RESUMEN
Bioprinting is a promising alternative method to generate skin substitutes because it can replicate the structural organization of the skin into biomimetic layers in vitro. In this study, six primary human skin cell types were used to bioprint a trilayer skin construct consisting of epidermis, dermis, and hypodermis. Transplantation of the bioprinted skin with human cells onto full-thickness wounds of nu/nu mice promoted rapid vascularization and formation of epidermal rete ridges analogous to the native human epidermis, with a normal-looking extracellular matrix. Cell-specific staining confirmed the integration of the implanted cells into the regenerated skin. Using a similar approach, a 5 centimeter-by-5 centimeter bioprinted autologous porcine skin graft was transplanted onto full-thickness wounds in a porcine excisional wound model. The bioprinted skin graft improved epithelialization, reduced skin contraction, and supported normal collagen organization with reduced fibrosis. Differential gene expression demonstrated pro-remodeling protease activity in wounds transplanted with bioprinted autologous skin grafts. These results demonstrate that bioprinted skin can support skin regeneration to allow for nonfibrotic wound healing and suggest that the skin bioprinting technology may be applicable for human clinical use.
Asunto(s)
Piel , Cicatrización de Heridas , Ratones , Humanos , Porcinos , Animales , Epidermis , Regeneración , Repitelización , Trasplante de PielRESUMEN
Cholestatic liver injury is frequently associated with drug inhibition of bile salt transporters, such as the bile salt export pump (BSEP). Reliable in silico models to predict BSEP inhibition directly from chemical structures would significantly reduce costs during drug discovery and could help avoid injury to patients. We report our development of classification and regression models for BSEP inhibition with substantially improved performance over previously published models. We assessed the performance effects of different methods of chemical featurization, data set partitioning, and class labeling and identified the methods producing models that generalized best to novel chemical entities.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Colestasis , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP , Humanos , Aprendizaje AutomáticoRESUMEN
The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs.
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Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/normas , Guías como Asunto , Técnicas In Vitro , Animales , Inteligencia Artificial , Automatización , Desarrollo de Medicamentos/métodos , Desarrollo de Medicamentos/normas , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Ensayos Analíticos de Alto Rendimiento , Humanos , Aprendizaje Automático , Modelos Moleculares , InvestigaciónRESUMEN
Tissue chips are poised to deliver a paradigm shift in drug discovery. By emulating human physiology, these chips have the potential to increase the predictive power of preclinical modeling, which in turn will move the pharmaceutical industry closer to its aspiration of clinically relevant and ultimately animal-free drug discovery. Despite the tremendous science and innovation invested in these tissue chips, significant challenges remain to be addressed to enable their routine adoption into the industrial laboratory. This article describes the main steps that need to be taken and highlights key considerations in order to transform tissue chip technology from the hands of the innovators into those of the industrial scientists. Written by scientists from 13 pharmaceutical companies and partners at the National Institutes of Health, this article uniquely captures a consensus view on the progression strategy to facilitate and accelerate the adoption of this valuable technology. It concludes that success will be delivered by a partnership approach as well as a deep understanding of the context within which these chips will actually be used. Impact statement The rapid pace of scientific innovation in the tissue chip (TC) field requires a cohesive partnership between innovators and end users. Near term uptake of these human-relevant platforms will fill gaps in current capabilities for assessing important properties of disposition, efficacy and safety liabilities. Similarly, these platforms could support mechanistic studies which aim to resolve challenges later in development (e.g. assessing the human relevance of a liability identified in animal studies). Building confidence that novel capabilities of TCs can address real world challenges while they themselves are being developed will accelerate their application in the discovery and development of innovative medicines. This article outlines a strategic roadmap to unite innovators and end users thus making implementation smooth and rapid. With the collective contributions from multiple international pharmaceutical companies and partners at National Institutes of Health, this article should serve as an invaluable resource to the multi-disciplinary field of TC development.
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Evaluación Preclínica de Medicamentos/métodos , Procedimientos Analíticos en Microchip/métodos , Microfluídica/métodos , Industria Farmacéutica , Humanos , Dispositivos Laboratorio en un ChipRESUMEN
Cell-based direct biofabrication and 3D bioprinting is becoming a dominant technological platform and is suggested as a new paradigm for twenty-first century tissue engineering. These techniques may be our next step in surpassing the hurdles and limitations of conventional scaffold-based tissue engineering, and may offer the industrial potential of tissue engineered products especially for load bearing tissues. Here we present a topically focused review regarding the fundamental concepts, state of the art, and perspectives of this new technology and field of biofabrication and 3D bioprinting, specifically focused on tissue engineering of load bearing tissues such as bone, cartilage, osteochondral and dental tissue engineering.
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Materiales Biocompatibles/metabolismo , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Soporte de Peso , Huesos/citología , Huesos/fisiología , Cartílago/citología , Cartílago/fisiología , Células Cultivadas , Humanos , Andamios del Tejido , Diente/citología , Diente/fisiologíaRESUMEN
Hyaluronic acid (HA)-poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA-PEG hydrogels that may differentially affect cellular "outcomes" of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA-PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and annulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network analysis to identify relationships between HA-PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA-SH) and PEG vinylsulfone (PEG-VS) macromers, and used as substrates for NP and AF cell culture in vitro. Hydrogel mechanical properties ranged from 70 to 489kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA-PEG hydrogels formed from the lower-molecular-weight HA, with evidence of higher sulfated glycosaminoglycan production also upon lower-HA-molecular-weight composite gels. All cells formed more multi-cell clusters upon any HA-PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.
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Ácido Hialurónico/química , Hidrogeles/química , Disco Intervertebral/citología , Disco Intervertebral/crecimiento & desarrollo , Redes Neurales de la Computación , Polietilenglicoles/química , Animales , Materiales Biocompatibles/síntesis química , Proliferación Celular/fisiología , Células Cultivadas , Técnicas Químicas Combinatorias/métodos , Ensayo de Materiales/métodos , Reconocimiento de Normas Patrones Automatizadas/métodos , Porcinos , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Andamios del Tejido , Resultado del TratamientoRESUMEN
Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to lower back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Previous studies have shown that NP cell-laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)-laminin 111 (PEG-LM111) hydrogel was developed. The mechanical properties of PEG-LM111 hydrogel could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, LM111-presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111-presenting and PEG-only gels. When cells were encapsulated in 3-D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1kPa) PEG-LM111 hydrogels. Overall, these findings suggest that soft, LM111-functionalized hydrogels may promote or maintain the expression of specific markers characteristic of an immature NP cell phenotype.
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Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Disco Intervertebral/fisiología , Laminina/farmacología , Luz , Polietilenglicoles/farmacología , Regeneración/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunohistoquímica , Disco Intervertebral/citología , Disco Intervertebral/efectos de los fármacos , Fenómenos Mecánicos , Fenotipo , Sus scrofaRESUMEN
As articular cartilage is avascular, and mature chondrocytes do not proliferate, cartilage lesions have a limited capacity for regeneration after severe damage. The treatment of such damage has been challenging due to the limited availability of autologous healthy cartilage and lengthy and expensive cell isolation and expansion procedures. Hence, the use of bone morphogenetic protein-2 (BMP-2), a potent regulator of chondrogenic expression, has received considerable attention in cartilage and osteochondral tissue engineering. However, the exact role of BMP-2 in cartilage repair has been postulated to promote both cartilage formation and subsequent cartilage degradation through hypertrophy and endochondral ossification. Furthermore, it is likely that the manner in which BMP-2 is presented to chondrocytes will influence the physiologic pathway (repair vs. degeneration). This study investigates the relative influence of BMP-2 on cartilage matrix and potential subsequent bone matrix production using primary chondrocytes seeded on designed 3D polycaprolactone (PCL) scaffolds with chemically conjugated BMP-2. The results show that chemically conjugated BMP-2 PCL scaffolds can promote significantly greater cartilage regeneration from seeded chondrocytes both in vitro and in vivo compared with untreated scaffolds. Furthermore, our results demonstrate that the conjugated BMP-2 does not particularly accelerate endochondral ossification even in a readily permissible and highly vascular in vivo environment compared with untreated PCL scaffolds. This study not only reveals the potential use of the BMP-2 conjugation delivery method for enhanced cartilage tissue formation but also gives new insights for the effects of conjugated BMP-2 on cartilage regeneration and osteochondral ossification.
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Proteína Morfogenética Ósea 2/farmacología , Cartílago/fisiología , Condrocitos/citología , Osteogénesis/efectos de los fármacos , Poliésteres/farmacología , Regeneración/efectos de los fármacos , Andamios del Tejido/química , Animales , Cartílago/citología , Cartílago/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , ADN/metabolismo , Sus scrofa , Microtomografía por Rayos XRESUMEN
The objective of this study was to evaluate the coupled effects of three-dimensional poly(1,8-octanediol-co-citrate) (POC) scaffold pore shape and permeability on chondrogenesis using primary chondrocytes in vivo. Chondrogenesis was characterized as cartilage matrix formation by sulfated glycosaminoglycan (sGAG) quantification, relative mRNA expression of the cartilage-related proteins collagen types I, II and X, aggrecan and matrix metalloproteinases 13 and 3 and the compressive mechanical properties of the tissue/scaffold construct. A low permeability design with a spherical pore shape showed a significantly greater increase in cartilage matrix formation over 6 weeks in vivo than a high permeability design with a cubical pore shape. This increase in cartilage matrix synthesis corresponded with increases in mechanical compressive nonlinear elastic properties and histological data demonstrating darker red Safranin-O staining. There was higher mRNA expression for both cartilage-specific proteins and matrix degradation proteins in the high permeability design, resulting in overall less sGAG retained in the high permeability scaffold compared with the low permeability scaffold. Controlled POC scaffolds with a spherical pore shape and low permeability correlated with significantly increased cartilage matrix production using primary seeded chondrocytes. These results indicate that the low permeability design with a spherical pore shape provided a better microenvironment for chondrogenesis than the high permeability design with a cubical pore shape. Thus, scaffold architecture and material design may have a significant impact on the success of matrix-based clinical cartilage repair strategies.
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Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Citratos/farmacología , Polímeros/farmacología , Tejido Subcutáneo/efectos de los fármacos , Andamios del Tejido/química , Animales , Células Cultivadas , Condrocitos/metabolismo , ADN/metabolismo , Módulo de Elasticidad/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Ensayo de Materiales , Ratones , Dinámicas no Lineales , Permeabilidad/efectos de los fármacos , Porosidad/efectos de los fármacos , Implantación de Prótesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Mecánico , Sus scrofaRESUMEN
Poly(1,8-octanediol-co-citrate) (POC) is a biocompatible, biodegradable elastomer with potential application for soft tissue applications such as cartilage. For chondrogenesis, permeability is a scaffold design target that may influence cartilage regeneration. Scaffold permeability is determined by many factors such as pore shape, pore size, pore interconnectivity, porosity, and so on. Our focus in this study was to examine the effects of pore shape and permeability of two different POC scaffold designs on matrix production, mRNA gene expression, and differentiation of chondrocytes in vitro and the consequent mechanical property changes of the scaffold/tissue constructs. Since type I collagen gel was used as a cell carrier in the POC scaffolds, we also examined the effects of collagen gel concentration on chondrogenesis. We found that lower collagen I gel concentration provides a favorable microenvironment for chondrocytes promoting better chondrogenic performance of chondrocytes. With regard to scaffold design, low permeability with a spherical pore shape better enhanced the chondrogenic performance of chondrocytes in terms of matrix production, and mRNA gene expressions in vitro compared to the highly permeable scaffold with a cubical pore shape.
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Condrogénesis/fisiología , Citratos/química , Polímeros/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Fenómenos Biomecánicos , Células Cultivadas , Condrocitos , Condrogénesis/genética , Reacción en Cadena de la Polimerasa , PorcinosRESUMEN
The goal of this study was to determine material effects on cartilage regeneration for scaffolds with the same controlled architecture. The 3D polycaprolactone (PCL), poly (glycerol sebacate) (PGS), and poly (1,8 octanediol-co-citrate) (POC) scaffolds of the same design were physically characterized and tissue regeneration in terms of cell phenotype, cellular proliferation and differentiation, and matrix production were compared to find which material would be most optimal for cartilage regeneration in vitro. POC provided the best support for cartilage regeneration in terms of tissue ingrowth, matrix production, and relative mRNA expressions for chondrocyte differentiation (Col2/Col1). PGS was seen as the least favorable material for cartilage based on its relatively high de-differentiation (Col1), hypertrophic mRNA expression (Col10) and high matrix degradation (MMP13, MMP3) results. PCL still provided microenvironments suitable for cells to be active yet it seemed to cause de-differentiation (Col1) of chondrocytes inside the scaffold while many cells migrated out, growing cartilage outside the scaffold.
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Cartílago , Condrocitos/fisiología , Citratos/química , Decanoatos/química , Glicerol/análogos & derivados , Poliésteres/química , Polímeros/química , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Cartílago/citología , Cartílago/fisiología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/citología , Citratos/metabolismo , Fuerza Compresiva , Decanoatos/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Glicerol/química , Glicerol/metabolismo , Ensayo de Materiales , Poliésteres/metabolismo , Polímeros/metabolismo , Estrés Mecánico , Propiedades de Superficie , Porcinos , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
Poly(1,8-octanediol-co-citric acid) (POC) is a synthetic biodegradable elastomer that can be processed into three-dimensional (3D) scaffolds for tissue engineering. We investigated the effect of designed porosity on the mechanical properties, permeability, and degradation profiles of the POC scaffolds. For mechanical properties, scaffold compressive data were fitted to a one-dimensional (1D) nonlinear elastic model, and solid tensile data were fitted to a Neohookean incompressible nonlinear elastic model. Chondrocytes were seeded on scaffolds to assess the biocompatibility of POC. Increased porosity was associated with increased degradation rate, increased permeability, and decreased mechanical stiffness, which also became less nonlinear. Scaffold characterization in this article will provide design guidance for POC scaffolds to meet the mechanical and biological parameters needed for engineering soft tissues such as cartilage.
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Citratos/química , Polímeros/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/química , Fenómenos Biomecánicos , Cartílago , Células Cultivadas , Condrocitos/citología , Diseño Asistido por Computadora , Tejido Conectivo , Elasticidad , Imagenología Tridimensional , Ensayo de Materiales , Dinámicas no Lineales , Permeabilidad , PorosidadRESUMEN
Non-invasively monitoring the extent of cell growth, scaffold degradation and tissue development will greatly help tissue engineers to monitor in vivo regenerate tissue function and scaffold degradation. Currently available methods for tissue and scaffold degradation analysis, such as histology and direct mechanical measurements, are not suitable for continuous monitoring of the same sample in vivo as they destroy cells, tissue matrix and scaffolds. In addition, different samples are prepared and measured at varying times, but high tissue growth deviation between specimens and the need for monitoring tissue growth and scaffold degradation at different times requires large sample numbers for statistical analysis. Ultrasound elasticity imaging (UEI) based on phase-sensitive speckle tracking can characterize the internal structural, compositional and functional change of biomaterial scaffolds and engineered tissues at high resolution. In this study, UEI resolution was 250 microm (axial) by 500 microm (lateral) using a commercial ultrasound transducer centered at 5 MHz. This method allows characterization of both globally and locally altered scaffold and engineered tissue elastic properties. Preliminary in vitro and in vivo results with poly(1,8-octanediol-co-citrate) scaffolds support the feasibility of UEI as a non-invasive quantitative monitoring tool for scaffold degradation and engineered tissue formation. This novel non-invasive monitoring tool will provide direct, time-dependent feedback on scaffold degradation and tissue ingrowth for tissue engineers to improve the design process.