Injectable neural hydrogel as in vivo therapeutic delivery vehicle.

Purpose: This study demonstrated in vivo delivery of a decellularized, injectable peripheral nerve (iPN) hydrogel and explored options for using iPN in combination with regenerative biomolecular therapies like stem cell secretome. Methods: Rat-derived iPN hydrogel solutions were combined with a dextran-dye before subcutaneous injection into adult Sprague Dawley rats. After injection, an in vivo imaging system (IVIS) was used to visualize hydrogels and quantify dextran-dye release over time. Poly(lactic-co-glycolic) acid (PLGA) was used to encapsulate the dextran-dye to prolong molecular release from the hydrogel scaffolds. Lastly, we investigated use of adipose-derived stem cell (ASC) secretome as a potential future combination strategy with iPN. ASC secretome was assessed for growth factor levels in response to media stimulation and was encapsulated in PLGA to determine loading efficiency. Results: Gelation of iPN hydrogels was successful upon subcutaneous injection. When combined with iPN, a 10 kDa dextran-dye was reduced to 54% its initial signal at 24 hours, while PLGA-encapsulated dextran-dye in iPN was only reduced to 78% by 24 hours. Modified media stimulation resulted in changes in ASC phenotype and dramatic upregulation of VEGF secretion. The PLGA encapsulation protocol was adapted for use with temperature sensitive biomolecules, however, considerations must be made with loading efficiency for cell secretome as the maximum efficiency was 28%. Conclusion: The results of this study demonstrated successful injection and subsequent gelation of our iPN hydrogel formulation in vivo. Biomolecular payloads can be encapsulated in PLGA to help prolong their release from the soft iPN hydrogels in future combination therapies. Lay Summary: We developed an injectable decellularized tissue scaffold from rat peripheral nerve tissue (called iPN), a potential minimally invasive therapeutic meant to fill lesion spaces after injury. This study was the first demonstration of iPN delivery to a living animal. The iPN solution was injected subcutaneously in a rat and properly formed a gelled material upon entering the body. Our results showed that encapsulating biomolecules in an FDA-approved polymer (PLGA) slowed the release of biomolecules from the iPN, which could allow therapeutics more time around the scaffold to help repair native tissue. Lastly, we investigated one potential avenue for combining iPN with other regenerative cues obtained from adipose-derived stem cells. Description of Future Works: Future work must focus on optimal loading conditions and release profiles from the iPN hydrogels. Next steps will be applying iPN in various combination therapies for spinal cord injury. We will focus efforts on developing a pro-regenerative secretome that directly promotes neurite extension and neural cell infiltration into iPN scaffolds upon transplantation in spinal cord.

Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight.

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.

Temporal characterization of hyaluronidases after peripheral nerve injury.

Hyaluronic acid (HA) is ubiquitously found in biological tissues and mediates wound healing mechanisms after injury by promoting cell migration and proliferation. With the development of tissue-engineered neural therapeutics, including off-the-shelf grafts for peripheral nerve repair, HA is an attractive material for clinical use because of its various biological roles. HA-based biomaterials have been carefully engineered to elicit specific in vivo host responses, however an important design feature that should be considered in these scaffolds is endogenous degradation. Hyaluronidases (HYALs) are the complementary enzymes that are responsible for HA turnover. Although HYAL expression has been widely characterized in various tissues, including the central nervous system, and for different pathologies, there remains a lack of knowledge of HYAL mediated turnover in peripheral nerve tissue. In this work, gene expression of two hyaluronidases, HYAL1 and HYAL2, and HA-binding receptor, CD44, were studied in two injury models: rat sciatic nerve crush and critical gap transection. HYAL2 and CD44 were shown to be upregulated 3 days after crush injury, whereas HYAL1 was upregulated at 3 weeks, which collectively demonstrate temporal patterning of HA breakdown. Additionally, differences were observed between HYAL and HA expression at 3 weeks when compared for both nerve injury models. The activity of HYAL in peripheral nerve tissue was determined to be approximately 0.11 µmol/min, which could be used to further model HA-based biomaterial breakdown for peripheral nerve applications. Overall, this work provides a landscape of HA turnover in peripheral nerve that can be used for future neural applications.

Development of a bioactive tunable hyaluronic-protein bioconjugate hydrogel for tissue regenerative applications.

Every year, there are approximately 500 000 peripheral nerve injury (PNI) procedures due to trauma in the US alone. Autologous and acellular nerve grafts are among current clinical repair options; however, they are limited largely by the high costs associated with donor nerve tissue harvesting and implant processing, respectively. Therefore, there is a clinical need for an off-the-shelf nerve graft that can recapitulate the native microenvironment of the nerve. In our previous work, we created a hydrogel scaffold that incorporates mechanical and biological cues that mimic the peripheral nerve microenvironment using chemically modified hyaluronic acid (HA). However, with our previous work, the degradation profile and cell adhesivity was not ideal for tissue regeneration, in particular, peripheral nerve regeneration. To improve our previous hydrogel, HA was conjugated with fibrinogen using Michael-addition to assist in cell adhesion and hydrogel degradability. The addition of the fibrinogen linker was found to contribute to faster scaffold degradation via active enzymatic breakdown, compared to HA alone. Additionally, cell count and metabolic activity was significantly higher on HA conjugated fibrinogen compared previous hydrogel formulations. This manuscript discusses the various techniques deployed to characterize our new modified HA fibrinogen chemistry physically, mechanically, and biologically. This work addresses the aforementioned concerns by incorporating controllable degradability and increased cell adhesivity while maintaining incorporation of hyaluronic acid, paving the pathway for use in a variety of applications as a multi-purpose tissue engineering platform.

Current State of 44Ti/44Sc Radionuclide Generator Systems and Separation Chemistry.

In recent years, there has been an increased interest in 44Ti/44Sc generators as an onsite source of 44Sc for medical applications without needing a proximal cyclotron. The relatively short half-life (3.97 hours) and high positron branching ratio (94.3%) of 44Sc make it a viable candidate for positron emission tomography (PET) imaging. This review discusses current 44Ti/44Sc generator designs, focusing on their chemistry, drawbacks, post-elution processing, and relevant preclinical studies of the 44Sc for potential PET radiopharmaceuticals.

Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Examining thein vivofunctionality of the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI).

Objective. Although neural-enabled prostheses have been used to restore some lost functionality in clinical trials, they have faced difficulty in achieving high degree of freedom, natural use compared to healthy limbs. This study investigated thein vivofunctionality of a flexible and scalable regenerative peripheral-nerve interface suspended within a microchannel-embedded, tissue-engineered hydrogel (the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI)) as a potential approach to improving current issues in peripheral nerve interfaces.Approach. Assembled MARTEENI devices were implanted in the gaps of severed sciatic nerves in Lewis rats. Both acute and chronic electrophysiology were recorded, and channel-isolated activity was examined. In terminal experiments, evoked activity during paw compression and stimulus response curves generated from proximal nerve stimulation were examined. Electrochemical impedance spectroscopy was performed to assess the complex impedance of recording sites during chronic data collection. Features of the foreign-body response (FBR) in non-functional implants were examined using immunohistological methods.Main results. Channel-isolated activity was observed in acute, chronic, and terminal experiments and showed a typically biphasic morphology with peak-to-peak amplitudes varying between 50 and 500µV. For chronic experiments, electrophysiology was observed for 77 days post-implant. Within the templated hydrogel, regenerating axons formed minifascicles that varied in both size and axon count and were also found to surround device threads. No axons were found to penetrate the FBR. Together these results suggest the MARTEENI is a promising approach for interfacing with peripheral nerves.Significance. Findings demonstrate a high likelihood that observed electrophysiological activity recorded from implanted MARTEENIs originated from neural tissue. The variation in minifascicle size seen histologically suggests that amplitude distributions observed in functional MARTEENIs may be due to a combination of individual axon and mini-compound action potentials. This study provided an assessment of a functional MARTEENI in anin vivoanimal model for the first time.

Microphysiological system for studying contractile differences in young, active, and old, sedentary adult derived skeletal muscle cells.

Microphysiological systems (MPS), also referred to as tissue chips, incorporating 3D skeletal myobundles are a novel approach for physiological and pharmacological studies to uncover new medical treatments for sarcopenia. We characterize a MPS in which engineered skeletal muscle myobundles derived from donor-specific satellite cells that model aged phenotypes are encapsulated in a perfused tissue chip platform containing platinum electrodes. Our myobundles were derived from CD56+ myogenic cells obtained via percutaneous biopsy of the vastus lateralis from adults phenotyped by age and physical activity. Following 17 days differentiation including 5 days of a 3 V, 2 Hz electrical stimulation regime, the myobundles exhibited fused myotube alignment and upregulation of myogenic, myofiber assembly, signaling and contractile genes as demonstrated by gene array profiling and localization of key components of the sarcomere. Our results demonstrate that myobundles derived from the young, active (YA) group showed high intensity immunofluorescent staining of α-actinin proteins and responded to electrical stimuli with a ~1 µm displacement magnitude compared with non-stimulated myobundles. Myobundles derived from older sedentary group (OS) did not display a synchronous contraction response. Hypertrophic potential is increased in YA-derived myobundles in response to stimulation as shown by upregulation of insulin growth factor (IGF-1), α-actinin (ACTN3, ACTA1) and fast twitch troponin protein (TNNI2) compared with OS-derived myobundles. Our MPS mimics disease states of muscle decline and thus provides an aged system and experimental platform to investigate electrical stimulation mimicking exercise regimes and may be adapted to long duration studies of compound efficacy and toxicity for therapeutic evaluation against sarcopenia.

Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth-permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug-loaded microparticles for a combinatorial approach to neural injury therapies.

Towards the translation of electroconductive organic materials for regeneration of neural tissues.

Carbon-based conductive and electroactive materials (e.g., derivatives of graphene, fullerenes, polypyrrole, polythiophene, polyaniline) have been studied since the 1970s for use in a broad range of applications. These materials have electrical properties comparable to those of commonly used metals, while providing other benefits such as flexibility in processing and modification with biologics (e.g., cells, biomolecules), to yield electroactive materials with biomimetic mechanical and chemical properties. In this review, we focus on the uses of these electroconductive materials in the context of the central and peripheral nervous system, specifically recent studies in the peripheral nerve, spinal cord, brain, eye, and ear. We also highlight in vivo studies and clinical trials, as well as a snapshot of emerging classes of electroconductive materials (e.g., biodegradable materials). We believe such specialized electrically conductive biomaterials will clinically impact the field of tissue regeneration in the foreseeable future. STATEMENT OF SIGNIFICANCE: This review addresses the use of conductive and electroactive materials for neural tissue regeneration, which is of significant interest to a broad readership, and of particular relevance to the growing community of scientists, engineers and clinicians in academia and industry who develop novel medical devices for tissue engineering and regenerative medicine. The review covers the materials that may be employed (primarily focusing on derivatives of fullerenes, graphene and conjugated polymers) and techniques used to analyze materials composed thereof, followed by sections on the application of these materials to nervous tissues (i.e., peripheral nerve, spinal cord, brain, optical, and auditory tissues) throughout the body.

Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields.

Exogenous electrical fields have been explored in regenerative medicine to increase cellular expression of pro-regenerative growth factors. Adipose-derived stem cells (ASCs) are attractive for regenerative applications, specifically for neural repair. Little is known about the relationship between low-level electrical stimulation (ES) and ASC regenerative potentiation. In this work, patterns of ASC expression and secretion of growth factors (i.e., secretome) were explored across a range of ES parameters. ASCs were stimulated with low-level stimulation (20 mV/mm) at varied pulse frequencies, durations, and with alternating versus direct current. Frequency and duration had the most significant effects on growth factor expression. While a range of stimulation frequencies (1, 20, 1000 Hz) applied intermittently (1 h × 3 days) induced upregulation of general wound healing factors, neural-specific factors were only increased at 1 Hz. Moreover, the most optimal expression of neural growth factors was achieved when ASCs were exposed to 1 Hz pulses continuously for 24 h. In evaluation of secretome, apparent inconsistencies were observed across biological replications. Nonetheless, ASC secretome (from 1 Hz, 24 h ES) caused significant increase in neurite extension compared to non-stimulated control. Overall, ASCs are sensitive to ES parameters at low field strengths, notably pulse frequency and stimulation duration.

Development of a magnetically aligned regenerative tissue-engineered electronic nerve interface for peripheral nerve applications.

Peripheral nerve injuries can be debilitating to motor and sensory function, with severe cases often resulting in complete limb amputation. Over the past two decades, prosthetic limb technology has rapidly advanced to provide users with crude motor control of up to 20° of freedom; however, the nerve-interfacing technology required to provide high movement selectivity has not progressed at the same rate. The work presented here focuses on the development of a magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI) that combines polyimide "threads" encapsulated within a magnetically aligned hydrogel scaffold. The technology exploits tissue-engineered strategies to address concerns over traditional peripheral nerve interfaces including poor axonal sampling through the nerve and rigid substrates. A magnetically templated hydrogel is used to physically support the polyimide threads while also promoting regeneration in close proximity to the electrode sites on the polyimide. This work demonstrates the utility of magnetic templating for use in tuning the mechanical properties of hydrogel scaffolds to match the stiffness of native nerve tissue while providing an aligned substrate for Schwann cell migration in vitro. MARTEENI devices were fabricated and implanted within a 5-mm-long rat sciatic-nerve transection model to assess regeneration at 6 and 12 weeks. MARTEENI devices do not disrupt tissue remodeling and show axon densities equivalent to fresh tissue controls around the polyimide substrates. Devices are observed to have attenuated foreign-body responses around the polyimide threads. It is expected that future studies with functional MARTEENI devices will be able to record and stimulate single axons with high selectivity and low stimulation regimes.

Chondroitinase ABC/galectin-3 fusion proteins with hyaluronan-based hydrogels stabilize enzyme and provide targeted enzyme activity for neural applications.

Objective. Chondroitinase ABC (ChABC) has emerged as a promising therapeutic agent for central nervous system regeneration. Despite multiple beneficial outcomes for regeneration, translation of this enzyme is challenged by poor pharmacokinetics, localization, and stability.Approach. This study explored the function andin vitroapplication of engineered ChABC fused to galectin-3 (Gal3). Two previously developed ChABC-Gal3 oligomers (monomeric and trimeric) were evaluated for functionality and kinetics, then applied to anin vitrocellular outgrowth model using dorsal root ganglia (DRGs). The fusions were combined with two formulations of hyaluronan (HA)-based scaffolds to determine the extent of active enzyme release compared to wild type (WT) ChABC.Main Results. Monomeric and trimeric ChABC-Gal3 maintained digestive capabilities with kinetic properties that were substrate-dependent for chondroitin sulfates A, B, and C. The fusions had longer half-lives at 37 °C on the order of seven fold for monomer and twelve fold for trimer compared to WT. Both fusions were also effective at restoring DRG outgrowthin vitro. To create a combination approach, two triple-component hydrogels containing modified HA were formulated to match the mechanical properties of native spinal cord tissue and to support astrocyte viability (>80%) and adhesion. The hydrogels included collagen-I and laminin mixed with either 5 mg ml-1of glycidyl methacrylate HA or 3 mg ml-1Hystem. When combined with scaffolds, ChABC-Gal3 release time was lengthened compared to WT. Both fusions had measurable enzymatic activity for at least 10 d when incorporated in gels, compared to WT that lost activity after 1 d. These longer term release products from gels maintained adequate function to promote DRG outgrowth.Significance. Results of this study demonstrated cohesive benefits of two stabilized ChABC-Gal3 oligomers in combination with HA-based scaffolds for neural applications. Significant improvements to ChABC stability and release were achieved, meriting future studies of ChABC-Gal3/hydrogel combinations to target neural regeneration.

Development of novel apoptosis-assisted lung tissue decellularization methods.

Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications. The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with patient-derived cells prior to transplantation, or digested to create thermally-gelling ECM hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergent-based decellularization protocol. Furthermore, thermally-gelling lung ECM hydrogels supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling purposes.

Microtopographical patterns promote different responses in fibroblasts and Schwann cells: A possible feature for neural implants.

The chronic reliability of bioelectronic neural interfaces has been challenged by foreign body reactions (FBRs) resulting in fibrotic encapsulation and poor integration with neural tissue. Engineered microtopographies could alleviate these challenges by manipulating cellular responses to the implanted device. Parallel microchannels have been shown to modulate neuronal cell alignment and axonal growth, and Sharklet™ microtopographies of targeted feature sizes can modulate bio-adhesion of an array of bacteria, marine organisms, and epithelial cells due to their unique geometry. We hypothesized that a Sharklet™ micropattern could be identified that inhibited fibroblasts partially responsible for FBR while promoting Schwann cell proliferation and alignment. in vitro cell assays were used to screen the effect of Sharklet™ and channel micropatterns of varying dimensions from 2 to 20 µm on fibroblast and Schwann cell metrics (e.g., morphology/alignment, nuclei count, metabolic activity), and a hierarchical analysis of variance was used to compare treatments. In general, Schwann cells were found to be more metabolically active and aligned than fibroblasts when compared between the same pattern. 20 µm wide channels spaced 2 µm apart were found to promote Schwann cell attachment and alignment while simultaneously inhibiting fibroblasts and warrant further in vivo study on neural interface devices. No statistically significant trends between cellular responses and geometrical parameters were identified because mammalian cells can change their morphology dependent on their environment in a manner dissimilar to bacteria. Our results showed although surface patterning is a strong physical tool for modulating cell behavior, responses to micropatterns are highly dependent on the cell type.

Three-Dimensional Bioprinted Hyaluronic Acid Hydrogel Test Beds for Assessing Neural Cell Responses to Competitive Growth Stimuli.

Hyaluronic acid (HA) is an abundant extracellular matrix (ECM) component in soft tissues throughout the body and has found wide adoption in tissue engineering. This study focuses on the optimization of methacrylated HA (MeHA) for three-dimensional (3D) bioprinting to create in vitro test beds that incorporate regeneration-promoting growth factors in neural repair processes. To evaluate MeHA as a potential bioink, rheological studies were performed with PC-12 cells to demonstrate shear thinning properties maintained when printing with and without cells. Next, an extrusion-based Cellink BIO X 3D printer was used to bioprint various MeHA solutions combined with collagen-I to determine which formulation was the most optimal for creating 3D features. Results indicated that MeHA (10 mg/mL) with collagen-I (3 mg/mL) was most suitable. As Schwann cells (SCs) are a critical component of neural repair and regeneration, SC adhesion assessment via integrin ß1 immunostaining indicated that the bioink candidate adequately supported SC adhesion and migration when compared to Col-I, a highly cell-adhesive ECM component. MeHA/collagen-I bioink was adapted for neural specific applications by printing with the neural growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). These test beds were conducive for SC infiltration and presented differential migration responses. Finally, a two-chamber in vitro test bed design was created to study competitive biochemical cues. Dorsal root ganglia were seeded in test beds and demonstrated directional neurite extension (measured by ß-III tubulin and GAP43 immunostaining) in response to NGF and GDNF. Overall, the selected MeHA/collagen-I bioink was bioprintable, improved cell viability compared to molded controls, and was conducive for cell adhesion, growth factor sequestration, and neural cell infiltration. MeHA is a suitable bioink candidate for extrusion-based bioprinting and will be useful in future development of spatially complex test beds to advance in vitro models as an alternative to common in vivo tests for neural repair applications.

Extracellular Matrix Disparities in an Nkx2-5 Mutant Mouse Model of Congenital Heart Disease.

Congenital heart disease (CHD) affects almost one percent of all live births. Despite diagnostic and surgical reparative advances, the causes and mechanisms of CHD are still primarily unknown. The extracellular matrix plays a large role in cell communication, function, and differentiation, and therefore likely plays a role in disease development and pathophysiology. Cell adhesion and gap junction proteins, such as integrins and connexins, are also essential to cellular communication and behavior, and could interact directly (integrins) or indirectly (connexins) with the extracellular matrix. In this work, we explore disparities in the expression and spatial patterning of extracellular matrix, adhesion, and gap junction proteins between wild type and Nkx2-5 +/R52G mutant mice. Decellularization and proteomic analysis, Western blotting, histology, immunostaining, and mechanical assessment of embryonic and neonatal wild type and Nkx2-5 mutant mouse hearts were performed. An increased abundance of collagen IV, fibronectin, and integrin ß-1 was found in Nkx2-5 mutant neonatal mouse hearts, as well as increased expression of connexin 43 in embryonic mutant hearts. Furthermore, a ventricular noncompaction phenotype was observed in both embryonic and neonatal mutant hearts, as well as spatial disorganization of ECM proteins collagen IV and laminin in mutant hearts. Characterizing such properties in a mutant mouse model provides valuable information that can be applied to better understanding the mechanisms of congenital heart disease.

Bench-to-Bedside Lessons Learned: Commercialization of an Acellular Nerve Graft.

Peripheral nerve injury can result in debilitating outcomes including loss of function and neuropathic pain. Although nerve repair research and therapeutic development are widely studied, translation of these ideas into clinical interventions has not occurred at the same rate. At the turn of this century, approaches to peripheral nerve repair have included microsurgical techniques, hollow conduits, and autologous nerve grafts. These methods provide satisfactory results; however, they possess numerous limitations that can prevent effective surgical treatment. Commercialization of Avance, a processed nerve allograft, sought to address limitations of earlier approaches by providing an off-the-shelf alternative to hollow conduits while maintaining many proregenerative properties of autologous grafts. Since its launch in 2007, Avance has changed the landscape of the nerve repair market and is used to treat tens of thousands of patients. Although Avance has become an important addition to surgeon and patient clinical options, the product's journey from bench to bedside took over 20 years with many research and commercialization challenges. This article reviews the events that have brought a processed nerve allograft from the laboratory bench to the patient bedside. Additionally, this review provides a perspective on lessons and considerations that can assist in translation of future medical products.

Decellularized tissues as platforms for in vitro modeling of healthy and diseased tissues.

Biomedical engineers are at the forefront of developing novel treatments to improve human health, however, many products fail to translate to clinical implementation. In vivo pre-clinical animal models, although the current best approximation of complex disease conditions, are limited by reproducibility, ethical concerns, and poor accurate prediction of human response. Hence, there is a need to develop physiologically relevant, low cost, scalable, and reproducible in vitro platforms to provide reliable means for testing drugs, biomaterials, and tissue engineered products for successful clinical translation. One emerging approach of developing physiologically relevant in vitro models utilizes decellularized tissues/organs as biomaterial platforms for 2D and 3D models of healthy and diseased tissue. Decellularization is a process that removes cellular content and produces tissue-specific extracellular matrix scaffolds that can more accurately recapitulate an organ/tissue's native microenvironment compared to other natural or synthetic materials. Decellularized tissues hold enormous potential for in vitro modeling of various disease phenotypes and tissue responses to drugs or external conditions such as aging, toxin exposure, or even implantation. In this review, we highlight the need for in vitro models, the advantages and limitations of implementing decellularized tissues, and considerations of the decellularization process. We discuss current research efforts towards applying decellularized tissues as platforms to generate in vitro models of healthy and diseased tissues, and where we foresee the field progressing. A variety of organs/tissues are discussed, including brain, heart, kidney, large intestine, liver, lung, skeletal muscle, skin, and tongue. STATEMENT OF SIGNIFICANCE: Many biomedical products fail to reach clinical translation due to animal model limitations. Development of physiologically relevant in vitro models can provide a more economic, scalable, and reproducible means of testing drugs/therapeutics for successful clinical translation. The use of decellularized tissues as platforms for in vitro models holds promise, as these scaffolds can effectively replicate native tissue complexity, but is not widely explored. This review discusses the need for in vitro models, the promise of decellularized tissues as biomaterial substrates, and the current research applying decellularized tissues towards the creation of in vitro models. Further, this review provides insights into the current limitations and future of such in vitro models.

Integration of flexible polyimide arrays into soft extracellular matrix-based hydrogel materials for a tissue-engineered electronic nerve interface (TEENI).

BACKGROUND: Biomimetic hydrogels used in tissue engineering can improve tissue regeneration and enable targeted cellular behavior; there is growing interest in combining hydrogels with microelectronics to create new neural interface platforms to help patient populations. However, effective processes must be developed to integrate flexible but relatively stiff (e.g., 1-10 GPa) microelectronic arrays within soft (e.g., 1-10 kPa) hydrogels. NEW METHOD: Here, a novel method for integrating polyimide microelectrode arrays within a biomimetic hydrogel scaffold is demonstrated for use as a tissue-engineered electronic nerve interface (TEENI). Tygon tubing and a series of 3D printed molds were used to facilitate hydrogel fabrication and device assembly. COMPARISON WITH EXISTING METHODS: Other comparable regenerative peripheral nerve interface technologies do not utilize the flexible microelectrode array design nor the hydrogel scaffold described here. These methods typically use stiff electrode arrays that are affixed to a similarly stiff implantable tube serving as the nerve guidance conduit. RESULTS: Our results indicate that there is a substantial mechanical mismatch between the flexible microelectronic arrays and the soft hydrogel. However, using the methods described here, there is consistent fabrication of these regenerative peripheral nerve interfaces suitable for implantation. CONCLUSIONS: The assembly process that was developed resulted in repeatable and consistent integration of microelectrode arrays within a soft tissue-engineered hydrogel. As reported elsewhere, these devices have been successfully implanted in a rat sciatic nerve model and yielded neural recordings. This process can be adapted for other applications and hydrogels in which flexible electronic materials are combined with soft regenerative scaffolds.