The influence of microbial colonization on inflammatory versus pro-healing trajectories in combat extremity wounds.

A combination of improved body armor, medical transportation, and treatment has led to the increased survival of warfighters from combat extremity injuries predominantly caused by blasts in modern conflicts. Despite advances, a high rate of complications such as wound infections, wound failure, amputations, and a decreased quality of life exist. To study the molecular underpinnings of wound failure, wound tissue biopsies from combat extremity injuries had RNA extracted and sequenced. Wounds were classified by colonization (colonized vs. non-colonized) and outcome (healed vs. failed) status. Differences in gene expression were investigated between timepoints at a gene level, and longitudinally by multi-gene networks, inferred proportions of immune cells, and expression of healing-related functions. Differences between wound outcomes in colonized wounds were more apparent than in non-colonized wounds. Colonized/healed wounds appeared able to mount an adaptive immune response to infection and progress beyond the inflammatory stage of healing, while colonized/failed wounds did not. Although, both colonized and non-colonized failed wounds showed increasing inferred immune and inflammatory programs, non-colonized/failed wounds progressed beyond the inflammatory stage, suggesting different mechanisms of failure dependent on colonization status. Overall, these data reveal gene expression profile differences in healing wounds that may be utilized to improve clinical treatment paradigms.

In vitro dose-dependent effects of matrix metalloproteinases on ECM hydrogel biodegradation.

Matrix metalloproteinases (MMPs) cause proteolysis of extracellular matrix (ECM) in tissues affected by stroke. However, little is known about how MMPs degrade ECM hydrogels implanted into stroke cavities to regenerate lost tissue. To establish a structure-function relationship between different doses of individual MMPs and isolate their effects in a controlled setting, an in vitro degradation assay quantified retained urinary bladder matrix (UBM) hydrogel mass as a measure of degradation across time. A rheological characterization indicated that lower ECM concentrations (<4 mg/mL) did not cure completely at 37 °C and had a high fraction of mobile proteins that were easily washed-out. Hydrolysis by dH2O caused a steady 2 % daily decrease in hydrogel mass over 14 days. An acceleration of degradation to 6 % occurred with phosphate buffered saline and artificial cerebrospinal fluid. MMPs induced a dose-dependent increase and within 14 days almost completely (>95 %) degraded the hydrogel. MMP-9 exerted the most significant biodegradation, compared to MMP-3 and -2. To model the in vivo exposure of hydrogel to MMPs, mixtures of MMP-2, -3, and -9, present in the cavity at 14-, 28-, or 90-days post-stroke, revealed that 14- and 28-days mixtures achieved an equivalent biodegradation, but a 90-days mixture exhibited a slower degradation. These results revealed that hydrolysis, in addition to proteolysis, exerts a major influence on the degradation of hydrogels. Understanding the mechanisms of ECM hydrogel biodegradation is essential to determine the therapeutic window for bioscaffold implantation after a stroke, and they are also key to determine optimal degradation kinetics to support tissue regeneration. STATEMENT OF SIGNIFICANCE: After implantation into a stroke cavity, extracellular matrix (ECM) hydrogel promotes tissue regeneration through the degradation of the bioscaffold. However, the process of degradation of an ECM hydrogel remains poorly understood. We here demonstrated in vitro under highly controlled conditions that hydrogel degradation is very dependent on its protein concentration. Lower protein concentration hydrogels were weaker in rheological measurements and particularly susceptible to hydrolysis. The proteolytic degradation of tissue ECM after a stroke is caused by matrix metalloproteinases (MMPs). A dose-dependent MMP-driven biodegradation of ECM hydrogel exceeded the effects of hydrolysis. These results highlight the importance of in vitro testing of putative causes of degradation to gain a better understanding of how these factors affect in vivo biodegradation.

Antiviral epithelial-macrophage crosstalk permits secondary bacterial infections.

IMPORTANCE: Miscommunication of antiviral and antibacterial immune signals drives worsened morbidity and mortality during respiratory viral-bacterial coinfections. Extracellular vesicles (EVs) are a form of intercellular communication with broad implications during infection, and here we show that epithelium-derived EVs released during the antiviral response impair the antibacterial activity of macrophages, an innate immune cell crucial for bacterial control in the airway. Macrophages exposed to antiviral EVs display reduced clearance of Staphylococcus aureus as well as altered inflammatory signaling and anti-inflammatory metabolic reprogramming, thus revealing EVs as a source of dysregulated epithelium-macrophage crosstalk during coinfection. As effective epithelium-macrophage communication is critical in mounting an appropriate immune response, this novel observation of epithelium-macrophage crosstalk shaping macrophage metabolism and antimicrobial function provides exciting new insight and improves our understanding of immune dysfunction during respiratory coinfections.

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.

Creating and Transferring an Innervated, Vascularized Muscle Flap Made from an Elastic, Cellularized Tissue Construct Developed In Situ.

Reanimating facial structures following paralysis and muscle loss is a surgical objective that would benefit from improved options for harvesting appropriately sized muscle flaps. The objective of this study is to apply electrohydrodynamic processing to generate a cellularized, elastic, biocomposite scaffold that could develop and mature as muscle in a prepared donor site in vivo, and then be transferred as a thin muscle flap with a vascular and neural pedicle. First, an effective extracellular matrix (ECM) gel type is selected for the biocomposite scaffold from three types of ECM combined with poly(ester urethane)urea microfibers and evaluated in rat abdominal wall defects. Next, two types of precursor cells (muscle-derived and adipose-derived) are compared in constructs placed in rat hind limb defects for muscle regeneration capacity. Finally, with a construct made from dermal ECM and muscle-derived stem cells, protoflaps are implanted in one hindlimb for development and then microsurgically transferred as a free flap to the contralateral limb where stimulated muscle function is confirmed. This construct generation and in vivo incubation procedure may allow the generation of small-scale muscle flaps appropriate for transfer to the face, offering a new strategy for facial reanimation.

Mitigation of influenza-mediated inflammation by immunomodulatory matrix-bound nanovesicles.

Cytokine storm describes a life-threatening, systemic inflammatory syndrome characterized by elevated levels of proinflammatory cytokines and immune cell hyperactivation associated with multi-organ dysfunction. Matrix-bound nanovesicles (MBV) are a subclass of extracellular vesicle shown to down-regulate proinflammatory immune responses. The objective of this study was to assess the efficacy of MBV in mediating influenza-induced acute respiratory distress syndrome and cytokine storm in a murine model. Intravenous administration of MBV decreased influenza-mediated total lung inflammatory cell density, proinflammatory macrophage frequencies, and proinflammatory cytokines at 7 and 21 days following viral inoculation. MBV decreased long-lasting alveolitis and the proportion of lung undergoing inflammatory tissue repair at day 21. MBV increased the proportion of activated anti-viral CD4+ and CD8+ T cells at day 7 and memory-like CD62L+ CD44+, CD4+, and CD8+ T cells at day 21. These results show immunomodulatory properties of MBV that may benefit the treatment of viral-mediated pulmonary inflammation with applicability to other viral diseases such as SARS-CoV-2.

A Comparative Study of the Resorption and Immune Response for Two Starch-Based Hemostat Powders.

INTRODUCTION: Powder hemostats are valuable adjuncts to minimize intraoperative and postoperative complications. In addition to promotion of rapid coagulation, resorption, and biocompatibility are desirable attributes. Plant starch-based polysaccharide hemostat powders are effective and widely used hemostatic agents, however their source and/or processing can affect characteristics such as in vivo degradability. For example, Arista is a purified/hydrolyzed starch powder that is rapidly resorbed in vivo; whereas PerClot shows slow resorption and preservation of a crystalline form. MATERIALS AND METHODS: In the present study, we compared the cellular response to the hemostatic agents PerClot and Arista both in vitro and in vivo, and used potato starch and urinary bladder extracellular matrix (UBM-ECM) as high crystallinity/slowly resorbable and prohealing controls, respectively. RESULTS: All test articles and their degradation products were cytocompatible in vitro as measured by cell viability and metabolic activity of bone-marrow macrophages. PerClot induced a stronger proinflammatory, M1-like macrophage response in vitro (P < 0.001) than Arista, likely due to differences in source composition. Histologic examination of the in vivo surgical site showed the almost complete degradation of Arista after 12 h (day 0), whereas both PerClot and potato starch were still present at 28 d with crystals identifiable with polarized light microscopy and periodic acid Schiff (PAS) staining. Macrophage phenotype in vivo showed no differences between PerClot and Arista. Collagen deposition and mononuclear cell accumulation consistent with an early foreign body response were present around PerClot and potato starch crystals, whereas no such cell or connective tissue deposition was noted at the site of Arista or UBM-ECM placement.

Silk fibroin bioscaffold from Bombyx mori and Antheraea assamensis elicits a distinct host response and macrophage activation paradigm in vivo and in vitro.

Biomaterials composed of silk fibroin from both mulberry and non-mulberry silkworm varieties have been investigated for their utility in tissue engineering and drug delivery, but these studies have largely excluded any evaluation of host immune response. The present study compares the macrophage activation response towards mulberry (Bombyx mori, BM) and non-mulberry (Antheraea assamensis, AA) silk types, individually and as a blend (BA) in a partial thickness rat abdominal wall defect model and in vitro primary murine bone marrow-derived macrophage (BMDM) assay. Biologic materials composed of liver extracellular matrix (LECM) and small intestinal submucosa (SIS) ECM that are recognized for constructive tissue remodeling, and polypropylene mesh that is associated with pro-inflammatory macrophage phenotype activation are used as controls in the animal model. The AA silk graft shows a host response similar to SIS with few foreign body multinucleate giant cells, vascularization, high CD206 expression, and high M2-like: M1-like macrophage phenotype ratio. Exposure to AA silk degradation products in vitro induces a higher arginase: iNOS ratio in both naive BMDM and pro-inflammatory activated BMDM; and higher Fizz1: iNOS ratio in pro-inflammatory activated BMDM. These data suggest that the AA silk supports a pro-remodeling macrophage response with potential therapeutic applications.

Biocompatibility and biodistribution of matrix-bound nanovesicles in vitro and in vivo.

Matrix-bound nanovesicles (MBV) are a distinct subtype of extracellular vesicles that are firmly embedded within biomaterials composed of extracellular matrix (ECM). MBV both store and transport a diverse, tissue specific portfolio of signaling molecules including proteins, miRNAs, and bioactive lipids. MBV function as a key mediator in ECM-mediated control of the local tissue microenvironment. One of the most important mechanisms by which MBV in ECM bioscaffolds support constructive tissue remodeling following injury is immunomodulation and, specifically, the promotion of an anti-inflammatory, pro-remodeling immune cell activation state. Recent in vivo studies have shown that isolated MBV have therapeutic efficacy in rodent models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV administered independent of the rest of the ECM, the in vitro and in vivo safety and biodistribution profile of MBV remain uncharacterized. The purpose of the present study was to thoroughly characterize the pre-clinical safety profile of MBV through a combination of in vitro cytotoxicity and MBV uptake studies and in vivo toxicity, immunotoxicity, and imaging studies. The results showed that MBV isolated from porcine urinary bladder are well-tolerated and are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive compared to the potent immunosuppressive drug cyclophosphamide. Furthermore, this safety profile was sustained across a wide range of MBV doses. STATEMENT OF SIGNIFICANCE: Matrix-bound nanovesicles (MBV) are a distinct subtype of bioactive extracellular vesicles that are embedded within biomaterials composed of extracellular matrix (ECM). Recent studies have shown therapeutic efficacy of MBV in models of both retinal damage and rheumatoid arthritis through the targeted immunomodulation of pro-inflammatory macrophages towards an anti-inflammatory activation state. While these results show the therapeutic potential of MBV, the in vitro and in vivo biocompatibility and biodistribution profile of MBV remain uncharacterized. The results of the present study showed that MBV are a well-tolerated ECM-derived therapy that are not cytotoxic in cell culture, are non-toxic to the whole organism, and are not immunosuppressive. Collectively, these data highlight the translational feasibility of MBV therapeutics across a wide variety of clinical applications.

3D Bioprinted Patient-Specific Extracellular Matrix Scaffolds for Soft Tissue Defects.

Soft tissue injuries such as volumetric muscle loss (VML) are often too large to heal normally on their own, resulting in scar formation and functional deficits. Decellularized extracellular matrix (dECM) scaffolds placed into these wounds have shown the ability to modulate the immune response and drive constructive healing. This provides a potential solution for functional tissue regeneration, however, these acellular dECM scaffolds are challenging to fabricate into complex geometries. 3D bioprinting is uniquely positioned to address this, being able to create patient-specific scaffolds based on clinical 3D imaging data. Here, a process to use freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting and computed tomography (CT) imaging to build large volume, patient-specific dECM patches (≈12 × 8 × 2 cm) for implantation into canine VML wound models is developed. Quantitative analysis shows that these dECM patches are dimensionally accurate and conformally adapt to the surface of complex wounds. Finally, this approach is extended to a human VML injury to demonstrate the fabrication of clinically relevant dECM scaffolds with precise control over fiber alignment and micro-architecture. Together these advancements represent a step towards an improved, clinically translatable, patient-specific treatment for soft tissue defects from trauma, tumor resection, and other surgical procedures.

Matrix Bound Nanovesicles Have Tissue-Specific Characteristics That Suggest a Regulatory Role.

Recent studies have identified an extracellular vesicle population that is tightly anchored within the extracellular matrix (ECM) of tissues and organs until released by matrix turnover events. Evidence suggests that these matrix-bound nanovesicles (MBVs) are a ubiquitous component of the ECM, raising questions regarding their tissue-specific identity and their biologic function(s). The primary objective of this study was to examine MBVs isolated from six different tissues and compare their physical and compositional characteristics to determine the common and differentially expressed features. Accordingly, the results of this characterization show that while MBVs are a ubiquitous component of the ECM, they contain a protein and microRNA cargo that is tissue specific. The results furthermore suggest that MBVs have an important role in regulating tissue homeostasis.

Transcriptomic, Proteomic, and Morphologic Characterization of Healing in Volumetric Muscle Loss.

Skeletal muscle has a robust, inherent ability to regenerate in response to injury from acute to chronic. In severe trauma, however, complete regeneration is not possible, resulting in a permanent loss of skeletal muscle tissue referred to as volumetric muscle loss (VML). There are few consistently reliable therapeutic or surgical options to address VML. A major limitation in investigation of possible therapies is the absence of a well-characterized large animal model. In this study, we present results of a comprehensive transcriptomic, proteomic, and morphologic characterization of wound healing following VML in a novel canine model of VML which we compare to a nine-patient cohort of combat-associated VML. The canine model is translationally relevant as it provides both a regional (spatial) and temporal map of the wound healing processes that occur in human VML. Collectively, these data show the spatiotemporal transcriptomic, proteomic, and morphologic properties of canine VML healing as a framework and model system applicable to future studies investigating novel therapies for human VML. Impact Statement The spatiotemporal transcriptomic, proteomic, and morphologic properties of canine volumetric muscle loss (VML) healing is a translational framework and model system applicable to future studies investigating novel therapies for human VML.

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.

Adventitia-derived extracellular matrix hydrogel enhances contractility of human vasa vasorum-derived pericytes via α2 ß1 integrin and TGFß receptor.

Pericytes are essential components of small blood vessels and are found in human aortic vasa vasorum. Prior work uncovered lower vasa vasorum density and decreased levels of pro-angiogenic growth factors in adventitial specimens of human ascending thoracic aortic aneurysm. We hypothesized that adventitial extracellular matrix (ECM) from normal aorta promotes pericyte function by increasing pericyte contractile function through mechanisms deficient in ECM derived from aneurysmal aortic adventitia. ECM biomaterials were prepared as lyophilized particulates from decellularized adventitial specimens of human and porcine aorta. Immortalized human aortic adventitia-derived pericytes were cultured within Type I collagen gels in the presence or absence of human or porcine adventitial ECMs. Cell contractility index was quantified by measuring the gel area immediately following gelation and after 48 h of culture. Normal human and porcine adventitial ECM increased contractility of pericytes when compared with pericytes cultured in absence of adventitial ECM. In contrast, aneurysm-derived human adventitial ECM failed to promote pericyte contractility. Pharmacological inhibition of TGFßR1 and antibody blockade of α2 ß1 integrin independently decreased porcine adventitial ECM-induced pericyte contractility. By increasing pericyte contractility, adventitial ECM may improve microvascular function and thus represents a candidate biomaterial for less invasive and preventative treatment of human ascending aortic disease.

Continuous Microfiber Wire Mandrel-Less Biofabrication for Soft Tissue Engineering Applications.

Suture materials are the most common bioimplants in surgical and clinical practice, playing a crucial role in wound healing and tendon and ligament repair. Despite the assortment available on the market, sutures are still affected by significant disadvantages, including failure in mimicking the mechanical properties of the tissue, excessive fibrosis, and inflammation. This study introduces a mandrel-less electrodeposition apparatus to fabricate continuous microfiber wires of indefinite length. The mandrel-less biofabrication produces wires, potentially used as medical fibers, with different microfiber bundles, that imitate the hierarchical organization of native tissues, and tailored mechanical properties. Microfiber wire morphology and mechanical properties are characterized by scanning electron microscopy, digital image processing, and uniaxial tensile test. Wires are tested in vitro on monocyte/macrophage stimulation and in vivo on a rat surgical wound model. The wires produced by mandrel-less deposition show an increased M2 macrophage phenotype in vitro. The in vivo assessment demonstrates that microfiber wires, compared to the medical fibers currently used, reduce pro-inflammatory macrophage response and preserve their mechanical properties after 30 days of use. These results make this microfiber wire an ideal candidate as a suture material for soft tissue surgery, suggesting a crucial role of microarchitecture in more favorable host response.

Immunomodulatory matrix-bound nanovesicles mitigate acute and chronic pristane-induced rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints affecting ~7.5 million people worldwide. Disease pathology is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells, especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles (MBV) embedded within ECM. Isolated MBV was delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-induced arthritis (PIA). The results of MBV administration were compared to intraperitoneal (i.p.) administration of methotrexate (MTX), the clinical standard of care. Relative to the diseased animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both acute and chronic pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint remodeling, and reduced the ratio of synovial and splenic M1 to M2 macrophages (p < 0.05). Both p.a. and i.v. MBV reduced the serum concentration of RA and PIA biomarkers CXCL10 and MCP-3 in the acute and chronic phases of disease (p < 0.05). Flow-cytometry revealed the presence of a systemic CD43hi/His48lo/CD206+, immunoregulatory monocyte population unique to p.a. and i.v. MBV treatment associated with disease resolution. The results show that the therapeutic efficacy of MBV is equal to that of MTX for the management of acute and chronic pristane-induced arthritis and, further, this effect is associated with modulation of local synovial macrophages and systemic myeloid populations.

A liquid fraction of extracellular matrix inhibits glioma cell viability in vitro and in vivo.

Suppressive effects of extracellular matrix (ECM) upon various cancers have been reported. Glioblastoma multiforme has poor prognosis and new therapies are desired. This work investigated the effects of a saline-soluble fraction of urinary bladder ECM (ECM-SF) upon glioma cells. Viability at 24 hours in 1, 5, or 10 mg/mL ECM-SF-spiked media was evaluated in primary glioma cells (0319, 1015, 1119), glioma cell lines (A172, T98G, U87MG, C6), and brain cell lines (HCN-2, HMC3). Viability universally decreased at 5 and 10 mg/mL with U87MG, HCN-2, and HCM3 being least sensitive. Apoptosis in 0319 and 1119 cells was confirmed via NucView 488. Bi-weekly intravenous injection of ECM-SF (120 mg/kg) for 10 weeks in Sprague-Dawley rats did not affect weight, temperature, complete blood count, or multi-organ histology (N = 5). Intratumoral injection of ECM-SF (10 uL of 30 mg/mL) at weeks 2-4 post C6 inoculation in Wistar rats increased median survival from 24.5 to 51 days (hazard ratio for death 0.22) and decreased average tumor volume at time of death from 349 mm3 to 90 mm3 over 10 weeks (N = 6). Mass spectrometry identified 2,562 protein species in ECM-SF, parent ECM, and originating tissue. These results demonstrate the suppressive effects of ECM on glioma and warrant further study.

Mapping the acute time course of immune cell infiltration into an ECM hydrogel in a rat model of stroke using 19F MRI.

Extracellular matrix (ECM) hydrogel implantation into a stroke-induced tissue cavity invokes a robust cellular immune response. However, the spatio-temporal dynamics of immune cell infiltration into peri-infarct brain tissues versus the ECM-bioscaffold remain poorly understood. We here tagged peripheral immune cells using perfluorocarbon (PFC) nanoemulsions that afford their visualization by 19F magnetic resonance imaging (MRI). Prior to ECM hydrogel implantation, only blood vessels could be detected using 19F MRI. Using "time-lapse" 19F MRI, we established the infiltration of immune cells into the peri-infarct area occurs 5-6 h post-ECM implantation. Immune cells also infiltrated through the stump of the MCA, as well as a hydrogel bridge that formed between the tissue cavity and the burr hole in the skull. Tissue-based migration into the bioscaffold was observed between 9 and 12 h with a peak signal measured between 12 and 18 h post-implantation. Fluorescence-activated cell sorting of circulating immune cells revealed that 9% of cells were labeled with PFC nanoemulsions, of which the vast majority were neutrophils (40%) or monocytes (48%). Histology at 24 h post-implantation, in contrast, indicated that macrophages (35%) were more numerous in the peri-infarct area than neutrophils (11%), whereas the vast majority of immune cells within the ECM hydrogel were neutrophils (66%). Only a small fraction (12%) of immune cells did not contain PFC nanoemulsions, indicating a low type II error for 19F MRI. 19F MRI hence provides a unique tool to improve our understanding of the spatio-temporal dynamics of immune cells invading bioscaffolds and effecting biodegradation.

Inductive Remodeling of Extracellular Matrix Scaffolds in the Temporomandibular Joint of Pigs.

The temporomandibular joint (TMJ) disc is a fibrocartilaginous tissue located between the condyle of the mandible and glenoid fossa and articular eminence of the temporal bone. Damage or derangement of the TMJ disc can require surgical removal (discectomy) to restore function. Removal of the TMJ disc, however, leaves the joint space vulnerable to condylar remodeling and degradation, potentially leading to long-term complications. No consistently effective clinical option exists for repair or replacement of the disc following discectomy. This study investigates the use of an acellular scaffold composed of extracellular matrix (ECM) derived from small intestinal submucosa (SIS) as a regenerative template for the TMJ disc in a porcine model. Acellular SIS ECM scaffolds were implanted following discectomy and allowed to remodel for 2, 4, 12, and 24 weeks postimplantation. Remodeling of the implanted device was assessed by longitudinal magnetic resonance imaging (MRI) over the course of 6 months, as well as gross morphologic, histologic, biochemical, and biomechanical analysis (tension and compression) of explanted tissues (disc and condyle) at the time of sacrifice. When the scaffold remained in the joint space, longitudinal MRI demonstrated that the scaffolds promoted new tissue formation within the joint space throughout the study period. The scaffolds were rapidly populated with host-derived cells and remodeled with formation of new, dense, aligned fibrocartilage resembling native tissue as early as 1 month postimplantation. De-novo formation of peripheral muscular and tendinous attachments resembling those in native tissue was also observed. The remodeled scaffolds approached native disc biochemical composition and compressive modulus, and possessed 50% of the tensile modulus within 3 months postimplantation. No degradation of the condylar surface was observed. These results suggest that this acellular bioscaffold fills a medical need for which there is currently no effective treatment and may represent a clinically relevant "off-the-shelf" implant for reconstruction of the TMJ disc.