Transient nuclear deformation primes epigenetic state and promotes cell reprogramming.

Cell reprogramming has wide applications in tissue regeneration, disease modelling and personalized medicine. In addition to biochemical cues, mechanical forces also contribute to the modulation of the epigenetic state and a variety of cell functions through distinct mechanisms that are not fully understood. Here we show that millisecond deformation of the cell nucleus caused by confinement into microfluidic channels results in wrinkling and transient disassembly of the nuclear lamina, local detachment of lamina-associated domains in chromatin and a decrease of histone methylation (histone H3 lysine 9 trimethylation) and DNA methylation. These global changes in chromatin at the early stage of cell reprogramming boost the conversion of fibroblasts into neurons and can be partially reproduced by inhibition of histone H3 lysine 9 and DNA methylation. This mechanopriming approach also triggers macrophage reprogramming into neurons and fibroblast conversion into induced pluripotent stem cells, being thus a promising mechanically based epigenetic state modulation method for cell engineering.

Co-Electrospun Silk Fibroin and Gelatin Methacryloyl Sheet Seeded with Mesenchymal Stem Cells for Tendon Regeneration.

Silk fibroin (SF) is a promising biomaterial for tendon repair, but its relatively rigid mechanical properties and low cell affinity have limited its application in regenerative medicine. Meanwhile, gelatin-based polymers have advantages in cell attachment and tissue remodeling but have insufficient mechanical strength to regenerate tough tissue such as tendons. Taking these aspects into account, in this study, gelatin methacryloyl (GelMA) is combined with SF to create a mechanically strong and bioactive nanofibrous scaffold (SG). The mechanical properties of SG nanofibers can be flexibly modulated by varying the ratio of SF and GelMA. Compared to SF nanofibers, mesenchymal stem cells (MSCs) seeded on SG fibers with optimal composition (SG7) exhibit enhanced growth, proliferation, vascular endothelial growth factor production, and tenogenic gene expression behavior. Conditioned media from MSCs cultured on SG7 scaffolds can greatly promote the migration and proliferation of tenocytes. Histological analysis and tenogenesis-related immunofluorescence staining indicate SG7 scaffolds demonstrate enhanced in vivo tendon tissue regeneration compared to other groups. Therefore, rational combinations of SF and GelMA hybrid nanofibers may help to improve therapeutic outcomes and address the challenges of tissue-engineered scaffolds for tendon regeneration.

Bioengineered Multicellular Liver Microtissues for Modeling Advanced Hepatic Fibrosis Driven Through Non-Alcoholic Fatty Liver Disease.

Despite considerable efforts in modeling liver disease in vitro, it remains difficult to recapitulate the pathogenesis of the advanced phases of non-alcoholic fatty liver disease (NAFLD) with inflammation and fibrosis. Here, a liver-on-a-chip platform with bioengineered multicellular liver microtissues is developed, composed of four major types of liver cells (hepatocytes, endothelial cells, Kupffer cells, and stellate cells) to implement a human hepatic fibrosis model driven by NAFLD: i) lipid accumulation in hepatocytes (steatosis), ii) neovascularization by endothelial cells, iii) inflammation by activated Kupffer cells (steatohepatitis), and iv) extracellular matrix deposition by activated stellate cells (fibrosis). In this model, the presence of stellate cells in the liver-on-a-chip model with fat supplementation showed elevated inflammatory responses and fibrosis marker up-regulation. Compared to transforming growth factor-beta-induced hepatic fibrosis models, this model includes the native pathological and chronological steps of NAFLD which shows i) higher fibrotic phenotypes, ii) increased expression of fibrosis markers, and iii) efficient drug transport and metabolism. Taken together, the proposed platform will enable a better understanding of the mechanisms underlying fibrosis progression in NAFLD as well as the identification of new drugs for the different stages of NAFLD.

Cell type-specific extracellular matrix guided the differentiation of human mesenchymal stem cells in 3D polymeric scaffolds.

The tissue microenvironment has profound effects on tissue-specific regeneration. The 3-dimensional extracellular matrix (ECM) niche influences the linage-specific differentiation of stem cells in tissue. To understand how ECM guides tissue-specific regeneration, we established a series of 3D composite scaffolds containing ECMs derived from different primary cells isolated from a single animal species and assessed their impact on the differentiation of human mesenchymal stem cells (hMSCs). Synthetic microfiber scaffolds (fiber mats) were fabricated by electrospinning tyrosine-derived polycarbonates (pDTEC). The bovine primary fibroblasts, chondrocytes and osteoblasts cultured on the fiber mats produced and assembled their ECMs, infiltrating the pores of the fibrous scaffold. The composite scaffolds were decellularized to remove cellular components, preserve ECM and minimally affect polymer integrity. Characterization of the ECMs derived from different primary cells in the composite scaffolds showed overlapping but distinct compositions. The chondrogenic and osteogenic differentiation of hMSCs on the different composite scaffolds were compared. Our results showed that ECM derived from chondrocytes cultured in synthetic fiber mats promoted the chondrogenic differentiation of hMSC in the presence or absence of soluble inducing factors. ECM derived from co-culture of osteoblasts and chondrocytes promoted osteogenic differentiation in hMSCs better than ECM derived from chondrocytes. This study demonstrated that decellularized ECMs derived from different cell types formed within synthetic fiber scaffolds guide the tissue-specific differentiation of hMSCs. These composite scaffolds may be developed into models to study the mechanisms of ECM-induced tissue regeneration.

Viscoelasticity in 3D Cell Culture and Regenerative Medicine: Measurement Techniques and Biological Relevance.

The field of mechanobiology is gaining prominence due to recent findings that show cells sense and respond to the mechanical properties of their environment through a process called mechanotransduction. The mechanical properties of cells, cell organelles, and the extracellular matrix are understood to be viscoelastic. Various technologies have been researched and developed for measuring the viscoelasticity of biological materials, which may provide insight into both the cellular mechanisms and the biological functions of mechanotransduction. Here, we explain the concept of viscoelasticity and introduce the major techniques that have been used to measure the viscoelasticity of various soft materials in different length- and timescale frames. The topology of the material undergoing testing, the geometry of the probe, the magnitude of the exerted stress, and the resulting deformation should be carefully considered to choose a proper technique for each application. Lastly, we discuss several applications of viscoelasticity in 3D cell culture and tissue models for regenerative medicine, including organoids, organ-on-a-chip systems, engineered tissue constructs, and tunable viscoelastic hydrogels for 3D bioprinting and cell-based therapies.

Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity.

Living tissue and extracellular matrices possess viscoelastic properties, but understanding how viscoelastic matrix regulates chromatin and the epigenome is limited. Here, we find that the regulation of the epigenetic state by the viscoelastic matrix is more pronounced on softer matrices. Cells on viscoelastic matrices exhibit larger nuclei, increased nuclear lamina ruffling, loosely organized chromatin, and faster chromatin dynamics, compared to those on elastic matrices. These changes are accompanied by a global increase in euchromatic marks and a local increase in chromatin accessibility at the cis -regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic matrices enhanced the efficiency of reprogramming fibroblasts into neurons and induced pluripotent stem cells, respectively. Together, our findings demonstrate the key roles of matrix viscoelasticity in the regulation of epigenetic state, and uncover a new mechanism of biophysical regulation of chromatin and cell reprogramming, with implications for the design of smart materials to engineer cell fate.

Biphasic regulation of epigenetic state by matrix stiffness during cell reprogramming.

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Engineering programmable material-to-cell pathways via synthetic notch receptors to spatially control differentiation in multicellular constructs.

Synthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs.

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.

Reduction of Intracellular Tension and Cell Adhesion Promotes Open Chromatin Structure and Enhances Cell Reprogramming.

The role of transcription factors and biomolecules in cell type conversion has been widely studied. Yet, it remains unclear whether and how intracellular mechanotransduction through focal adhesions (FAs) and the cytoskeleton regulates the epigenetic state and cell reprogramming. Here, it is shown that cytoskeletal structures and the mechanical properties of cells are modulated during the early phase of induced neuronal (iN) reprogramming, with an increase in actin cytoskeleton assembly induced by Ascl1 transgene. The reduction of actin cytoskeletal tension or cell adhesion at the early phase of reprogramming suppresses the expression of mesenchymal genes, promotes a more open chromatin structure, and significantly enhances the efficiency of iN conversion. Specifically, reduction of intracellular tension or cell adhesion not only modulates global epigenetic marks, but also decreases DNA methylation and heterochromatin marks and increases euchromatin marks at the promoter of neuronal genes, thus enhancing the accessibility for gene activation. Finally, micro- and nano-topographic surfaces that reduce cell adhesions enhance iN reprogramming. These novel findings suggest that the actin cytoskeleton and FAs play an important role in epigenetic regulation for cell fate determination, which may lead to novel engineering approaches for cell reprogramming.

A Replication of DiMaggio et al. (2020) in Phoenix, AZ.

This research replicates in Phoenix, Arizona a study originally conducted by DiMaggio et al. (2020) that investigated the associations between positive COVID-19 tests and demographic, socioeconomic, and racial characteristics in New York City at the ZIP Code Tabulation Area level. We extend that work through a conceptual replication that introduces covariates appropriate to Phoenix, AZ. Our direct replication, which focuses on that city's first wave of COVID-19 (May 31, 2020 to August 1, 2020), demonstrates that the framework used by DiMaggio et al. can be transferred across cities, but also identifies specification decisions that need careful consideration. Our conceptual replication identifies the proportion of Hispanic residents, rather than that of Black/African American residents, to be a key predictor of positive COVID-19 testing. This finding sheds light on the dynamics of race during the pandemic.

Application of lung microphysiological systems to COVID-19 modeling and drug discovery: a review.

There is a pressing need for effective therapeutics for coronavirus disease 2019 (COVID-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The process of drug development is a costly and meticulously paced process, where progress is often hindered by the failure of initially promising leads. To aid this challenge, in vitro human microphysiological systems need to be refined and adapted for mechanistic studies and drug screening, thereby saving valuable time and resources during a pandemic crisis. The SARS-CoV-2 virus attacks the lung, an organ where the unique three-dimensional (3D) structure of its functional units is critical for proper respiratory function. The in vitro lung models essentially recapitulate the distinct tissue structure and the dynamic mechanical and biological interactions between different cell types. Current model systems include Transwell, organoid and organ-on-a-chip or microphysiological systems (MPSs). We review models that have direct relevance toward modeling the pathology of COVID-19, including the processes of inflammation, edema, coagulation, as well as lung immune function. We also consider the practical issues that may influence the design and fabrication of MPS. The role of lung MPS is addressed in the context of multi-organ models, and it is discussed how high-throughput screening and artificial intelligence can be integrated with lung MPS to accelerate drug development for COVID-19 and other infectious diseases.

Biomaterial-based immunoengineering to fight COVID-19 and infectious diseases.

Infection by SARS-CoV-2 virus often induces the dysregulation of immune responses, tissue damage, and blood clotting. Engineered biomaterials from the nano- to the macroscale can provide targeted drug delivery, controlled drug release, local immunomodulation, enhanced immunity, and other desirable functions to coordinate appropriate immune responses and to repair tissues. Based on the understanding of COVID-19 disease progression and immune responses to SARS-CoV-2, we discuss possible immunotherapeutic strategies and highlight biomaterial approaches from the perspectives of preventive immunization, therapeutic immunomodulation, and tissue healing and regeneration. Successful development of biomaterial platforms for immunization and immunomodulation will not only benefit COVID-19 patients, but also have broad applications for a variety of infectious diseases.

Key components of engineering vascularized 3-dimensional bioprinted bone constructs.

Vascularization has a pivotal role in engineering successful tissue constructs. However, it remains a major hurdle of bone tissue engineering, especially in clinical applications for the treatment of large bone defects. Development of vascularized and clinically-relevant engineered bone substitutes with sufficient blood supply capable of maintaining implant viability and supporting subsequent host tissue integration remains a major challenge. Since only cells that are 100-200 µm from blood vessels can receive oxygen through diffusion, engineered constructs that are thicker than 400 µm face a challenging oxygenation problem. Following implantation in vivo, spontaneous ingrowth of capillaries in thick engineered constructs is too slow. Thus, it is critical to provide optimal conditions to support vascularization in engineered bone constructs. To achieve this, an in-depth understanding of the mechanisms of angiogenesis and bone development is required. In addition, it is also important to mimic the physiological milieu of native bone to fabricate more successful vascularized bone constructs. Numerous applications of engineered vascularization with cell-and/or microfabrication-based approaches seek to meet these aims. Three-dimensional (3D) printing promises to create patient-specific bone constructs in the future. In this review, we discuss the major components of fabricating vascularized 3D bioprinted bone constructs, analyze their related challenges, and highlight promising future trends.

Rhodamine Conjugated Gelatin Methacryloyl Nanoparticles for Stable Cell Imaging.

Fluorescent nanomaterials have been widely used in biological imaging due to their selectivity, sensitivity, and noninvasive nature. These characteristics make the materials suitable for real-time and in situ imaging. However, further development of highly biocompatible nanosystems with long-lasting fluorescent intensity and photostability is needed for advanced bioimaging. We have used electrospraying to generate gelatin methacryloyl (GelMA)-based fluorescent nanoparticles (NPs) with chemically conjugated rhodamine B (RB). The extent of conjugation can be controlled by varying the mass ratio of RB and GelMA precursors to obtain RB-conjugated GelMA (RB-GelMA) NPs with optimal fluorescent properties and particle size. These NPs exhibited superior biocompatibility when compared with pure RB in in vitro cell viability and proliferation assays using multiple cell types. Moreover, RB-GelMA NPs showed enhanced cell internalization and improved brightness compared with unconjugated RB. Our experiments demonstrate that engineered RB-GelMA NPs can be used as a biocompatible fluorescent label for bioimaging.

Chasing the Paradigm: Clinical Translation of 25 Years of Tissue Engineering.

IMPACT STATEMENT: In this Perspective, we discuss the impact of the past 25 years of tissue engineering on the development of clinical therapies. Based on their success and other significant research accomplishments, platforms of innovation were identified. Their discoveries will enable tissue engineering inspired therapies to meet the requirements necessary for large-scale manufacturing and Food and Drug Administration (FDA) approval for a diverse range of indications.

Endogenous viable cells in lyopreserved amnion retain differentiation potential and anti-fibrotic activity in vitro.

Human amniotic membrane (AM) has intrinsic anti-inflammatory, anti-fibrotic and antimicrobial properties. Tissue preservation methods have helped to overcome the short shelf life of fresh AM allowing "on demand" use of AM grafts. Cryopreserved AM that retains all native tissue components, including viable cells, has clinical benefits in treating chronic wounds. However, cryopreservation requires ultra-low temperature storage, limiting the use of cryopreserved products. To overcome this limitation, a new lyopreservation method has been developed for ambient storage of living tissues. The goal of this study was to investigate the viability and functionality of AM cells following lyopreservation. Fresh AM and devitalized lyopreserved AM (DLAM) served as positive and negative controls, respectively. Using live/dead staining, we confirmed the presence of living cells in viable lyopreserved AM (VLAM) and showed that these cells persisted up to 21 days in culture medium. The functionality of cells in VLAM was assessed by their differentiation potential and anti-fibrotic activity in vitro. With osteogenic induction, cells in VLAM deposited calcium within the membrane, a marker of osteogenic cells, in a time-dependent manner. The migration of human lung fibrotic fibroblasts in a scratch wound assay was reduced significantly in the presence of VLAM-derived conditioned medium. Quantitative PCR analyses indicated that VLAM reduced the expression of pro-fibrotic factors such as type I collagen and increased the expression of anti-fibrotic factors such as hepatocyte growth factor and anti-fibrotic microRNA in fibrotic fibroblasts. Taken together, these results demonstrate that endogenous cells in VLAM remain viable and functional post-lyophilization. STATEMENT OF SIGNIFICANCE: This study, for the first time, provides direct evidence showing that tissue viability and functional cells can be preserved by lyophilization. Similar to fresh amniotic membrane (AM), viable lyopreserved AM (VLAM) retains viable cells for extended periods of time. More importantly, these cells are functional and maintain their osteogenic differentiation potential and anti-fibrotic activity. Our results confirmed that the novel lyophilization method preserves tissue viability.

A Viable Lyopreserved Amniotic Membrane Modulates Diabetic Wound Microenvironment and Accelerates Wound Closure.

Objective: Wound healing is a complex process involving the dynamic interplay of various types of cells and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of the process, resulting in non-healing chronic wounds. Human amniotic membrane (AM) has been shown to be effective in the management of chronic wounds. Recently, a viable lyopreserved AM (VLAM) has been developed. The VLAM retains the structural, molecular, and functional properties of fresh AM with the advantage of a long shelf life for living tissue at ambient temperatures. The objective of this study was to evaluate the effects of VLAM on the impaired wound microenvironment and wound closure in db/db mice. Approach: VLAM or saline gel (control) was applied weekly to 7-mm excisional wounds in diabetic (db/db) mice. Wound appearance and size were assessed weekly. Inflammation and redox state in wounds were tested by cytokine gene and protein expression, and by catalase and glutathione peroxidase activities, respectively. Wound tissue granulation and neovascularization were assessed histologically. Results: Diabetic wounds treated with VLAM closed faster than control wounds. On an average, VLAM-treated wounds closed 4 days faster than the control wounds, with a significantly faster rate of closure at days 7 and 14 as compared with control wounds. The faster closure correlated with a decrease in the expression of proinflammatory factors and oxidative stress, and an increase in angiogenesis and dermal thickness. Innovation: Effects of VLAM on a chronic wound microenvironment and underlying molecular mechanisms were investigated for the first time. Conclusion: VLAM accelerates wound closure in db/db mice by decreasing inflammation and oxidative stress and supporting wound tissue granulation, neovascularization, and re-epithelialization.

Viable cryopreserved umbilical tissue (vCUT) reduces post-operative adhesions in a rabbit abdominal adhesion model.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) "point of care" allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at -80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.