Safety and efficacy of an injectable nerve-specific hydrogel in a rodent crush injury model.

INTRODUCTION/AIMS: While the peripheral nervous system has the inherent ability to recover following injury, results are often unsatisfactory, resulting in permanent functional deficits and disability. Therefore, methods that enhance regeneration are of significant interest. The present study investigates an injectable nerve-tissue-specific hydrogel as a biomaterial for nerve regeneration in a rat nerve crush model. METHODS: Nerve-specific hydrogels were injected into the subepineurial space in both uninjured and crushed sciatic nerves of rats to assess safety and efficacy, respectively. The animals were followed longitudinally for 12 wk using sciatic functional index and kinematic measures. At 12 wk, electrophysiologic examination was also performed, followed by nerve and muscle histologic assessment. RESULTS: When the hydrogel was injected into an uninjured nerve, no differences in sciatic functional index, kinematic function, or axon counts were observed. A slight reduction in muscle fiber diameter was observed in the hydrogel-injected animals, but overall muscle area and kinematic function were not affected. Hydrogel injection following nerve crush injury resulted in multiple modest improvements in sciatic functional index and kinematic function with an earlier return to normal function observed in the hydrogel treated animals as compared to untreated controls. While no improvements in supramaximal compound motor action potential were observed in hydrogel treated animals, increased axon counts were observed on histologic assessment. DISCUSSION: These improvements in functional and histologic outcomes in a rapidly and fully recovering model suggest that injection of a nerve-specific hydrogel is safe and has the potential to improve outcomes following nerve injury.

Inflammation and Ectopic Fat Deposition in the Aging Murine Liver Is Influenced by CCR2.

Aging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month-old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month-old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand-CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients.

Decellularization techniques and their applications for the repair and regeneration of the nervous system.

A variety of surgical and non-surgical approaches have been used to address the impacts of nervous system injuries, which can lead to either impairment or a complete loss of function for affected patients. The inherent ability of nervous tissues to repair and/or regenerate is dampened due to irreversible changes that occur within the tissue remodeling microenvironment following injury. Specifically, dysregulation of the extracellular matrix (i.e., scarring) has been suggested as one of the major factors that can directly impair normal cell function and could significantly alter the regenerative potential of these tissues. A number of tissue engineering and regenerative medicine-based approaches have been suggested to intervene in the process of remodeling which occurs following injury. Decellularization has become an increasingly popular technique used to obtain acellular scaffolds, and their derivatives (hydrogels, etc.), which retain tissue-specific components, including critical structural and functional proteins. These advantageous characteristics make this approach an intriguing option for creating materials capable of stimulating the sensitive repair mechanisms associated with nervous system injuries. Over the past decade, several diverse decellularization methods have been implemented specifically for nervous system applications in an attempt to carefully remove cellular content while preserving tissue morphology and composition. Each application-based decellularized ECM product requires carefully designed treatments that preserve the unique biochemical signatures associated within each tissue type to stimulate the repair of brain, spinal cord, and peripheral nerve tissues. Herein, we review the decellularization techniques that have been applied to create biomaterials with the potential to promote the repair and regeneration of tissues within the central and peripheral nervous system.

Effects of age-related shifts in cellular function and local microenvironment upon the innate immune response to implants.

The host macrophage response is now well recognized as a predictor of the success or failure of biomaterial implants following placement. More specifically, shifts from an "M1" pro-inflammatory towards a more "M2-like" anti-inflammatory macrophage polarization profile have been shown to result in enhanced material integration and/or tissue regeneration downstream. As a result, a number of biomaterials-based approaches to controlling macrophage polarization have been developed. However, the ability to promote such activity is predicated upon an in-depth, context-dependent understanding of the host response to biomaterials. Recent work has shown the impacts of both tissue location and tissue status (i.e. underlying pathology) upon the host innate immune response to implants, representing a departure from a focus upon implant material composition and form. Thus, the ideas of "biocompatibility," the host macrophage reaction, and ideal material requirements and modification strategies may need to be revisited on a patient, tissue, and disease basis. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that the host response to biomaterials in aged individuals should differ from that in younger individuals. However, despite the increasing usage of implantable medical devices in aged patients, few studies examining the effects of aging upon the host response to biomaterials and the implications of this response for long-term integration and function have been performed. The objective of the present manuscript is to review the putative effects of aging upon the host response to implanted materials and to advance the hypothesis that age-related changes in the local microenvrionement, with emphasis on the extracellular matrix, play a previously unrecognized role in determining the host response to implants.

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.

Properties of the Temporomandibular Joint in Growing Pigs.

A subset of temporomandibular joint (TMJ) disorders is attributed to joint degeneration. The pig has been considered the preferred in vivo model for the evaluation of potential therapies for TMJ disorders, and practical considerations such as cost and husbandry issues have favored the use of young, skeletally immature animals. However, the effect of growth on the biochemical and biomechanical properties of the TMJ disk and articulating cartilage has not been examined. The present study investigates the effect of age on the biochemical and biomechanical properties of healthy porcine TMJs at 3, 6, and 9 months of age. DNA, hydroxyproline, and glycosaminoglycan (GAG) content were determined and the disks and condyles were tested in uniaxial unconfined stress relaxation compression from 10% to 30% strain. TMJ disks were further assessed with a tensile test to failure technique, which included the ability to test multiple samples from the same region of an individual disk to minimize the intraspecimen variation. No differences in biochemical properties for the disk or compressive properties at 30% stress relaxation in the disk and condylar cartilage were found. In tension, no differences were observed for peak stress and tensile modulus. The collagen content of the condyle was higher at 9 months than 3 months (p < 0.05), and the GAG content was higher at 9 months than 6 months (p < 0.05). There was a trend of increased compressive instantaneous modulus with age. As such, age-matched controls for growing pigs are probably appropriate for most parameters measured.

Host response to synthetic mesh in women with mesh complications.

BACKGROUND: Despite good anatomic and functional outcomes, urogynecologic polypropylene meshes that are used to treat pelvic organ prolapse and stress urinary incontinence are associated with significant complications, most commonly mesh exposure and pain. Few studies have been performed that specifically focus on the host response to urogynecologic meshes. The macrophage has long been known to be the key cell type that mediates the foreign body response. Conceptually, macrophages that respond to a foreign body can be dichotomized broadly into M1 proinflammatory and M2 proremodeling subtypes. A prolonged M1 response is thought to result in chronic inflammation and the formation of foreign body giant cells with potential for ongoing tissue damage and destruction. Although a limited M2 predominant response is favorable for tissue integration and ingrowth, excessive M2 activity can lead to accelerated fibrillar matrix deposition and result in fibrosis and encapsulation of the mesh. OBJECTIVE: The purpose of this study was to define and compare the macrophage response in patients who undergo mesh excision surgery for the indication of pain vs a mesh exposure. STUDY DESIGN: Patients who were scheduled to undergo a surgical excision of mesh for pain or exposure at Magee-Womens Hospital were offered enrollment. Twenty-seven mesh-vagina complexes that were removed for the primary complaint of a mesh exposure (n = 15) vs pain in the absence of an exposure (n = 12) were compared with 30 full-thickness vaginal biopsy specimens from women who underwent benign gynecologic surgery without mesh. Macrophage M1 proinflammatory vs M2 proremodeling phenotypes were examined via immunofluorescent labeling for cell surface markers CD86 (M1) vs CD206 (M2) and M1 vs M2 cytokines via enzyme-linked immunosorbent assay. The amount of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) proteolytic enzymes were quantified by zymography and substrate degradation assays, as an indication of tissue matrix degradation. Statistics were performed with the use of 1-way analysis of variance with appropriate post hoc tests, t-tests, and Fisher's Exact test. RESULTS: Twenty-seven mesh-vaginal tissue complexes were excised from 27 different women with mesh complications: 15 incontinence mid urethral slings and 12 prolapse meshes. On histologic examination, macrophages surrounded each mesh fiber in both groups, with predominance of the M1 subtype. M1 and M2 cytokines/chemokines, MMP-9 (pro- and active), and MMP-2 (active) were increased significantly in mesh-vagina explants, as compared with vagina without mesh. Mesh explants that were removed for exposure had 88.4% higher pro-MMP-9 (P = .035) than those removed for pain. A positive correlation was observed between the profibrotic cytokine interleukin-10 and the percentage of M2 cells (r = 0.697; P = .037) in the pain group. CONCLUSION: In women with complications, mesh induces a proinflammatory response that persists years after implantation. The increase in MMP-9 in mesh explants that were removed for exposure indicates degradation; the positive association between interleukin-10 and M2 macrophages in mesh explants that are removed for pain is consistent with fibrosis.

Distinct macrophage phenotype and collagen organization within the intraluminal thrombus of abdominal aortic aneurysm.

OBJECTIVE: Little is known about the etiologic factors that lead to the occurrence of intraluminal thrombus (ILT) during abdominal aortic aneurysm (AAA) development. Recent work has suggested that macrophages may play an important role in progression of a number of other vascular diseases, including atherosclerosis; however, whether these cells are present within the ILT of a progressing AAA is unknown. The purpose of this work was to define the presence, phenotype, and spatial distribution of macrophages within the ILT excised from six patients. We hypothesized that the ILT contains a population of activated macrophages with a distinct, nonclassical phenotypic profile. METHODS: ILT samples were examined using histologic staining and immunofluorescent labeling for multiple markers of activated macrophages (cluster of differentiation [CD]45, CD68, human leukocyte antigen-DR, matrix metalloproteinase 9) and the additional markers α-smooth muscle actin, CD34, CD105, fetal liver kinase-1, and collagen I and III. RESULTS: Histologic staining revealed a distinct laminar organization of collagen within the shoulder region of the ILT lumen and a spatially heterogeneous cell composition within the ILT. Most of the cellular constituents of the ILT were in the luminal region and predominantly expressed markers of activated macrophages but also concurrently expressed α-smooth muscle actin, CD105, and synthesized collagen I and III. CONCLUSIONS: This report presents evidence for the presence of a distinct macrophage population within the luminal region of AAA ILT. These cells express a set of markers indicative of a unique population of activated macrophages. The exact contributions of these previously unrecognized cells to ILT formation and AAA pathobiology remains unknown.

Characterization of the host inflammatory response following implantation of prolapse mesh in rhesus macaque.

OBJECTIVE: We sought to determine the predominant cell type (macrophage, T lymphocyte, B lymphocyte, mast cell) within the area of implantation of the prototypical polypropylene mesh, Gynemesh PS (Ethicon, Somerville, NJ); and to determine the phenotypic profile (M1 proinflammatory, M2 antiinflammatory) of the macrophage response to 3 different polypropylene meshes: Gynemesh PS (Ethicon), and 2 lower-weight, higher-porosity meshes, UltraPro (Ethicon) and Restorelle (Coloplast, Humblebaek, Denmark). STUDY DESIGN: Sacrocolpopexy was performed following hysterectomy in rhesus macaques. Sham-operated animals served as controls. At 12 weeks postsurgery, the vagina-mesh complex was excised and the host inflammatory response was evaluated. Hematoxylin and eosin was used to perform routine histomorphologic evaluation. Identification of leukocyte (CD45(+)) subsets was performed by immunolabeling for CD68 (macrophage), CD3 (T lymphocyte), CD20 (B lymphocyte), and CD117 (mast cell). M1 and M2 macrophage subsets were identified using immunolabeling (CD86(+) and CD206(+), respectively), and further evaluation was performed using enzyme-linked immunosorbent assay for 2 M1 (tumor necrosis factor-alpha and interleukin [IL]-12) and 2 M2 (IL-4 and IL-10) cytokines. RESULTS: Histomorphologic evaluation showed a dense cellular response surrounding each mesh fiber. CD45(+) leukocytes accounted for 21.4 ± 5.4% of total cells within the perimesh area captured in a ×20 field, with macrophages as the predominant leukocyte subset (10.5 ± 3.9% of total cells) followed by T lymphocytes (7.3 ± 1.7%), B lymphocytes (3.0 ± 1.2%), and mast cells (0.2 ± 0.2%). The response was observed to be more diffuse with increasing distance from the fiber surface. Few leukocytes of any type were observed in sham-operated animals. Immunolabeling revealed polarization of the macrophage response toward the M1 phenotype in all mesh groups. However, the ratio of M2:M1 macrophages was increased in the fiber area in UltraPro (P = .033) and Restorelle (P = .016) compared to Gynemesh PS. In addition, a shift toward increased expression of the antiinflammatory cytokine IL-10 was observed in Restorelle as compared to Gynemesh PS (P = .011). CONCLUSION: The host response to mesh consists predominantly of activated, proinflammatory M1 macrophages at 12 weeks postsurgery. However, this response is attenuated with implantation of lighter-weight, higher-porosity mesh. While additional work is required to establish causal relationships, these results suggest a link among the host inflammatory response, mesh textile properties, and clinical outcomes in the repair of pelvic organ prolapse.

Porcine Acellular Nerve-Derived Hydrogel Improves Outcomes of Direct Muscle Neurotization in Rats.

Background: The ability to reinnervate a muscle in the absence of a viable nerve stump is a challenging clinical scenario. Direct muscle neurotization (DMN) is an approach to overcome this obstacle; however, success depends on the formation of new muscle endplates, a process, which is often limited due to lack of appropriate axonal pathfinding cues. Objective: This study explored the use of a porcine nerve extracellular matrix hydrogel as a neuroinductive interface between nerve and muscle in a rat DMN model. The goal of the study was to establish whether such hydrogel can be used to improve neuromuscular function in this model. Materials and Methods: A common peroneal nerve-to-gastrocnemius model of DMN was developed. Animals were survived for 2 or 8 weeks following DMN with or without the addition of the hydrogel at the site of neurotization. Longitudinal postural thrust, terminal electrophysiology, and muscle weight assessments were performed to qualify and quantify neuromuscular function. Histological assessments were made to qualify the host response at the DMN site, and to quantify neuromuscular junctions (NMJs) and muscle fiber diameter. Results: The hydrogel-treated group showed a 132% increase in postural thrust at 8 weeks compared with that of the DMN alone group. This was accompanied by an 80% increase in the number of NMJs at 2 weeks, and 26% increase in mean muscle fiber diameter at 8 weeks. Conclusions: These results suggest that a nerve-derived hydrogel may improve the neuromuscular outcome following DNM.

A bioengineered in situ ovary (ISO) supports follicle engraftment and live-births post-chemotherapy.

Female cancer patients who have undergone chemotherapy have an elevated risk of developing ovarian dysfunction and failure. Experimental approaches to treat iatrogenic infertility are evolving rapidly; however, challenges and risks remain that hinder clinical translation. Biomaterials have improved in vitro follicle maturation and in vivo transplantation in mice, but there has only been marginal success for early-stage human follicles. Here, we developed methods to obtain an ovarian-specific extracellular matrix hydrogel to facilitate follicle delivery and establish an in situ ovary (ISO), which offers a permissive environment to enhance follicle survival. We demonstrate sustainable follicle engraftment, natural pregnancy, and the birth of healthy pups after intraovarian microinjection of isolated exogenous follicles into chemotherapy-treated (CTx) mice. Our results confirm that hydrogel-based follicle microinjection could offer a minimally invasive delivery platform to enhance follicle integration for patients post-chemotherapy.

Peripheral Nerve Matrix Hydrogel Promotes Recovery after Nerve Transection and Repair.

BACKGROUND: Nerve transection is the most common form of peripheral nerve injury. Treatment of peripheral nerve injury has primarily focused on stabilization and mechanical cues to guide extension of the regenerating growth cone across the site of transection. The authors investigated the effects of a peripheral nerve matrix (PNM) hydrogel on recovery after nerve transection. METHODS: The authors used rodent models to determine the effect of PNM on axon extension, electrophysiologic nerve conduction, force generation, and neuromuscular junction formation after nerve transection and repair. The authors complemented this work with in vivo and in vitro fluorescence-activated cell sorting and immunohistochemistry approaches to determine the effects of PNM on critical cell populations early after repair. RESULTS: Extension of axons from the proximal stump and overall green fluorescent protein-positive axon volume within the regenerative bridge were increased in the presence of PNM compared with an empty conduit ( P < 0.005) 21 days after repair. PNM increased electrophysiologic conduction (compound muscle action potential amplitude) across the repair site ( P < 0.05) and neuromuscular junction formation ( P = 0.04) 56 days after repair. PNM produced a shift in macrophage phenotype in vitro and in vivo ( P < 0.05) and promoted regeneration in a murine model used to characterize the early immune response to PNM ( P < 0.05). CONCLUSION: PNM, delivered by subepineural injection, promoted recovery after nerve transection with immediate repair, supporting a beneficial macrophage response, axon extension, and downstream remodeling using a range of clinically relevant outcome measures. CLINICAL RELEVANCE STATEMENT: This article describes an approach for subepineural injection at the site of nerve coaptation to modulate the response to injury and improve outcomes.

Inductive, scaffold-based, regenerative medicine approach to reconstruction of the temporomandibular joint disk.

PURPOSE: A device composed of extracellular matrix (ECM) was investigated as an inductive template in vivo for reconstruction of the temporomandibular joint (TMJ) disk after discectomy. MATERIALS AND METHODS: A scaffold material composed of porcine-derived ECM was configured to mimic the shape and size of the TMJ. This device was implanted in a canine model of bilateral TMJ discectomy. After discectomy, 1 side was repaired with an ECM scaffold material and the contralateral side was left empty as a control. At 6 months after implantation, the joint space was opened, the joints were evaluated for signs of gross pathologic degenerative changes, and newly formed tissue was excised for histologic, biochemical, and biomechanical analysis. RESULTS: The results showed that implantation of an initially acellular material supported the formation of site-appropriate, functional host tissue that resembled that of the native TMJ disk. Furthermore, this prevented gross degenerative changes in the temporal fossa and mandibular condyle. No tissue formation and mild to severe gross pathologic changes were observed in the contralateral controls. CONCLUSIONS: These results suggest that an ECM-based bioscaffold could represent an off-the-shelf solution for TMJ disk replacement.

Decellularized small intestine submucosa device for temporomandibular joint meniscus repair: Acute timepoint safety study.

Temporomandibular joint (TMJ) Meniscus removal is an option for the patient to regain full range of motion if the disc is irreversibly damaged or unable to be reduced. However, this procedure leaves the joint vulnerable to condylar remodeling and degeneration. We have shown that extracellular matrix (ECM) scaffolds remodel into a tissue with near native TMJ meniscus in previous studies. The next step towards clinical translation is to manufacture the ECM scaffold as a device under good manufacturing practices (GMP) and test it in a pre-clinical animal study under good laboratory practices (GLP). The primary objective of this study was to evaluate the in-vivo histopathological response to a Prototype GMP manufactured device made of decellularized porcine small intestinal submucosa (SIS), by observing for signs of surrounding tissue reaction to the device that are indicative of an adverse host response in comparison to an empty control at 21 days post-surgical implantation in a canine TMJ meniscus removal and implant model in a GLP setting. The conclusive findings were that the ECM device is safe for placement in the TMJ. After 21 days post implantation, histology of tissue surrounding the device and draining lymph nodes showed that the Prototype GMP device had no negative effects compared to the empty site (as evaluated by the board-certified veterinary pathologist). Furthermore, there was a lack of negative findings for clinical pathology (hematology and clinical chemistry), mortality, and body weight/weight change. Future studies will go to one year after implantation to show that the remodel device remains as a viable tissue with near native mechanical properties.

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.

Development of an acellular nerve cap xenograft for neuroma prevention.

Neuroma formation following limb amputation is a prevalent and debilitating condition that can deeply affect quality of life and productivity. Several approaches exist to prevent or treat neuromas; however, no approach is either consistently reliable or surgically facile, with high rates of neuroma occurrence and/or recurrence. The present study describes the development and testing of a xenogeneic nerve cap graft made from decellularized porcine nerve. The grafts were tested in vitro for cellular removal, cytotoxicity, mechanical properties, and morphological characteristics. The grafts were then tested in rat sciatic nerve gap reconstruction and nerve amputation models for 8 weeks. Gross morphology, electrophysiology, and histopathology assessments were performed to determine the ability of the grafts to limit pathologic nerve regrowth. In vitro testing showed well decellularized and demyelinated nerve cap graft structures without any cytotoxicity from residual reagents. The grafts had a proximal socket for the proximal nerve stump and longitudinally oriented internal pores. Mechanical and surgical handling properties suggested suitability for implantation as a nerve graft. Following 8 weeks in vivo, the grafts were well integrated with the proximal and distal nerve segments without evidence of fibrotic adhesions to the surrounding tissues or bulbous outgrowth of the nerve. Electrophysiology revealed absence of nerve conduction within the remodeled nerve cap grafts and significant downstream muscle atrophy. Histologic evaluation showed well organized but limited axonal regrowth within the grafts without fibrous overgrowth or neuromatous hypercellularity. These results provide proof of concept for a novel xenograft-based approach to neuroma prevention.

Extracellular matrix as an inductive template for temporomandibular joint meniscus reconstruction: a pilot study.

PURPOSE: A device consisting of powdered porcine urinary bladder extracellular matrix (UBM-ECM) encapsulated within sheets of the same material was investigated as a scaffold for temporomandibular joint (TMJ) meniscus reconstruction. MATERIALS AND METHODS: Five dogs underwent unilateral resection of the native meniscus and replacement with a UBM-ECM device. Necropsies were performed at 3, 4, 8, 12, and 24 weeks. Two additional dogs underwent bilateral resection of the meniscus with replacement with a UBM-ECM device on 1 side, leaving the contralateral side empty as a control. Necropsies were performed at 24 weeks for bilaterally treated animals. RESULTS: Macroscopically, the UBM-ECM implants were remodeled rapidly and were indistinguishable from newly deposited host tissue at all time points. Microscopically, remodeling was characterized by a dense infiltration of predominantly CD68(+) mononuclear cells and smooth muscle actin-positive fibroblast-like cells at early time points changing with time to a sparse population of smooth muscle actin-negative spindle-shaped cells resembling those of the native fibrocartilaginous TMJ meniscus. Furthermore, the remodeling process showed deposition of predominantly type I collagen, the density and organization of which resembled those of the native meniscus by the 24-week time point. Ingrowth of calsequestrin-positive skeletal muscle tissue was also observed at the periphery of the remodeled UBM-ECM device and was similar to that found at the attachment site of the native meniscus to the surrounding soft tissues. Histologic results were identical for samples excised from both unilaterally and bilaterally treated animals. No adverse changes in the articulating surfaces of the condyle or fossa were observed in UBM-ECM-implanted joints. In the bilaterally treated animals, the unimplanted control side was characterized by degeneration and pitting of the articulating surfaces of both the condyle and the fossa, with disorganized bands of fibrous connective tissue observed within the joint space. CONCLUSION: Results of this study suggest that the UBM-ECM device provides an effective interpositional material while serving as an inductive template for reconstruction of the TMJ meniscus.

Macrophage phenotype and function are dependent upon the composition and biomechanics of the local cardiac tissue microenvironment.

Macrophage accumulation and nitrosative stress are known mechanisms underlying age-related cardiovascular pathology and functional decline. The cardiac muscle microenvironment is known to change with age, yet the direct effects of these changes have yet to be studied in-depth. The present study sought to better elucidate the role that biochemical and biomechanical alterations in cardiac tissue have in the altered phenotype and functionality of cardiac resident macrophages observed with increasing age. To accomplish this, naïve bone marrow derived macrophages from young mice were seeded onto either functionalized poly-dimethyl-siloxane hydrogels ranging in stiffness from 2kPA to 64kPA or onto tissue culture plastic, both of which were coated with either young or aged solubilized mouse cardiac extracellular matrix (cECM). Both biomechanical and biochemical alterations were found to have a significant effect on macrophage polarization and function. Increased substrate stiffness was found to promote macrophage morphologies associated with pro-inflammatory macrophage activation, increased expression of pro-inflammatory inducible nitric oxide synthase protein with increased nitric oxide secretion, and attenuated arginase activity and protein expression. Additionally, exposure to aged cECM promoted attenuated responsivity to both canonical pro-inflammatory and anti-inflammatory cytokine signaling cues when compared to young cECM treated cells. These results suggest that both biomechanical and biochemical changes in the cardiovascular system play a role in promoting the age-related shift towards pro-inflammatory macrophage populations associated with cardiovascular disease development.

An Exploratory Study into the Implantation of Arytenoid Cartilage Scaffold in the Horse.

Respiratory function in the horse can be severely compromised by arytenoid chondritis, or arytenoid chondropathy, a pathologic condition leading to deformity and dysfunction of the affected cartilage. Current treatment in cases unresponsive to medical management is removal of the cartilage, which can improve the airway obstruction, but predisposes the patient to other complications like tracheal penetration of oropharyngeal content and dynamic collapse of the now unsupported soft tissue lateral to the cartilage. A tissue engineering approach to reconstructing the arytenoid cartilage would represent a significant advantage in the management of arytenoid chondritis. In this study, we explored if decellularized matrix could potentially be incorporated into the high motion environment of the arytenoid cartilages of horses. Equine arytenoid cartilages were decellularized and a portion of the resultant acellular scaffolds was implanted in a full-thickness defect created in the arytenoids of eight horses. The implantation was performed bilaterally in each horse, with one side randomly selected to receive an implant seeded with autologous bone marrow-derived nucleated cells (BMNCs). Arytenoids structure and function were monitored up to 4 months. In vivo assessments included laryngeal ultrasound, and laryngeal endoscopy at rest and during exercise on a high-speed treadmill. Histologic evaluation of the arytenoids was performed postmortem. Implantation of the cartilaginous graft had no adverse effect on laryngeal respiratory function or swallowing, despite induction of a transient granuloma on the medial aspect of the arytenoids. Ultrasonographic monitoring detected a postoperative increase in the thickness and cross-sectional area of the arytenoid body that receded faster in the arytenoids not seeded with BMNCs. The explanted tissue showed epithelialization of the mucosal surface, integration of the implant into the native arytenoid, with minimal adverse cellular reaction. Remodeling of the scaffold material was evident by 2 months after implantation. Preseeding the scaffold with BMNCs increased the rate of scaffold degradation and incorporation. Replacement of arytenoid portion with a tissue-engineered cartilaginous graft preseeded with BMNCs is surgically feasible in the horse, is well tolerated, and results in appropriate integration within the native tissue, also preventing laryngeal tissue collapse during exercise.

Decellularizing the Porcine Optic Nerve Head: Toward a Model to Study the Mechanobiology of Glaucoma.

Purpose: Studying the extracellular matrix (ECM) remodeling of the lamina cribrosa in vivo can be extremely challenging and costly. There exist very few options for studying optic nerve head (ONH) mechanobiology in vitro that are able to reproduce the complex anatomic and biomechanical environment of the ONH. Herein, we have developed a decellularization procedure that will enable more anatomically relevant and cost-efficient future studies of ECM remodeling of the ONH. Methods: Porcine posterior poles were decellularized using a detergent and enzyme-based decellularization protocol. DNA quantification and histology were used to investigate the effectiveness of the protocol. We subsequently investigated the ability of a polyethylene glycol (PEG)-based hydrogel to restore the ONH's ability to hold pressure following decellularization. Anterior-posterior displacement of the decellularized and PEG treated ONH in a pressure bioreactor was used to evaluate the biomechanical response of the ONH. Results: DNA quantification and histology confirmed decellularization using Triton X-100 at low concentration for 48 hours successfully reduced the cellular content of the tissue by 94.9% compared with native tissue while preserving the ECM microstructure and basal lamina of the matrix. Infiltrating the decellularized tissues with PEG 6000 and PEG 10,000 hydrogel restored their ability to hold pressure, producing displacements similar to those measured for the non-decellularized control samples. Conclusions: Our decellularized ONH model is capable of producing scaffolds that are cell-free and maintain the native ECM microstructure. Translational Relevance: This model represents a platform to study the mechanobiology in the ONH and potentially for glaucoma drug testing.