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Extracellular adenosine plays a key role in promoting bone tissue formation. Local delivery of adenosine could be an effective therapeutic strategy to harness the beneficial effect of extracellular adenosine on bone tissue formation following injury. Herein, we describe the development of an injectable in situ curing scaffold containing microgel-based adenosine delivery units. The two-component scaffold includes adenosine-loaded microgels and functionalized hyaluronic acid (HA) molecules. The microgels were generated upon copolymerization of 3-acrylamidophenylboronic acid (3-APBA)- and 2-aminoethylmethacrylamide (2-AEMA)-conjugated HA (HA-AEMA) in an emulsion suspension. The PBA functional groups were used to load the adenosine molecules. Mixing of the microgels with the HA polymers containing clickable groups, dibenzocyclooctyne (DBCO) and azide (HA-DBCO and HA-Azide), resulted in a 3D scaffold embedded with adenosine delivery units. Application of the in situ curing scaffolds containing adenosine-loaded microgels following tibial fracture injury showed improved bone tissue healing in a mouse model as demonstrated by the reduced callus size, higher bone volume, and increased tissue mineral density compared to those treated with the scaffold without adenosine. Overall, our results suggest that local delivery of adenosine could potentially be an effective strategy to promote bone tissue repair.

Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. Ex vivo platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell–monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment.Significance:This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment.

Figure S6 shows the TALL-104 cell distribution within the bi-layer GelMA hydrogels loaded with MCF-7 cancer cells and monocytes.

Figure captions for the Supplementary Figures.

Supplementary Movie 2

Materials and Methods

pH-Sensitive nanocarriers deliver adenosine to the bone tissue and mitigate osteoporotic-mediated bone loss through the promotion of osteoblastogenesis and inhibition of osteoclastogenesis.

Systemic sclerosis (SSc) is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as SSc has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (Spondin-2) in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Nociceptor sensory neurons play a key role in eliciting pain. An active crosstalk between nociceptor neurons and the vascular system at the molecular and cellular level is required to sense and respond to noxious stimuli. Besides nociception, interaction between nociceptor neurons and vasculature also contributes to neurogenesis and angiogenesis. In vitro models of innervated vasculature can greatly help delineate these roles while facilitating disease modeling and drug screening. Herein, we report the development of a microfluidic-assisted tissue model of nociception in the presence of microvasculature. The self-assembled innervated microvasculature was engineered using endothelial cells and primary dorsal root ganglion (DRG) neurons. The sensory neurons and the endothelial cells displayed distinct morphologies in presence of each other. The neurons exhibited an elevated response to capsaicin in the presence of vasculature. Concomitantly, increased transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor expression was observed in the DRG neurons in presence of vascularization. Finally, we demonstrated the applicability of this platform for modeling nociception associated with tissue acidosis. While not demonstrated here, this platform could also serve as a tool to study pain resulting from vascular disorders while also paving the way towards the development of innervated microphysiological models.

Skeletal injuries are a major cause of morbidities worldwide with bone fractures accounting for a substantial portion. Patients suffering from bone fractures and undergoing surgery experience different levels of pain throughout the healing process requiring pain-mitigating interventions. Furthermore, a considerable number of bone fractures suffer from delayed healing, and unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids, however, these analgesics have substantial drawbacks including delaying healing, systemic side effects, and potential for addiction. Hence, a therapeutic approach that concomitantly attenuates pain locally and actively promotes healing would address a significant clinical problem and improve the overall functional outcome for patients. Herein, we tested the hypothesis that the purine molecule, adenosine, could simultaneously alleviate fracture pain and promote healing by targeting different adenosine receptor subtypes in different cell populations. Our results demonstrate that local delivery of adenosine inhibited nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigates pain. Concurrently, localization of adenosine at the fracture site also promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) and improved bone healing. Although further work is needed to extend the findings to human patients, this study provides evidence that the unique functional properties of adenosine along with its local delivery could provide an innovative, safe, and translatable therapeutic strategy to treat bone trauma and associated pain.One Sentence SummaryAdenosine as a therapeutic for fracture pain and healing

Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvβ3 expression, and ETS family transcription factor binding. Here we characterize these changes in vitro, in vivo, and in a dataset of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X alpha thalassemia mental retardation syndrome.

Meniscus injuries are common and a major cause of long-term joint degeneration and disability. Current treatment options are limited, so novel regenerative therapies or tissue engineering strategies are urgently needed. The development of new therapies is hindered by a lack of knowledge regarding the cellular biology of the meniscus and a lack of well-established methods for studying meniscus cells in vitro. The goals of this study were to (1) establish baseline expression profiles and dedifferentiation patterns of inner and outer zone primary meniscus cells, and (2) evaluate the utility of poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA) polymer hydrogels to reverse dedifferentiation trends for long-term meniscus cell culture. Using reverse transcription-quantitative polymerase chain reaction, we measured expression levels of putative meniscus phenotype marker genes in freshly isolated meniscus tissue, tissue explant culture, and monolayer culture of inner and outer zone meniscus cells from porcine knees to establish baseline dedifferentiation characteristics, and then compared these expression levels to PEGDA/GelMA embedded passaged meniscus cells. COL1A1 showed robust upregulation, while CHAD, CILP, and COMP showed downregulation with monolayer culture. Expression levels of COL2A1, ACAN, and SOX9 were surprisingly similar between inner and outer zone tissue and were found to be less sensitive as markers of dedifferentiation. When embedded in PEGDA/GelMA hydrogels, expression levels of meniscus cell phenotype genes were significantly modulated by varying the ratio of polymer components, allowing these materials to be tuned for phenotype restoration, meniscus cell culture, and tissue engineering applications.

Recruitment of immune cells to a tumor is determined by the complex interplay between cellular and noncellular components of the tumor microenvironment. Ex vivo platforms that enable identification of key components that promote immune cell recruitment to the tumor could advance the field significantly. Herein, we describe the development of a perfusable multicellular tumor-on-a-chip platform involving different cell populations. Cancer cells, monocytes, and endothelial cells were spatially confined within a gelatin hydrogel in a controlled manner by using 3D photopatterning. The migration of the encapsulated endothelial cells against a chemokine gradient created an endothelial layer around the constructs. Using this platform, we examined the effect of cancer cell–monocyte interaction on T-cell recruitment, where T cells were dispersed within the perfused media and allowed to infiltrate. The hypoxic environment in the spheroid cultures recruited more T cells compared with dispersed cancer cells. Moreover, the addition of monocytes to the cancer cells improved T-cell recruitment. The differences in T-cell recruitment were associated with differences in chemokine secretion including chemokines influencing the permeability of the endothelial barrier. This proof-of-concept study shows how integration of microfabrication, microfluidics, and 3D cell culture systems could be used for the development of tumor-on-a-chip platforms involving heterotypic cells and their application in studying recruitment of cells by the tumor-associated microenvironment. Significance: This study describes how tumor-on-chip platforms could be designed to create a heterogeneous mix of cells and noncellular components to study the effect of the tumor microenvironment on immune cell recruitment.

Fibrosis is a result of chronically activated fibroblasts leading to the overproduction of extracellular matrix (ECM), causing tissue hardening and loss of organ function. Systemic sclerosis (SSc) is a fibrotic skin disease marked by inflammation, autoimmunity and vasculopathy along with progressive fibrosis of the skin and internal organs. A major bottleneck in understanding the etiology of SSc has been the lack of a holistic animal model that can mimic the human SSc disease. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce hallmark clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing inflammatory cytokine and collagen production in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

The gas exchange units of the lung, the alveoli, are mechanically active and undergo cyclic deformation during breathing. The epithelial cells that line the alveoli contribute to lung function by reducing surface tension

Uncontrolled growth of tumor cells is a key contributor to cancer-associated mortalities. Tumor growth is a biomechanical process whereby the cancer cells displace the surrounding matrix that provides mechanical resistance to the growing cells. The process of tumor growth and remodeling is regulated by material properties of both the cancer cells and their surrounding matrix, yet the mechanical interdependency between the two entities is not well understood. Herein, this work develops a microfluidic platform that precisely positions tumor spheroids within a hydrogel and mechanically probes the growing spheroids and surrounding matrix simultaneously. By using hydrostatic pressure to deform the spheroid-laden hydrogel along with confocal imaging and finite element (FE) analysis, this work deduces the material properties of the spheroid and the matrix in situ. For spheroids embedded within soft hydrogels, decreases in the Young's modulus of the matrix are detected at discrete locations accompanied by localized tumor growth. Contrastingly, spheroids within stiff hydrogels do not significantly decrease the Young's modulus of the surrounding matrix, despite exhibiting growth. Spheroids in stiff matrices leverage their high bulk modulus to grow and display a uniform volumetric expansion. Collectively, a quantitative platform is established and new insights into tumor growth within a stiff 3D environment are provided.

Convergent advances in the field of soft matter, macromolecular chemistry, and engineering have led to the development of biomaterials that possess autonomous, adaptive, and self-healing characteristics similar to living systems. These rationally designed biomaterials can surpass the capabilities of their parent material. Herein, the modification of hyaluronic acid (HA) to exhibit self-healing properties is described, and its physical and biological function both in vitro and in vivo is studied. The in vitro findings showed that self-healing HA designed to undergo self-repair improves lubrication, enhances free radical scavenging, and attenuates enzymatic degradation compared to unmodified HA. Longitudinal imaging following intraarticular injection of self-healing HA shows improved in vivo retention despite its low molecular weight. Concomitant with these functions, intraarticular injection of self-healing HA mitigates anterior cruciate ligament injury-mediated cartilage degeneration in rodents. This proof-of-concept study shows how incorporation of functional properties such as self-healing can be used to surpass the existing capabilities of biolubricants.

Convergent advances in the field of soft matter, macromolecular chemistry, and engineering have led to the development of biomaterials that possess autonomous, adaptive, and self-healing characteristics similar to living systems. These rationally designed biomaterials could surpass the capabilities of their parent material. Herein, we describe the modification of hyaluronic acid (HA) molecules to exhibit self-healing properties and studied its physical and biological function both in vitro and in vivo. Our in vitro findings showed that self-healing HA designed to undergo autonomous repair improved lubrication, enhanced free radical scavenging, and resisted enzymatic degradation compared to unmodified HA. Longitudinal imaging following intra-articular injection of self-healing HA showed improved in vivo retention despite the low molecular weight. Concomitant with these functions, intra-articular injection of self-healing HA mitigated anterior cruciate ligament injury-mediated cartilage degeneration in rodents. This proof-of-concept study shows how incorporation of functional properties like self-healing can be used to surpass the existing capabilities of biolubricants.

Impaired bone healing occurs in 5-10% of cases following injury, leading to a significant economic and clinical impact. While an inflammatory response upon injury is necessary to facilitate healing, its resolution is critical for bone tissue repair as elevated acute or chronic inflammation is associated with impaired healing in patients and animal models. This process is governed by important crosstalk between immune cells through mediators that contribute to resolution of inflammation in the local healing environment. Approaches modulating the initial inflammatory phase followed by its resolution leads to a pro-regenerative environment for bone regeneration. In this review, we discuss the role of inflammation in bone repair, the negative impact of dysregulated inflammation on bone tissue regeneration, and how timely resolution of inflammation is necessary to achieve normal healing. We will discuss applications of biomaterials to treat large bone defects with a specific focus on resolution of inflammation to modulate the immune environment following bone injury, and their observed functional benefits. We conclude the review by discussing future strategies that could lead to the realization of anti-inflammatory therapeutics for bone tissue repair.

Recent advancements in soft robotics have led to the development of compliant robots that can exhibit complex motions driven by living cells, chemical reactions, or electronics. Further innovations are, however, needed to create the next generation of soft robots that can carry out advanced functions and exhibit complex locomotion. Material designs that incorporate “smart” functional properties can contribute to the development of robotic systems with in‐built mechanical responsiveness and functions. Herein, a simple material design that integrates stimuli‐responsive self‐healing and microarchitectural features to control locomotion of soft robots is reported. By employing these material designs along with hyperelastic soft actuators to control propellant dispersion and direction, a circuitry of pneumatic and microfluidic logic is created within a dragonfly‐shaped body that enables the robot to undergo user‐ and environment‐controlled locomotion over water surface. In addition to steering the robot to skim, the material properties are also leveraged to detect water acidification, temperature changes, and hydrophobic impurities such as oil. The design, fabrication, and integration strategies demonstrated herein pave a way for developing futuristic multifunctional soft robots, biomedical devices, and environmental monitoring probe.

Although bone tissues possess an intrinsic capacity for repair, there are cases where bone healing is either impaired or insufficient, such as fracture non-union, osteoporosis, osteomyelitis, and cancers. In these cases, treatments like surgical interventions are used, either alone or in combination with bioactive agents, to promote tissue repair and manage associated clinical complications. Improving the efficacy of bioactive agents often requires carriers, with biomaterials being a pivotal player. In this review, we discuss the role of biomaterials in realizing the local and systemic delivery of biomolecules to the bone tissue. The versatility of biomaterials enables design of carriers with the desired loading efficiency, release profile, and on-demand delivery. Besides local administration, systemic administration of drugs is necessary to combat diseases like osteoporosis, warranting bone-targeting drug delivery systems. Thus, chemical moieties with the affinity towards bone extracellular matrix components like apatite minerals have been widely utilized to create bone-targeting carriers with better biodistribution, which cannot be achieved by the drugs alone. Bone-targeting carriers combined with the desired drugs or bioactive agents have been extensively investigated to enhance bone healing while minimizing off-target effects. Herein, these advancements in the field have been systematically reviewed. STATEMENT OF SIGNIFICANCE: Drug delivery is imperative when surgical interventions are not sufficient to address various bone diseases/defects. Biomaterial-assisted delivery systems have been designed to provide drugs with the desired loading efficiency, sustained release, and on-demand delivery to enhance bone healing. By surveying recent advances in the field, this review outlines the design of biomaterials as carriers for the local and systemic delivery of bioactive agents to the bone tissue. Particularly, biomaterials that bear chemical moieties with affinity to bone are attractive, as they can present the desired bioactive agents to the bone tissue efficiently and thus enhance the drug efficacy for bone repair.

Recent advances in materials development and tissue engineering has resulted in a substantial number of bioinspired materials that recapitulate cardinal features of bone extracellular matrix (ECM) such as dynamic inorganic and organic environment(s), hierarchical organization, and topographical features. Bone mimicking materials, as defined by its self-explanatory term, are developed based on the current understandings of the natural bone ECM during development, remodeling, and fracture repair. Compared to conventional plastic cultures, biomaterials that resemble some aspects of the native environment could elicit a more natural molecular and cellular response relevant to the bone tissue. Although current bioinspired materials are mainly developed to assist tissue repair or engineer bone tissues, such materials could nevertheless be applied to model various skeletal diseases in vitro. This review summarizes the use of bioinspired materials for bone tissue engineering, and their potential to model diseases of bone development and remodeling ex vivo. We largely focus on biomaterials, designed to re-create different aspects of the chemical and physical cues of native bone ECM. Employing these bone-inspired materials and tissue engineered bone surrogates to study bone diseases has tremendous potential and will provide a closer portrayal of disease progression and maintenance, both at the cellular and tissue level. We also briefly touch upon the application of patient-derived stem cells and introduce emerging technologies such as organ-on-chip in disease modeling. Faithful recapitulation of disease pathologies will not only offer novel insights into diseases, but also lead to enabling technologies for drug discovery and new approaches for cell-based therapies.

We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications.

Mechanisms of myogenic commitment are deciphered through omics analysis of hiPSC-specific lineage specification measurements., Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Furthermore, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that β-catenin transcriptional cofactors mediate cross-talk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.

Stimuli-responsive (smart) hydrogels have attracted widespread attention as biomimetic systems due to their ability to respond to subtle changes in external and internal stimuli ranging from physical triggers such as temperature and electric field to chemical triggers like glucose and pH. Besides their intriguing behavior, the main interest in such smart hydrogels lies in their potential industrial and biomedical applications. Some of these applications include injectable biomaterials, tunable surfaces for cell sheet engineering, sensors, and actuators. In this review, we discuss the fundamental principles underlying the stimuli-responsive behavior of hydrogels and how these properties have led to major technological innovations. We also review recent advancements in the field of hydrogels, including self-healing and stimuli-responsive degradation in hydrogels. We conclude by providing a perspective on the potential use of smart hydrogels as multifunctional, bioactuating systems for cell and tissue engineering.

Recent advancements in soft robotics have led to the development of compliant robots that can exhibit complex motions driven by living cells(1,2), chemical reactions(3), or electronics(4). Further innovations are however needed to create the next generation of soft robots that can carry out advanced functions beyond locomotion. Here we describeDraBot—a dragonfly-inspired, entirely soft, multifunctional robot that combines long-term locomotion over water surface with sensing, responding, and adaptation capabilities. By integrating soft actuators, stimuli-responsive materials, and microarchitectural features, we created a circuitry of pneumatic and microfluidic logic that enabled the robot to undergo user- and environment-controlled (pH) locomotion, including navigating hazardous (acidic) conditions. DraBot was also engineered to sense additional environmental perturbations (temperature) and detect and clean up chemicals (oil). The design, fabrication, and integration strategies demonstrated here pave a way for developing futuristic soft robots that can acclimatize and adapt to harsh conditions while carrying out complex tasks such as exploration, environmental remediation, and health care in complex environments.

Osteochondral tissue repair remains a significant challenge in orthopedic surgery. Tissue engineering of osteochondral tissue has transpired as a potential therapeutic solution as it can effectively regenerate bone, cartilage, and the bone-cartilage interface. While advancements in scaffold fabrication and stem cell engineering have made significant progress towards the engineering of composite tissues, such as osteochondral tissue, new approaches are required to improve the outcome of such strategies. Herein, we discuss the use of a single-unit trilayer scaffold with depth-varying pore architecture and mineral environment to engineer osteochondral tissues in vivo. The trilayer scaffold includes a biomineralized bottom layer mimicking the calcium phosphate (CaP)-rich bone microenvironment, a cryogel middle layer with anisotropic pore architecture, and a hydrogel top layer. The mineralized bottom layer was designed to support bone formation, while the macroporous middle layer and hydrogel top layer were designed to support cartilage tissue formation. The bottom layer was kept acellular and the top two layers were loaded with cells prior to implantation. When implanted in vivo, these trilayer scaffolds resulted in the formation of osteochondral tissue with a lubricin-rich cartilage surface. The osteochondral tissue formation was a result of continuous differentiation of the transplanted cells to form cartilage tissue and recruitment of endogenous cells through the mineralized bottom layer to form bone tissue. Our results suggest that integrating exogenous cell-based cartilage tissue engineering along with scaffold-driven in situ bone tissue engineering could be a powerful approach to engineer analogs of osteochondral tissue. In addition to offering new therapeutic opportunities, such approaches and systems could also advance our fundamental understanding of osteochondral tissue regeneration and repair. Statement of Significance In this work, we describe the use of a single-unit trilayer scaffold with depth-varying pore architecture and mineral environment to engineer osteochondral tissues in vivo. The trilayer scaffold was designed to support continued differentiation of the donor cells to form cartilage tissue while supporting bone formation through recruitment of endogenous cells. When implanted in vivo, these trilayer scaffolds partially loaded with cells resulted in the formation of osteochondral tissue with a lubricin-rich cartilage surface. Approaches such as the one presented here that integrates ex vivo tissue engineering along with endogenous cell-mediated tissue engineering can have a significant impact in tissue engineering composite tissues with diverse cell populations and functionality.

Given the shortage of available organs for whole or partial liver transplantation, hepatocyte cell transplantation has long been considered a potential strategy to treat patients suffering from various liver diseases. Some of the earliest approaches that attempted to deliver hepatocytes via portal vein or spleen achieved little success due to poor engraftment. More recent efforts include transplantation of cell sheets or thin hepatocyte-laden synthetic hydrogels. However, these implants must remain sufficiently thin to ensure that nutrients can diffuse into the implant.To circumvent these limitations, we investigated the use of a vascularizable dual-compartment hydrogel system for minimally invasive transplantation of primary hepatocytes. The dual-compartment system features a macroporous outer polyethylene glycol diacrylate/hyaluronic acid methacrylate hydrogel compartment for seeding supportive cells and facilitating host cell infiltration and vascularization and a hollow inner core to house the primary human hepatocytes.We show that the subcutaneous implantation of these cell-loaded devices in NOD/SCID mice facilitated vascular formation while supporting viability of the transplanted cells. Furthermore, the presence of human serum albumin in peripheral blood and the immunostaining of excised implants indicated that the hepatocytes maintained function in vivo for at least 1 month, the longest assayed time point.Cell transplantation devices that assist the anastomosis of grafts with the host can be potentially used as a minimally invasive ectopic liver accessory to augment liver-specific functions as well as potentially treat various pathologies associated with compromised functions of liver, such as hemophilia B or alpha-1 antitrypsin deficiency.

Development of efficacious invivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted invivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from invivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for invivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner.

Synthetic biomimetic matrices with osteoconductivity and osteoinductivity have been developed to regenerate bone tissues. However, whether such systems harbor donor marrow in vivo and support mixed chimerism remains unknown. We devised a strategy to engineer bone tissues with a functional bone marrow (BM) compartment in vivo by using a synthetic biomaterial with spatially differing cues. Specifically, we have developed a synthetic matrix recapitulating the dual-compartment structures by modular assembly of mineralized and nonmineralized macroporous structures. Our results show that these matrices incorporated with BM cells or BM flush transplanted into recipient mice matured into functional bone displaying the cardinal features of both skeletal and hematopoietic compartments similar to native bone tissue. The hematopoietic function of bone tissues was demonstrated by its support for a higher percentage of mixed chimerism compared with i.v. injection and donor hematopoietic cell mobilization in the circulation of nonirradiated recipients. Furthermore, hematopoietic cells sorted from the engineered bone tissues reconstituted the hematopoietic system when transplanted into lethally irradiated secondary recipients. Such engineered bone tissues could potentially be used as ectopic BM surrogates for treatment of nonmalignant BM diseases and as a tool to study hematopoiesis, donor–host cell dynamics, tumor tropism, and hematopoietic cell transplantation.

Engineered skeletal muscle tissues can be used for in vitro studies that require physiologically relevant models of native tissues. Herein, we describe the development of a three-dimensional (3D) skeletal muscle tissue that recapitulates the architectural and structural complexities of muscle within a microfluidic device. Using a 3D photo-patterning approach, we spatially confined a cell-laden gelatin network around two bio-inert hydrogel pillars, which induce uniaxial alignment of the cells and serve as anchoring sites for the encapsulated cells and muscle tissues as they form and mature. We have characterized the tissue morphology and strain profile during differentiation of the cells and skeletal muscle tissue formation by using a combination of fluorescence microscopy and computational tools. The time-dependent strain profile suggests the existence of individual cells within the gelatin matrix, which differentiated to form a multinucleated skeletal muscle tissue bundle as a function of culture time. We have also developed a method to calculate the passive tension generated by the engineered muscle tissue bundles suspended between two pillars. Finally, as a proof-of-concept we have examined the applicability of the skeletal muscle-on-chip system as a screening platform and in vitro muscle injury model. We studied the dose-dependent effect of cardiotoxin on the engineered muscle tissue architecture and its subsequent effect on the passive tension. This simple yet effective tool can be appealing for studies that necessitate the analysis of skeletal muscle structure and function, including preclinical drug discovery and development.