From Promise to Practice: Recent Growth in 30 Years of Tissue Engineering Commercialization.

This perspective, marking the 30th anniversary of the Tissue Engineering journal, discusses the exciting trends in the global commercialization of tissue engineering technology. Within a historical context, we present an evolution of challenges and a discussion of the last 5 years of global commercial successes and emerging market trends, highlighting the continued expansion of the field in the northeastern United States. This leads to an overview of the last 5 years' progress in clinical trials for tissue-engineered therapeutics, including an analysis of trends in success and failure. Finally, we provide a broad overview of preclinical research and a perspective on where the state-of-the-art lies on the horizon.

Designing Biomimetic 3D-Printed Osteochondral Scaffolds for Enhanced Load-Bearing Capacity.

Osteoarthritis is a debilitating chronic joint disorder that affects millions of people worldwide. Since palliative and surgical treatments cannot completely regenerate hyaline cartilage within the articulating joint, osteochondral (OC) tissue engineering has been explored to heal OC defects. Utilizing computational simulations and three-dimensional (3D) printing, we aimed to build rationale around fabricating OC scaffolds with enhanced biomechanics. First, computational simulations revealed that interfacial fibrils within a bilayer alter OC scaffold deformation patterns by redirecting load-induced stresses toward the top of the cartilage layer. Principal component analysis revealed that scaffolds with 800 µm long fibrils (scaffolds 8A-8H) possessed optimal biomechanical properties to withstand compression and shear forces. While compression testing indicated that OC scaffolds with 800 µm fibrils did not have greater compressive moduli than other scaffolds, interfacial shear tests indicated that scaffold 8H possessed the greatest shear strength. Lastly, failure analysis demonstrated that yielding or buckling models describe interfacial fibril failure depending on fibril slenderness S. Specifically for scaffolds with packing density n = 6 and n = 8, the yielding failure model fits experimental loads with S < 10, while the buckling model fitted scaffolds with S < 10 slenderness. The research presented provides critical insights into designing 3D printed interfacial scaffolds with refined biomechanics toward improving OC tissue engineering outcomes.

Development of a patient specific cartilage graft using magnetic resonance imaging and 3D printing.

OBJECTIVES: The goal of this project was to develop and validate a patient-specific, anatomically correct graft for cartilage restoration using magnetic resonance imaging (MRI) data and 3-dimensional (3D) printing technology. The specific aim was to test the accuracy of a novel method for 3D printing and implanting individualized, anatomically shaped bio-scaffolds to treat cartilage defects in a human cadaveric model. We hypothesized that an individualized, anatomic 3D-printed scaffold designed from MRI data would provide a more optimal fill for a large cartilage defect compared to a generic flat scaffold. METHODS: Four focal cartilage defects (FCDs) were created in paired human cadaver knees, age <40 years, in the weight-bearing surfaces of the medial femoral condyle (MFC), lateral femoral condyle (LFC), patella, and trochlea of each knee. MRIs were obtained, anatomic grafts were designed and 3D printed for the left knee as an experimental group, and generic flat grafts for the right knee as a control group. Grafts were implanted into corresponding defects and fixed using tissue adhesive. Repeat post-implant MRIs were obtained. Graft step-off was measured as the distance in mm between the surface of the graft and the native cartilage surface in a direction perpendicular to the subchondral bone. Graft contour was measured as the gap between the undersurface of the graft and the subchondral bone in a direction perpendicular to the joint surface. RESULTS: Graft step-off was statistically significantly better for the anatomic grafts compared to the generic grafts in the MFC (0.0 â€‹± â€‹0.2 â€‹mm vs. 0.7 â€‹± â€‹0.5 â€‹mm, p â€‹< â€‹0.001), LFC (0.1 â€‹± â€‹0.3 â€‹mm vs. 1.0 â€‹± â€‹0.2 â€‹mm, p â€‹< â€‹0.001), patella (-0.2 â€‹± â€‹0.3 â€‹mm vs. -1.2 â€‹± â€‹0.4 â€‹mm, p â€‹< â€‹0.001), and trochlea (-0.4 â€‹± â€‹0.3 vs. 0.4 â€‹± â€‹0.7, p â€‹= â€‹0.003). Graft contour was statistically significantly better for the anatomic grafts in the LFC (0.0 â€‹± â€‹0.0 â€‹mm vs. 0.2 â€‹± â€‹0.4 â€‹mm, p â€‹= â€‹0.022) and trochlea (0.0 â€‹± â€‹0.0 â€‹mm vs. 1.4 â€‹± â€‹0.7 â€‹mm, p â€‹< â€‹0.001). The anatomic grafts had an observed maximum step-off of -0.9 â€‹mm and a maximum contour mismatch of 0.8 â€‹mm. CONCLUSION: This study validates a process designed to fabricate anatomically accurate cartilage grafts using MRI and 3D printing technology. Anatomic grafts demonstrated superior fit compared to generic flat grafts. LEVEL OF EVIDENCE: Level IV.

A novel perfusion bioreactor promotes the expansion of pluripotent stem cells in a 3D-bioprinted tissue chamber.

While the field of tissue engineering has progressed rapidly with the advent of 3D bioprinting and human induced pluripotent stem cells (hiPSCs), impact is limited by a lack of functional, thick tissues. One way around this limitation is to 3D bioprint tissues laden with hiPSCs. In this way, the iPSCs can proliferate to populate the thick tissue mass prior to parenchymal cell specification. Here we design a perfusion bioreactor for an hiPSC-laden, 3D-bioprinted chamber with the goal of proliferating the hiPSCs throughout the structure prior to differentiation to generate a thick tissue model. The bioreactor, fabricated with digital light projection, was optimized to perfuse the interior of the hydrogel chamber without leaks and to provide fluid flow around the exterior as well, maximizing nutrient delivery throughout the chamber wall. After 7 days of culture, we found that intermittent perfusion (15 s every 15 min) at 3 ml min-1provides a 1.9-fold increase in the density of stem cell colonies in the engineered tissue relative to analogous chambers cultured under static conditions. We also observed a more uniform distribution of colonies within the tissue wall of perfused structures relative to static controls, reflecting a homogeneous distribution of nutrients from the culture media. hiPSCs remained pluripotent and proliferative with application of fluid flow, which generated wall shear stresses averaging ∼1.0 dyn cm-2. Overall, these promising outcomes following perfusion of a stem cell-laden hydrogel support the production of multiple tissue types with improved thickness, and therefore increased function and utility.

4D Bioprinting via Molecular Network Contraction for Membranous Tissue Fabrication.

Generation of thin membranous tissues (TMT), such as the cornea, epidermis, and periosteum, presents a difficult fabrication challenge in tissue engineering (TE). TMTs consist of several cell layers that are less than 100 µm in thickness per layer. While traditional methods provide the necessary resolution for TMT fabrication, they require significant handling and incorporation of several layers is limited. Extrusion bioprinting offers precise control over deposition of different biomaterials and cell populations within the same construct but lacks the resolution to generate biomimetic TMTs. For the first time, a 4D bioprinting strategy that allows for the generation of cell-laden TMTs is developed. Anionic gelatin methacrylate (GelMA) hydrogels are treated with cationic poly-l-lysine (PLL), which induces charge attraction, microscale network collapse, and macroscale hydrogel shrinking. The impact of shrinking on hydrogel properties, print resolution, and cell viability is presented. Additionally, this work suggests that a novel mechanism is occurring, where PLL exhibits a contractile force on GelMA and PLL molecular weight drives GelMA shrinking capabilities. Finally, it is shown that this phenomenon can occur while maintaining an encapsulated cell population. These findings address a critical barrier by generating macroscale tissue structures with their microscale TMT counterparts in the same print.

Fabrication Strategies for Engineered Thin Membranous Tissues.

Thin membranous tissues (TMTs) are anatomical structures consisting of multiple stratified cell layers, each less than 100 µm in thickness. While these tissues are small in scale, they play critical roles in normal tissue function and healing. Examples of TMTs include the tympanic membrane, cornea, periosteum, and epidermis. Damage to these structures can be caused by trauma or congenital disabilities, resulting in hearing loss, blindness, dysfunctional bone development, and impaired wound repair, respectively. While autologous and allogeneic tissue sources for these membranes exist, they are significantly limited by availability and patient complications. Tissue engineering has therefore become a popular strategy for TMT replacement. However, due to their complex microscale architecture, TMTs are often difficult to replicate in a biomimetic manner. The critical challenge in TMT fabrication is balancing fine resolution with the ability to mimic complex target tissue anatomy. This Review reports existing TMT fabrication strategies, their resolution and material capabilities, cell and tissue response, and the advantages and disadvantages of each technique.

Retraction of "Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial".

[This retracts the article DOI: 10.1021/acscentsci.8b00050.].

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

3D printable phantom for mimicking electrical properties of dermal tissue.

Skin cancer is one of the most ubiquitous forms of cancer that is often overdiagnosed or missed by traditional diagnostic techniques. Bioimpedance spectroscopy (BIS) is a technology that aims to take advantage of the variations in electrical properties of tissue to identify ectopic formations. It is difficult to develop BIS technologies without obtaining tumor tissue samples. One solution is to use a "tissue phantom," a synthetic structure that mimics the properties of tissue. Current solutions using natural biomaterials, such as gelatin, have not been able to create complex tissue geometries that are vital to honing BIS diagnostics. However, semi-synthetic polymers, such has gelatin methacrylate (GelMA), offer the benefits of possessing similar electrical properties to their respective source biomaterial while being 3D printable. In this work, we first measured the impedance of porcine dermal tissue. We then applied these impedance measurements to create an electrically accurate tissue phantom using a photocurable hydrogel, GelMA, and varying concentrations of NaCl, aluminum powder, and titanium dioxide powder.

Hyperglycemic Conditions Enhance the Mechanosensitivity of Proinflammatory RAW264.7 Macrophages.

Macrophages are a primary contributor to the orchestration and severity of the foreign body response. As phagocytes and antigen-presenting cells, macrophages engage foreign objects, producing chemokines, degrading enzymes, and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Encapsulated islet transplantation (EIT) is a return of function therapy in which donor insulin-secreting cells are encased in a biomaterial and implanted into a diabetic patient to regulate blood glucose levels. However, the foreign body response by macrophages to the encapsulated islet allograft may cause rejection. Recent studies have shown that substrate stiffness affects macrophage activity, which can inform EIT capsule design. However, due to the dysregulation of glucose maintenance in diabetic patients, varying from normoglycemic to hypoglycemic or hyperglycemic conditions, it is imperative to determine if glucose dysregulation affects macrophage mechanosensitivity to EIT biomaterials. This study explores the relationship between glucose metabolism and mechanosensitivity and the ultimate impact on proinflammatory macrophage function in static hyperglycemic and normoglycemic conditions. Using a 2-dimensional (2D) polyacrylamide model of 3-order magnitude in stiffness, 2, 15, and 274 kPa Young's moduli, the effect of glycemic condition on the mechanosensitive characteristics of unstimulated and proinflammatory RAW264.7 macrophage function in vitro using lipopolysaccharide (LPS) was examined. Hyperglycemic conditions were found to impact macrophage response to substrate stiffness significantly. Notably, TNF-α secretion was significantly reduced as substrate stiffness increased in LPS-stimulated hyperglycemic conditions, whereas normoglycemic macrophages held similar secretion across all stiffnesses. Stiffness-influenced differences in cytokine secretion were also induced in IL-6 secretion by hyperglycemic conditions. Hyperglycemic conditions promoted a biphasic trend in IL-6 cytokine secretion and gene expression by proinflammatory macrophages with significantly decreased production when cultured on 15 kPa compared to production on 2 and 274 kPa. Although hyperglycemic conditions drastically increased IL-10 secretion, stiffness-influenced differences were not shown when compared to the same glycemic condition. Furthermore, under LPS stimulation, lactate secretion had an inverse relationship to TNF-α secretion. However, no significant stiffness-influenced difference was demonstrated in glucose transporter 1 (GLUT1) expression, glucose uptake, or GAPDH. These findings suggest that hyperglycemic conditions enhance the mechanosensitivity of proinflammatory macrophages and should be explored further. Impact statement The work presented increases our understanding of the effect of glycemic condition on macrophage mechanosensitivity related to substrate stiffness. This has ramifications on the design of material-based therapies, such as encapsulated islet transplantation, for type 1 diabetic patients who experience glycemic dysregulation.

A Case for Material Stiffness as a Design Parameter in Encapsulated Islet Transplantation.

Diabetes is a disease that plagues over 463 million people globally. Approximately 40 million of these patients have type 1 diabetes mellitus (T1DM), and the global incidence is increasing by up to 5% per year. T1DM is where the body's immune system attacks the pancreas, specifically the pancreatic beta cells, with antibodies to prevent insulin production. Although current treatments such as exogenous insulin injections have been successful, exorbitant insulin costs and meticulous administration present the need for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to diabetes. Donor islets are encapsulated in a semipermeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration and antibody access to the transplanted cells. Although successful in small animal models, EIT is still far from commercial use owing to necessary long-term systemic immunosuppressants and consistent immune rejection. Most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability. However, most studies have been limited in scope to biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages-primary foreign body response (FBR) orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. In this review, we explore strategies to improve the clinical viability of EIT. A brief overview of the immune system, the FBR, and current biochemical approaches will be elucidated throughout this exploration. Furthermore, an argument for using substrate stiffness as a capsule design parameter to increase EIT efficacy and clinical viability will be posed.

Smurf2 Regulates Inflammation and Collagen Processing in Cutaneous Wound Healing through Transforming Growth Factor-ß/Smad3 Signaling.

Wound healing is a highly conserved process that restores the integrity and functionality of injured tissues. Transforming growth factor (TGF)-ß is a master regulator of wound healing, whose signaling is attenuated by the E3 ubiquitin ligase Smurf2. Herein, the roles of Smurf2 in cutaneous wound healing were examined using a murine incisional cutaneous model. Loss of Smurf2 increased early inflammation in the wounds and led to narrower wounds with greater breaking strength. Loss of Smurf2 also led to more linearized collagen bundles in normal and wounded skin. Gene expression analyses by real-time quantitative PCR indicated that Smurf2-deficient fibroblasts had increased levels of TGF-ß/Smad3 signaling and changes in expression profile of genes related to matrix turnover. The effect of Smurf2 loss on wound healing and collagen bundling was attenuated by the heterozygous loss of Smad3. Together, these results show that Smurf2 affects inflammation and collagen processing in cutaneous wounds by down-regulating TGF-ß/Smad3 signaling.

An open-source bioink database for microextrusion 3D printing.

3D printing has rapidly become a critical enabling technology in tissue engineering and regenerative medicine for the fabrication of complex engineered tissues. 3D bioprinting, in particular, has advanced greatly to facilitate the incorporation of a broad spectrum of biomaterials along with cells and biomolecules of interest forin vitrotissue generation. The increasing complexity of novel bioink formulations and application-dependent printing conditions poses a significant challenge for replicating or innovating new bioprinting strategies. As the field continues to grow, it is imperative to establish a cohesive, open-source database that enables users to search through existing 3D printing formulations rapidly and efficiently. Through the efforts of the NIH/NIBIB Center for Engineering Complex Tissues, we have developed, to our knowledge, the first bioink database for extrusion-based 3D printing. The database is publicly available and allows users to search through and easily access information on biomaterials and cells specifically used in 3D printing. In order to enable a community-driven database growth, we have established an open-source portal for researchers to enter their publication information for addition into the database. Although the database has a broad range of capabilities, we demonstrate its utility by performing a comprehensive analysis of the printability domains of two well-established biomaterials in the printing world, namely poly(ϵ-caprolactone) and gelatin methacrylate. The database allowed us to rapidly identify combinations of extrusion pressure, temperature, and speed that have been used to print these biomaterials and more importantly, identify domains within which printing was not possible. The data also enabled correlation analysis between all the printing parameters, including needle size and type, that exhibited compatibility for cell-based 3D printing. Overall, this database is an extremely useful tool for the 3D printing and bioprinting community to advance their research and is an important step towards standardization in the field.

Recent Advances in Senotherapeutics Delivery.

Accumulation of senescent cells (SnCs) in various tissue types has been connected to an occurrence of different age-related diseases that are indicated by its own tissue-specific hallmarks. Discovery of novel senolytic compounds that target major cellular mechanisms to inhibit the level of SnCs within the specific tissues or organs has been an emerging field in the age-related disease research. Although the positive effect of senolytics in global suppression of SnCs has been well studied in the past, effective tissue-specific delivery strategy of senotherapeutics before clinical application needs to be further investigated. In this review, we discuss the latest biological insights to currently available senotherapeutic options and explore the impactful in vitro tissue-engineered models possibly as a testbed for replicable testing of tissue-specific potency of senolytics. Impact statement Senotherapy, the inhibition of accumulated senescent cells, is recognized as a significantly impactful way to treat various human diseases. However, there is limited comprehensive reviews on this topic. This review provides in-depth discussion on diverse delivery strategies of senolytic agents and latest updates on a novel senotherapeutic research.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1ß mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Noncovalent reversible binding-enabled facile fabrication of leak-free PDMS microfluidic devices without plasma treatment for convenient cell loading and retrieval.

The conventional approach for fabricating polydimethylsiloxane (PDMS) microfluidic devices is a lengthy and inconvenient procedure and may require a clean-room microfabrication facility often not readily available. Furthermore, living cells can't survive the oxygen-plasma and high-temperature-baking treatments required for covalent bonding to assemble multiple PDMS parts into a leak-free device, and it is difficult to disassemble the devices because of the irreversible covalent bonding. As a result, seeding/loading cells into and retrieving cells from the devices are challenging. Here, we discovered that decreasing the curing agent for crosslinking the PDMS prepolymer increases the noncovalent binding energy of the resultant PDMS surfaces without plasma or any other treatment. This enables convenient fabrication of leak-free microfluidic devices by noncovalent binding for various biomedical applications that require high pressure/flow rates and/or long-term cell culture, by simply hand-pressing the PDMS parts without plasma or any other treatment to bind/assemble. With this method, multiple types of cells can be conveniently loaded into specific areas of the PDMS parts before assembly and due to the reversible nature of the noncovalent bonding, the assembled device can be easily disassembled by hand peeling for retrieving cells. Combining with 3D printers that are widely available for making masters to eliminate the need of photolithography, this facile yet rigorous fabrication approach is much faster and more convenient for making PDMS microfluidic devices than the conventional oxygen plasma-baking-based irreversible covalent bonding method.

Computational investigation of interface printing patterns within 3D printed multilayered scaffolds for osteochondral tissue engineering.

Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that commonly affects many joints. Repetitive joint overloading perpetuates the damage to the affected cartilage, which undermines the structural integrity of the osteochondral unit. Various tissue engineering strategies have been employed to design multiphasic osteochondral scaffolds that recapitulate layer-specific biomechanical properties, but the inability to fully satisfy mechanical demands within the joint has limited their success. Through computational modeling and extrusion-based bioprinting, we attempted to fabricate a biphasic osteochondral scaffold with improved shear properties and a mechanically strong interface. A 3D stationary solid mechanics model was developed to simulate the effect of lateral shear force on various thermoplastic polymer/hydrogel scaffolds with a patterned interface. Additionally, interfacial shear tests were performed on bioprinted polycaprolactone (PCL)/hydrogel interface scaffolds. The first simulation showed that the PCL/gelatin methacrylate (GelMA) and PCL/polyethylene glycol diacrylate (PEGDA) scaffolds interlocking hydrogel and PCL at interface in a 1:1 ratio possessed the largest average tensile (PCL/GelMA: 80.52 kPa; PCL/PEGDA: 79.75 kPa) and compressive stress (PCL/GelMA: 74.71 kPa; PCL/PEGDA: 73.83 kPa). Although there were significant differences in shear strength between PCL/GelMA and PCL/PEGDA scaffolds, no significant difference was observed among the treatment groups within both scaffold types. Lastly, the hypothetical simulations of potential biphasic 3D printed scaffolds showed that for every order of magnitude decrease in Young's modulus (E) of the soft bioink, all the scaffolds underwent an exponential increase in average displacement at the cartilage and interface layers. The following work provides valuable insights into the biomechanics of 3D printed osteochondral scaffolds, which will help inform future scaffold designs for enhanced regenerative outcomes.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.