RESUMEN
Debilitating chronic pain resulting from genetic predisposition, injury, or acquired neuropathy is becoming increasingly pervasive. Opioid analgesics remain the gold standard for intractable pain, but overprescription of increasingly powerful and addictive opioids has contributed to the current prescription drug abuse epidemic. There is a pressing need to screen experimental compounds more efficiently for analgesic potential that remains unmet by conventional research models. The spinal cord dorsal horn is a common target for analgesic intervention, where peripheral nociceptive signals are relayed to the central nervous system through synaptic transmission. Here, we demonstrate that coculturing peripheral and dorsal spinal cord nerve cells in a novel bioengineered microphysiological system facilitates self-directed emergence of native nerve tissue macrostructure and concerted synaptic function. The mechanistically distinct analgesics-morphine, lidocaine, and clonidine-differentially and predictably modulate this microphysiological synaptic transmission. Screening drug candidates for similar microphysiological profiles will efficiently identify therapeutics with analgesic potential.
Asunto(s)
Morfina , Nocicepción , Analgésicos/farmacología , Analgésicos Opioides/farmacología , Animales , Morfina/farmacología , Ratas , Médula Espinal , Transmisión Sináptica/fisiologíaRESUMEN
The high attrition rate of neuro-pharmaceuticals as they proceed to market necessitates the development of clinically-relevant in vitro neural microphysiological systems that can be utilized during the preclinical screening phase to assess the safety and efficacy of potential compounds. Historically, proposed models have adhered to two distinct approaches; those that are biologically relevant (e.g.-organoids, spheroids) or those that provide engineering control (e.g.-bioprinting, microfluidics). Separately, these approaches fail to fully recapitulate the complex hierarchical structure of the nervous system, limiting their clinical applications. Furthermore, the reliance on manual implementation present in many models fails to effectively scale up or satisfy the consistency standards required for widespread industry adoption. This work serves as a proof-of-concept for merging the two approaches to create a neural microphysiological system that overcomes their individual limitations. Spinal cord spheroids, fabricated using magnetic nanoparticles, are positioned in a three-dimensional hydrogel construct using magnetic bioprinting. Resulting constructs demonstrate both localized cell-cell interactions and long-distance projections that mimic in vivo structure. The use of magnetic nanoparticles for spheroid formation provides batch-to-batch consistency in size and shape and reduces the reliance on trained experimenters for accurate placing for culture. Taken together, this combination approach provides the first steps towards developing a simple approach for integrating spheroid, hydrogel culture, and bioprinting as an alternative to more specialized and expensive processes.
Asunto(s)
Bioimpresión/métodos , Médula Espinal/citología , Bioimpresión/instrumentación , Proliferación Celular , Humanos , Hidrogeles/química , Magnetismo , Nanopartículas/química , Organoides/química , Esferoides Celulares/química , Esferoides Celulares/citología , Médula Espinal/químicaRESUMEN
Photopolymerization provides a favorable method for hydrogel formation due to its simplicity, convenience, and versatility. However, the light exposure required to initiate photopolymerization is known to have a cytotoxic effect on encapsulated cells. Here, a 3D in vitro model of the nervous system microenvironment, micropatterned through the use of digital projection photolithography using a single hydrogel formulation that cross-links similarly under ultraviolet A (UVA, 315-400 nm) and visible light (400-700 nm) exposure, is presented. This setup allowed for the investigation of neuronal responses to different light wavelengths and exposure times during photoencapsulation, while ruling out effects due to the hydrogel formulation or photoinitiators used. Cellular studies-including neurite viability, DNA fragmentation, and neurite outgrowth for both UVA and visible light irradiation, the most common spectra used in biological photomicropatterning applications-were performed to assess the effect of light source on neuronal cultures. These studies indicated that while cell death occurs after exposure to either spectrum, visible light was less phototoxic than UVA, when using comparable levels of irradiation, and interestingly, glial cells were more susceptible to phototoxicity than neuronal cells. Thus, while utilizing visible light for micropatterning and cell encapsulation for nervous system applications is beneficial, it is helpful to keep the light exposure low to ensure optimal neuronal survival and growth. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 134-144, 2019.
Asunto(s)
Técnicas de Cultivo de Célula , Fragmentación del ADN/efectos de la radiación , Hidrogeles/química , Neuritas/metabolismo , Rayos Ultravioleta/efectos adversos , Animales , Ratas , Ratas Long-EvansRESUMEN
The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and ß-amyloid production play a central role in Alzheimer's disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Orientación del Axón/genética , Corteza Cerebral/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Embrión de Mamíferos , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/fisiología , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN/fisiología , Receptores Inmunológicos/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas RoundaboutRESUMEN
The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.