Tissue Engineering Part A

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen expression. Five conditions predicted to produce the greatest Luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen expression. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Youngs tensile modulus compared to engineered cartilage in basal media alone. Collagen cross-linking analysis confirmed formation of type II-type : II collagen and type II-type : IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure matrix-induced autologous chondrocyte implantation (MACI). Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to 1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists when expanded chondrocytes are transferred to a 3D culture; and 2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded chondrocytes. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0, 8 and 16 population doublings in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a three-dimensional (3D) hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for 16 population doublings and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Cardiac fibrosis is a pathological process in which the myocardium stiffens due to the overproduction of extracellular matrix (ECM) proteins. Cardiac fibroblasts activate to myofibroblasts in response to the inflammatory cytokine transforming growth factor beta1 (TGF-{beta}1) to promote fibrotic scarring. Biological sex also influences cardiac fibrosis progression and patient outcomes, where males exhibit increased fibrotic scarring after acute inflammation relative to females. At the cellular level, sex differences in TGF-{beta}1-mediated cardiac myofibroblast activation processes have not been clearly defined. We hypothesized that TGF-{beta}1 would cause sex-specific cardiac myofibroblast activation levels and alter the secretion of bioactive molecules to modulate sex differences in cardiac fibrosis. Primary left ventricle cardiac fibroblasts were isolated from male and female C57BL/6J mice and cultured on hydrogel biomaterials mimicking native myocardial ECM stiffness and treated with TGF-{beta}1 and/or the TGF-{beta}1 receptor inhibitor SD208. Male myofibroblasts exhibited increased -SMA stress fiber formation, increased SMAD2/3 localization, and greater resistance to SD208 inhibition compared to female myofibroblasts on hydrogels at various time points tested. Sex differences in relative secreted cytokine abundance were also determined, with male CFs secreting increased vascular endothelial growth factor (VEGF) and female CFs producing increased periostin and fibroblast growth factor 21 in response to TGF-{beta}1. Our findings establish that TGF-{beta}1 mediates sex differences in cardiac myofibroblast activation on hydrogels and secreted factors that may modulate the myocardial microenvironment. Our work underscores the importance of using hydrogels as cell culture platforms to recapitulate sex-specific cardiac fibrosis phenotypes as a steppingstone towards identifying sex-dependent therapeutic interventions for cardiac fibrosis.

Decellularized extracellular matrix (ECM) hydrogels present a novel, clinical intervention for a myriad of regenerative medicine applications. The source of ECM is typically the same tissue to which the treatment is applied; however, the need for tissue specific ECM sources has not been rigorously studied. We hypothesized that tissue specific ECM would improve regeneration through preferentially stimulating physiologically relevant processes (e.g. progenitor cell proliferation and differentiation). One of two decellularized hydrogels (tissue specific skeletal muscle or non mesoderm-derived lung) or saline were injected intramuscularly two days after notexin injection in mice (n=7 per time point) and muscle was harvested at days 5 and 14 for histological and gene expression analysis. Both injectable hydrogels were decellularized using the same detergent and were controlled for donor characteristics (i.e. species, age). At day 5, the skeletal muscle ECM hydrogel significantly increased the density of Pax7+ satellite cells in the muscle. Gene expression analysis at day 5 showed that skeletal muscle ECM hydrogels increased expression of genes implicated in muscle contractility. By day 14, skeletal muscle ECM hydrogels improved muscle regeneration over saline and lung ECM hydrogels as shown through a shift in fiber cross sectional area distribution towards larger fibers. This data indicates a potential role for muscle-specific regenerative capacity of decellularized, injectable muscle hydrogels. Further transcriptomic analysis of whole muscle mRNA indicates the mechanism of tissue specific ECM-mediated tissue repair may be immune and metabolism pathway-driven. Taken together, this suggests there is benefit in using tissue specific ECM for regenerative medicine applications.Competing Interest StatementKLC is co-founder, board member, consultant, receives income, and has equity interest in Ventrix, Inc.View Full Text

Controlling basic fibroblast growth factor (bFGF) signaling is important for both tissue-engineering purposes, controlling proliferation and differentiation potential, and for cancer biology, influencing tumor progression and metastasis. Here, we observed that human mesenchymal stromal cells (hMSCs) no longer responded to soluble or covalently bound bFGF when cultured on microfibrillar substrates, while fibroblasts did. This correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates, compared to hMSCs on conventional tissue culture plastic (TCP). hMSCs also expressed less SRF on ESP scaffolds, compared to TCP, while fibroblasts maintained high FGFR1 and SRF expression. Inhibition of actin-myosin tension or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 osteosarcoma cells. This downregulation was functional, as hMSCs became irresponsive to bFGF in the presence of MRTF/SRF inhibitor. Together, our data show that hMSCs, but not fibroblasts, are irresponsive to bFGF when cultured on microfibrillar susbtrates by downregulation of FGFR1 through the MRTF/SRF pathway. This is the first time FGFR1 expression has been shown to be mechanosensitive and adds to the sparse literature on FGFR1 regulation. These results could open up new targets for cancer treatments and could aid designing tissue engineering constructs that better control cell proliferation.

Cartilage tissue engineering using bone marrow-derived mesenchymal stem cells (BM-MSCs) is a growing technology for the repair of joint defects. Culturing BM-MSCs to over confluence has historically been avoided due to perceived risk to cell viability, growth inhibition and differentiation potential. Here we show that a simple change in culture practice, based on mimicking the condensation phase during embryonic cartilage development, results in biochemically and histologically superior cartilage tissue engineered constructs. Whole transcriptome analysis of the condensing cells revealed a phenotype associated with early commitment to chondrogenic precursors. This simple adjustment to the common stem cell culture technique would impact the quality of all cartilage tissue engineering modalities utilising these cells.

Estrogens are global regulators of cellular signaling pathways, impacting fundamental processes and phenotypes that are essential for tissue remodeling and homeostasis. Traditional cell culture media contains estrogen-mimetic compounds, including phenol red and endogenous estrogen in fetal bovine serum (FBS). However, the potential of these compounds to bias in vitro studies, particularly when considering sex as a biological variable, remains unclear. This gap in understanding critically impacts the culture of human mesenchymal stromal cells (hMSCs), whose basic functions and differentiation potential, central to cell therapy and tissue engineering, are sensitive to perturbations in the culture conditions. Despite this, the effect of estrogens from cell culture media on male and female hMSCs is not currently considered in cell processing for clinical trials. As such, a baseline understanding of these estrogen-mimetic media influences on hMSCs is critical for clinical efficacy and adequate study design in research. To this end, we investigated the effects of phenol red and fetal bovine serum on the proliferation, metabolism, senescence, and differentiation capacity of male and female hMSCs. Phenol red, FBS, donor sex, and 17{beta}-estradiol (E2) supplementation all had significant impacts on hMSC health and differentiation potential in culture. Notably, dosing with estrogen at the levels found in FBS did not recover most of the hMSC metrics tested. The only outcomes that were not significantly different based on donor sex were senescence and mRNA transcripts for RUNX2 and PPARG, transcriptional regulators for osteogenesis and adipogenesis. Overall, these findings reveal the sex-biased effects of estrogen and estrogen-mimetic compounds in traditional culture media, underscoring a current gap in considering sex as a biological variable in cell therapy and tissue engineering research and manufacturing.

Technological advancements have enabled the design of increasingly complex engineered tissue constructs, which better mimic native tissue cellularity. Therefore, dissecting the bi-directional interactions between distinct cell types in 3D is necessary to understand how heterotypic interactions at the single-cell level impact tissue-level properties. We systematically interrogated the interactions between cardiomyocytes (CMs) and cardiac non-myocytes in 3D self-assembled tissue constructs in an effort to determine the phenotypic and functional contributions of cardiac fibroblasts (CFs) and endothelial cells (ECs) to cardiac tissue properties. One week after tissue formation, cardiac microtissues containing CFs exhibited improved calcium handling function compared to microtissues comprised of CMs alone or CMs mixed with ECs, and CMs cultured with CFs exhibited distinct transcriptional profiles, with increased expression of cytoskeletal and ECM-associated genes. However, one month after tissue formation, functional and phenotypic differences between heterotypic tissues were mitigated, indicating diminishing impacts of non-myocytes on CM phenotype and function over time. The combination of single-cell RNA-sequencing and calcium imaging enabled the determination of reciprocal transcriptomic changes accompanying tissue-level functional properties in engineered heterotypic cardiac microtissues.

Human extracellular matrix (ECM) exhibits complex protein composition and architecture depending on tissue and disease state, which remains challenging to reverse engineer. One promising approach is based on cell-secreted ECM from human fibroblasts, which can then be decellularized into an acellular biomaterial. However, fibroblasts initially seeded on rigid tissue culture plastic or biomaterial scaffolds experience aberrant mechanical cues that influence ECM deposition. Here, we show that engineered microtissues of primary human fibroblasts seeded in low-adhesion microwells can be decellularized to produce human, tissue-specific ECM. We investigate: 1) cardiac fibroblasts, as well as 2) lung fibroblasts from healthy, idiopathic fibrosis and chronic obstructive pulmonary disease donors. We demonstrate optimized culture and decellularization conditions, then characterize gene expression and protein composition. We further characterize ECM microstructure and mechanical properties. We envision that this method could be utilized for biomanufacturing of patient and tissue-specific ECM for organoid drug screening as well as implantable scaffolds. ImpactIn this study, we demonstrate a method for preparing decellularized matrix using primary human fibroblasts with tissue and disease-specific features. We aggregate single cell dispersions into engineered tissues using low adhesion microwells and show culture conditions that promote ECM deposition. We demonstrate this approach for cardiac fibroblasts as well as lung fibroblasts (both normal and diseased). We systematically investigate tissue morphology, matrix architecture, and mechanical properties, along with transcriptomic and proteomic analysis. This approach should be widely applicable for generating personalized ECM with features of patient tissues and disease state, relevant for culturing patient cells ex vivo as well as implantation for therapeutic treatments.

BackgroundUnderstanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns. MethodsWe generated iPSCs with homozygous D65A cardiac troponin C (cTnC) mutations. This mutation prevents calcium binding to site II of cTnC, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. The iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via fluorescence imaging and calcium transient analysis. WT and mutant proteomes were examined via mass spectrometry throughout differentiation and maturation. We also replated partially matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus non-contractile cells. ResultsSurprisingly, we found that sarcomeres formed in the cTnC D65A cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. Mutant cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Plating D65A cardiomyocytes on nanopatterns improved structural and proteomic maturation. However, plating WT cardiomyocytes on nanopatterns led to a reduction in sarcomeric and oxidative phosphorylation protein content. ConclusionsCalcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In non-contractile, mutant cardiomyocytes, nanopatterns enhance maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate WT maturation and may have hindered it. In addition to these novel findings, these large mass spectrometry datasets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular community.

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies require two-dimensional (2D) cell expansion processes to achieve large cell populations critical for the repair of damaged (e.g. connective and musculoskeletal) tissues. However, the influence of mechanical memory on cell fate following expansion is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we show that the organization of histone H3 trimethylated at lysine 9 (H3K9me3) and expression of tissue-identifying genes in primary cartilage cells (chondrocytes) transferred to three-dimensional (3D) hydrogels depends on the number of previous population doublings on tissue culture plastic during 2D cell expansion. Decreased levels of H3K9me3 occupying promoters of dedifferentiation genes after the 2D culture were also retained in 3D culture. Suppression of H3K9me3 during expansion of cells isolated from a murine model similarly resulted in the loss of the chondrocyte phenotype and global remodeling of nuclear architecture. In contrast, increasing levels of H3K9me3 through inhibiting H3K9 demethylases partially rescued the chondrogenic nuclear architecture and gene expression, which has important implications for tissue repair therapies, where expansion of large numbers of phenotypically-suitable cells is required. Overall, our findings indicate mechanical memory in primary cells is encoded in the chromatin architecture, which impacts cell fate and the phenotype of expanded cells. SIGNIFICANCE STATEMENTTissue regeneration procedures, such as cartilage defect repair (e.g. Matrix-induced Autologous Chondrocyte Implantation) often require cell expansion processes to achieve sufficient cells to transplant into an in vivo environment. However, the chondrocyte cell expansion on 2D stiff substrates induces epigenetic changes that persist even when the chondrocytes are transferred to a different (e.g. 3D) or in vivo environment. Treatments to alter epigenetic gene regulation may be a viable strategy to improve existing cartilage defect repair procedures and other tissue engineering procedures that involve cell expansion.

Myocardial infarction can lead to the loss of billions of cardiomyocytes, and while cell-based therapies are a promising option, the immature nature of in vitro-generated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) is a significant roadblock to their development. Through the years, various approaches have emerged to improve iCM maturation, yet none could fully recapitulate the complexity of cardiac development and were not enough to achieve full cardiac maturity in vitro. Cardiac differentiation occurs at the early stages of development in a highly dynamic environment. Although significantly improved over the past two decades, small molecule-based iPSC differentiation protocols dont go beyond producing high purity fetal iCMs. Recently adult extracellular matrix (ECM) was shown to retain tissue memory and has shown some success in driving tissue-specific differentiation in unspecified cells in various organ systems. Therefore, here, we first characterized the adult human heart left ventricle components. We then investigated the effect of adult human heart-derived ECM on iPSC cardiac differentiation and subsequent maturation. By preconditioning iPSCs with ECM, we tested whether creating a cardiac environment around iPSCs would drive them toward cardiac fate before small molecule mediated differentiation. Ultimately, we investigated ECM components that might be responsible for the observed effects. We identified critical glycoproteins and proteoglycans involved in early cardiac development in the adult heart ECM. Namely, adult ECM had extracellular galactin-1, fibronectin, fibrillins, and basement-membrane-specific heparan-sulfate proteoglycan (HSPG), which have been implicated in normal heart development and associated with various embryonic developmental processes. Relatedly, we showed that preconditioning iPSCs with adult ECM resulted in enhanced cardiac differentiation, yielding iCMs with higher functional maturity. Further investigation revealed that a more developed mitochondrial network and coverage as well as enhanced metabolic maturity and a shift towards a more energetic profile allowed the observed functional enhancement in ECM pretreated iCMs. These findings demonstrate the potential of using cardiac ECM for promoting iCM maturation and suggest a promising strategy for improving the development of iCM-based therapies and in vitro cardiac disease modeling and drug screening studies. Upon manipulating ECM, such as heat denaturation and sonication to eliminate protein components and release ECM bound vesicle contents, respectively, we concluded that the beneficial effects that we observed are not solely due to the ECM proteins, and might be related to the decorative units attached to them.

As of yet, no standard of care incorporates biomaterials to treat traumatic spinal cord injury (SCI). However, intense development of biomaterials for treating SCI has focused on fabricating microscale channels to support the regrowth of axons while minimizing scar formation. We previously demonstrated that plant tissues could be decellularized and processed to form sterile, biocompatible and implantable biomaterials that support cell infiltration and vascularization in vivo. Vascularized plant tissues contain continuous microscale channels with geometries relevant for supporting neural regeneration. We hypothesized that decellularized vascular bundles would support neural regeneration and motor recovery in SCI. Sprague Dawley rats received a complete T8-T9 spinal cord transection and were implanted with acellular plant-derived scaffolds and allowed to recover over 28 weeks. Animals that received the scaffolds alone, with no other therapeutic compounds, demonstrated a significant and stable partial improvement in motor function compared to control animals as early as week 4 post-injury. Hind-limb motor function did not deteriorate over the remaining 28 weeks. Histological analysis revealed minimal astrocyte scarring at the spinal cord - scaffold interface, aligned axonal projection through the scaffolds, populations of serotonergic neurons and Schwann cells, laminin and collagen deposition and the presence of blood vessels. Axonal reconnection via the scaffold was also confirmed by Fluro-gold retrograde tracing. Taken together, our work defines a novel route for building upon naturally occurring plant microarchitectures to support the repair of the spinal cord post-injury. Notably, these results were achieved without the use of growth factors, stem/progenitor cells, or any other interventions.

There are limited biomaterials for skeletal muscle regeneration. This study aimed to apply a decellularization protocol in a muscle flap model and investigate its patency. Twenty-six gracilis-muscle (GM) flaps were harvested from 13 rats. GMs were divided into groups of either 1) normal (control), 2) perfusion with 1% sodium dodecyl sulfate or SDS for 48h, followed by Triton X-100 or TX100, or lastly, 3) perfusion with SDS for 72h, followed by TX100. The morphology, microcirculatory network patency, and residual DNA content (DNAC) were evaluated. Decellularized muscle (DM) for 72h was more translucent than DM-48h. Despite longer decellularization, the DM-72h microcirculatory network maintained its integrity, except when the dye infiltrated from the muscle edges. Compared to normal, all DM had significantly lower DNAC (normal of 1.44 g/mg vs. DM-48h of 0.37 g/mg vs. DM-72h of 0.089 g/mg; P < 0.001). The DNAC of the DM-72h group was significantly lower than DM-48h (P< 0.001). We report successful GM flap decellularization. Longer decellularization led to lower DNAC, which did not compromise circulation. Our protocol may be applicable as a free-flap scaffold model for transplantation in the future. Statement of clinical significanceThe impact of our work involves a reproducible skeletal muscle decellularization protocol to later apply in translational research.

ObjectivesCardiac regenerative therapy using human induced pluripotent stem cell (hiPSC)-derived tissues and organoids holds great promise for treating heart diseases. Successful clinical translation requires biomimetic cardiac tissues that not only recapitulate native myocardial architecture but also actively integrate with host vasculature. We aimed to engineer self-organized, vascularized cardiac microtissues (VCMs) and evaluate their therapeutic and regenerative potential in a rat model of myocardial infarction (MI). MethodsVCMs composed of hiPSC-derived cardiomyocytes, vascular endothelial cells, and vascular mural cells were cultured under dynamic conditions to promote self-organization and prevascular network formation. One week after MI induction by coronary artery ligation in athymic immunodeficient rats, VCMs were transplanted onto the infarcted myocardium. Cardiac function was assessed by echocardiography and magnetic resonance imaging. Three-dimensional host-graft vascular architecture was visualized by light-sheet fluorescence microscopy following tissue clearing, and functional perfusion was evaluated by intravenous DyLight 488-conjugated lectin injection via host systemic circulation prior to tissue harvest. ResultsVCM transplantation significantly improved cardiac function and reduced infarct size compared with controls. Histological analyses demonstrated enhanced graft survival and neovascularization. Three-dimensional imaging revealed human-derived self-organized vascular networks within engrafted VCMs. Lectin perfusion confirmed functionally perfused, reciprocal host-graft vascular integration, including extension of graft-derived vessels into host myocardium, accompanied by myocardial regeneration. Early graft engraftment was significantly higher in the VCM group than in non-prevascularized controls. ConclusionsSelf-organized prevascularization of hiPSC-derived cardiac microtissues enable active host-graft vascular integration through functional vascular networks, thereby enhancing myocardial regeneration and therapeutic efficacy. This strategy represents an advanced approach for cardiac regenerative medicine. SummaryThis study aimed to develop self-organized, vascularized cardiac microtissues (VCMs) derived from human induced pluripotent stem cells (hiPSCs) and to evaluate their myocardial regenerative potential in a rat model of myocardial infarction (MI). VCMs were engineered from hiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells and cultured under dynamic conditions to enable self-organization and prevascular network formation. One week after MI induction, VCMs were transplanted onto the infarcted myocardium. Cardiac function was evaluated using echocardiography and magnetic resonance imaging. Light-sheet fluorescence microscopy combined with tissue clearing was used to visualize three-dimensional vascular architecture and host-graft integration, while lectin perfusion analysis assessed functional blood flow. VCM transplantation significantly improved cardiac function, increased early graft engraftment, and enhanced neovascularization. Importantly, self-organized human-derived vascular networks within the VCMs actively integrated with the host vasculature, forming functional, perfused host-graft vascular connections. These findings indicate that prevascularized VCMs do not merely survive after transplantation but actively promote vascular integration and myocardial regeneration through functional vascular networks. Together, these results demonstrate that self-organized vascularization markedly enhances graft integration, survival, and therapeutic efficacy, underscoring the clinical potential of VCM-based strategies for cardiac regenerative therapy.

Myocardial Infarction (MI) is a major contributor to morbidity and mortality, wherein blood flow is blocked to a portion of the left ventricle and leads to myocardial necrosis and scar formation. Cardiac remodeling in response to MI is a major determinant of patient prognosis, so many therapies are under development to improve infarct healing. Part of this development involves in vitro therapy screening which can be accelerated by engineered heart tissues (EHTs). Unfortunately, EHTs often over-simplify the infarcted tissue microarchitecture by neglecting spatial variation found in infarcted ventricles. MI results in a spatially heterogeneous environment with an infarct zone composed mostly of extracellular matrix (ECM) and cardiac fibroblasts, contrasted with a remote (non-infarct) zone composed mostly of cardiomyocytes, and a border zone transitioning in between. The heterogeneous structure is accompanied by heterogeneous mechanics where the passive infarct zone is cyclically stretched under tension as the remote zone cyclically contracts with every heartbeat. We present an in vitro 3-dimensional tissue culture platform focused on mimicking the heterogeneous mechanical environment of post-infarct myocardium. Herein, EHTs were subjected to a cryowound injury to induce localized cell death in a central portion of beating tissues composed of neonatal rat cardiomyocytes and cardiac fibroblasts. After injury, the remote zone continued to contract (i.e., negative strains) while the wounded zone was cyclically stretched (i.e., positive tensile strains) with intermediate strains in the border zone. We also observed increased tissue stiffnesses in the wounded zone and border zone following injury, while the remote zone did not show the same stiffening. Collectively, this work establishes a novel in vitro platform for characterizing myocardial wound remodeling with both spatial and temporal resolution, contributing to a deeper understanding of MI and offering insights for potential therapeutic approaches.

BACKGROUNDCartilage damage affects 25 million people globally each year. Tissue engineering strategies such as microfracture and matrix induced autologous chondrocyte implantation (MACI) are currently being used in the clinic; however, they are accompanied by their own limitations such as donor site morbidity, rapid clearance from the injury site, and extensive cost. To overcome these limitations, the tissue engineering field has shown increasing interest in the use of decellularized extracellular matrix (dECM) biomaterials due to their heightened integration with native tissue and regeneration rates. METHODSThe Gottardi Lab has developed a new dECM material sourced from porcine meniscus decellularization (MEND), in which elastin fibers are removed via enzymatic digestion, resulting in channels that can be easily recellularized. RESULTSIn this work we demonstrate that MEND can be seeded with bone-marrow derived mesenchymal stem cells (MSCs), achieving a uniform distribution of cell nuclei throughout the cross section of the scaffold. We also show that MEND retains its native structure in the presence of MSCs and can support chondrogenesis comparably to other commonly used tissue engineering materials such as methacrylated type I collagen and gelatin/hyaluronic acid hydrogels. CONCLUSIONOverall, MEND is a promising new dECM biomaterial for cartilage regeneration.

Epicardial cells are a crucial component in constructing in vitro 3D tissue models of the human heart, contributing to the ECM environment and the resident mesenchymal cell population. Studying the human epicardium and its development from the proepicardial organ is difficult, but induced pluripotent stem cells can provide a source of human epicardial cells for developmental modeling and for biomanufacturing heterotypic cardiac tissues. This study shows that a robust population of epicardial cells (approx. 87.7% WT1+) can be obtained by small molecule modulation of the Wnt signaling pathway. The population maintains WT1 expression and characteristic epithelial morphology over successive passaging, but increases in size and decreases in cell number, suggesting a limit to their expandability in vitro. Further, low passage number epicardial cells formed into more robust 3D microtissues compared to their higher passage counterparts, suggesting that the ideal time frame for use of these epicardial cells for tissue engineering and modeling purposes is early on in their differentiated state. Additionally, the differentiated epicardial cells displayed two distinct morphologic sub populations with a subset of larger, more migratory cells which led expansion of the epicardial cells across various extracellular matrix environments. When incorporated into a mixed 3D co-culture with cardiomyocytes, epicardial cells promoted greater remodeling and migration without impairing cardiomyocyte function. This study provides an important characterization of stem cell-derived epicardial cells, identifying key characteristics that influence their ability to fabricate consistent engineered cardiac tissues.

Summary/AbstractRemodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use GelMA hydrogels with defined stiffness represent a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells. Workflow O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/583012v1_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@127453borg.highwire.dtl.DTLVardef@1325f8dorg.highwire.dtl.DTLVardef@1f2de0org.highwire.dtl.DTLVardef@fd0494_HPS_FORMAT_FIGEXP M_FIG C_FIG

Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. IMPACT STATEMENTLittle is known about the impact of donor age and time in culture on the contraction of the 3D hydrogel-based graft. These results show how cellular phenotypes differ based on donor age and time in culture, which could account for important inconsistencies in biomanufacturing of skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. Future in vivo studies could help determine the best donor age and time in culture for improved wound healing outcomes or more reproducible in vitro testing constructs.