion and reaction with primary amines in hydrated networks. The reaction rate of genipin into fibrin gels was measured via spectroscopy while the rate of marginal crosslink formation was measured by rheology. Covalent coupling between genipin and primary amines in fibrin gels obeys second-order kinetics in genipin concentration with an effective activation energy of -71.9 ± 3.2 kJ-mol-1. Genipin diffusion-reaction within fibrin gels exhibits Thiele moduli between 0.02-0.28, which suggests that the systems studied herein are reaction-limited. Genipin-crosslinked fibrin clots are resistant to fibrinolytic degradation as measured by rheology. Finally, active genipin can be delivered from poly(D,L-lactide-co-glycolide) matrices to gels at rates that are comparable to the characteristic rate of incorporation in fibrin networks. Taken together, this work establishes a quantitative framework to engineer controlled release systems for genipin delivery into protein-based hydrogel networks.

Photoresponsive hydrogel networks using melanin nanoparticle photothermal sensitizers.

Photoreconfigurable and photodegradable polymeric networks have broad utility as functional biomaterials for many applications in medicine and biotechnology. The vast majority of these functional polymers are synthesized using chemical moieties that may be cytotoxic in vivo. Materials synthesized from these substituents also pose unknown risk upon implantation and thus will encounter significant regulatory challenges prior to use in vivo. This work describes a strategy to prepare photodegradable hydrogel networks that are composed of well-characterized synthetic polymers and natural melanin pigments found within the human body. Self-assembled networks of poly(l-lactide-co-glycolide)-poly(ethylene glycol) ABA triblock copolymers are doped with melanin nanoparticles to produce reconfigurable networks based on photothermal phase transitions. Self-assembled hydrogel networks with melanin nanoparticles exhibit a storage modulus ranging from 1.5 ± 0.6 kPa to 8.0 ± 7.5 kPa as measured by rheology. The rate of UV-induced photothermal heating was non-monotonic and varied as a function of melanin nanoparticle loading. A maximum steady state temperature increase of 20.5 ± 0.30 °C was measured. Experimental heating rates were in close agreement with predictions based on attenuation of light in melanins via photothermal absorption and Mie scattering. The implications of melanin nanoparticles on hydrogel network formation and light-induced disintegration were also characterized by rheology and dynamic light scattering. Taken together, this class of photoreconfigurable hydrogels represents a potential strategy for photodegradable polymers with increased likelihood for clinical translation.