Tissue engineering has broad and diverse impacts on a variety of different applications from tissue regeneration to drug screening. While two-dimensional (2-D) cell culture platforms are suitable for ...tissue interfaces where planar surfaces are relevant, three dimensional (3-D) tissue models have enhanced relevance and sustainability over 2-D devices. The improvements between 2-D and 3-D functions and sustainability are related to the limitations of 2-D systems to support proper cellular morphology and signaling over time, resulting in cell overgrowth or changes in viability. For sustainable (long-term) cultures, 3-D silk protein scaffolds provide biocompatibility, porous features for transport, robust yet tunable mechanical properties, retain size and open porous structures for extended time frames due to slow proteolytic biodegradation, avoid specific cell signaling, and require no chemical cross-linking. Silk degradation can be extended for months to years without premature collapse of structures (that would result in necrosis) to support cell interactions during slow remodeling toward native tissue. Silk can also be fabricated into different material formats, such as hydrogels, tubes, sponges, composites, fibers, microspheres, and thin films, providing versatile platforms and interfaces for a variety of different applications. For sustainable tissue engineering applications, many formats have been used, including silk ionmer hydrogels that have been cultured for up to 8 weeks and porous silk scaffolds that have been cultured for up to 6 months. In this review, we highlight some of our tissue engineering work related to long-term in vitro cultures. While each tissue engineered system (adipose tissue, cortical brain tissue, intestine, kidney tissue, bone) is unique, they all use silk biomaterials as a base scaffolding material to achieve sustainable cultivation. Sustainability is important for studies that extend past a few weeks to study acute and chronic impacts of treatments, disease models, and other related applications in the field of tissue engineering.
Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive decline, memory loss, and inability to perform everyday functions. Hallmark features of AD-including generation of ...amyloid plaques, neurofibrillary tangles, gliosis, and inflammation in the brain-are well defined; however, the cause of the disease remains elusive. Growing evidence implicates pathogens in AD development, with herpes simplex virus type I (HSV-1) gaining increasing attention as a potential causative agent. Here, we describe a multidisciplinary approach to produce physiologically relevant human tissues to study AD using human-induced neural stem cells (hiNSCs) and HSV-1 infection in a 3D bioengineered brain model. We report a herpes-induced tissue model of AD that mimics human disease with multicellular amyloid plaque-like formations, gliosis, neuroinflammation, and decreased functionality, completely in the absence of any exogenous mediators of AD. This model will allow for future studies to identify potential downstream drug targets for treating this devastating disease.
Bioelectronic scaffolds that support devices while promoting tissue integration could enable tissue hybrids with augmented electronic capabilities. Here, we demonstrate a photo–cross-linkable silk ...fibroin (PSF) derivative and investigate its structural, electrical, and chemical properties. Lithographically defined PSF films offered tunable thickness and <1-μm spatial resolution and could be released from a relief layer yielding freestanding scaffolds with centimeter-scale uniformity. These constructs were electrically insulating; multielectrode arrays with PSF-passivated interconnects provided stable electrophysiological readouts from HL-1 cardiac model cells, brain slices, and hearts. Compared to SU8, a ubiquitous biomaterial, PSF exhibited superior affinity toward neurons which we attribute to its favorable surface charge and enhanced attachment of poly-D-lysine adhesion factors. This finding is of significant importance in bioelectronics, where tight junctions between devices and cell membranes are necessary for electronic communication. Collectively, our findings are generalizable to a variety of geometries, devices, and tissues, establishing PSF as a promising bioelectronic platform.
Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and ...the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery.
Designing biomimetic scaffolds with in vivo–like microenvironments using biomaterials is an essential component of successful tissue engineering approaches. The intestinal smooth muscle layers ...exhibit a complex tubular structure consisting of two concentric muscle layers in which the inner circular layer is orthogonally oriented to the outer longitudinal layer. Here, a 3D bi‐layered tubular scaffold is presented based on flexible, mechanically robust, and well aligned silk protein microfibers to mimic the native human intestinal smooth muscle structure. The scaffolds are seeded with primary human intestinal smooth muscle cells to replicate intestinal muscle tissues in vitro. Characterization of the tissue constructs reveals good biocompatibility and support for cell alignment and elongation in the different scaffold layers to enhance cell differentiation and functions. Furthermore, the engineered smooth muscle constructs support oriented neurite outgrowth, a requisite step to achieve functional innervation. These results suggest these microfiber scaffolds as functional templates for in vitro regeneration of human intestinal smooth muscle systems. The scaffolding provides a crucial step toward engineering functional human intestinal tissue in vitro, as well as engineering other types of smooth muscles in terms of their similar phenotypes. Such utility may lead to a better understanding of smooth muscle associated diseases and treatments.
A bi‐layered tubular microfiber scaffold is designed to reproduce the architecture of native human intestine smooth muscle for in vitro tissue engineering applications. The biomaterial scaffold demonstrates good cytocompatibility and enhances smooth muscle cell differentiation and neurite outgrowth in the different scaffold layers. This type of composite scaffold can be useful for engineering other types of tissues.
Brain extracellular matrix (ECM) is often overlooked in vitro brain tissue models, despite its instructive roles during development. Using developmental stage-sourced brain ECM in reproducible 3D ...bioengineered culture systems, we demonstrate enhanced functional differentiation of human induced neural stem cells (hiNSCs) into healthy neurons and astrocytes. Particularly, fetal brain tissue-derived ECM supported long-term maintenance of differentiated neurons, demonstrated by morphology, gene expression and secretome profiling. Astrocytes were evident within the second month of differentiation, and reactive astrogliosis was inhibited in brain ECM-enriched cultures when compared to unsupplemented cultures. Functional maturation of the differentiated hiNSCs within fetal ECM-enriched cultures was confirmed by calcium signaling and spectral/cluster analysis. Additionally, the study identified native biochemical cues in decellularized ECM with notable comparisons between fetal and adult brain-derived ECMs. The development of novel brain-specific biomaterials for generating mature in vitro brain models provides an important path forward for interrogation of neuron-glia interactions.
Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron ...generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.
•Human induced neural stem cell (hiNSC) lines can be passaged and cryopreserved•Rapid and robust media-independent differentiation in as few as 4 days•hiNSCs contribute to both central and peripheral nervous systems in vivo•Demonstration of utility in innervated muscle co-culture and 3D human brain model
In this article, Kaplan and colleagues describe the generation of human induced neural stem cell lines, which undergo rapid neurogenesis independently of media composition. They demonstrate utility in innervated co-culture and a 3D human brain model. This method provides an interdisciplinary tool for developing patient-specific disease models related to disorders of innervation and the brain.
Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism ...by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing.
•Non-associative learning responses were elicited in bioengineered neural tissues.•Upregulation of immediate early genes (IEG) were observed with distributed training.•Implications of studying ...fundamental features of cognition in vitro are discussed.
Though neuroscientists have historically relied upon measurement of established nervous systems, contemporary advances in bioengineering have made it possible to design and build artificial neural tissues with which to investigate normative and diseased states 1–5 however, their potential to display features of learning and memory remains unexplored. Here, we demonstrate response patterns characteristic of habituation, a form of non-associative learning, in 3D bioengineered neural tissues exposed to repetitive injections of current to elicit evoked-potentials (EPs). A return of the evoked response following rest indicated learning was transient and partially reversible. Applying patterned current as massed or distributed pulse trains induced differential expression of immediate early genes (IEG) that are known to facilitate synaptic plasticity and participate in memory formation 6,7. Our findings represent the first demonstration of a learning response in a bioengineered neural tissue in vitro.
Alzheimer's Disease (AD) is a neurodegenerative disorder that can cause life-altering and debilitating cognitive decline. AD's etiology is poorly understood, and no disease-modifying therapeutics ...exist. Here, we describe the use of 2D and 3D tissue culture models of herpesvirus-induced AD, which recapitulate hallmark disease features of plaque formation, gliosis, neuroinflammation, and impaired neuronal signaling, to screen a panel of 21 medications, supplements, and nutraceuticals with purported neuroprotective benefits. This screen identified green tea catechins and resveratrol as having strong anti-plaque properties, functional neuroprotective benefits, and minimal neurotoxicity, providing support for their further investigation as AD preventives and therapies. Two other candidates, citicoline and metformin, reduced plaque formation and were minimally toxic, but did not protect against virus-induced impairments in neuronal signaling. This study establishes a simple platform for rapidly screening and characterizing AD compounds of interest in 2D and 3D human cortical tissue models representing physiologically relevant disease features.
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•Viral infection in neural tissue cultures induces Alzheimer's disease phenotypes.•2D screen of compound library, with lead candidates screened in 3D.•Citicoline and metformin showed plaque suppression, but no functional protection.•Green tea catechins and resveratrol provided plaque and functional protection.
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