Tissue Engineering Part A

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.

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.

Hypertrophic cartilage is a promising bone repair strategy by producing a mineralizable matrix that transitions to bone through endochondral ossification. Current approaches require large cell numbers and costly recombinant factors to induce chondrogenesis. Here, we developed a composite granular scaffold using photocrosslinkable alginate microgels, cell-secreted decellularized extracellular matrix (dECM), and mesenchymal stromal cell (MSC) spheroids under dynamic compressive loading for hypertrophic cartilage formation. Incorporation of dECM into MSC spheroids enhanced expression of chondrogenic markers and supported the hypertrophic phenotype, evidenced by increased VEGFA and SPP1 expression and ALP activity. Dynamic loading further increased spheroid sprouting and scaffold mineralization. Histology confirmed mature hypertrophic cartilage conducive to bone formation. Upregulation of hypertrophic and osteogenic markers was associated with YAP1 activation, linking compressive loading to mechanotransduction to drive hypertrophic cartilage formation. These results demonstrate that dynamic compressive loading, cell aggregates, and scaffold granular macroporosity synergistically yield hypertrophic cartilage.

Critical sized craniomaxillofacial bone defects do not heal naturally and often exhibit chronic inflammatory responses that restrict regeneration. It is increasingly apparent that biomaterials must facilitate dynamic crosstalk between immune cells, such as macrophages, and osteoprogenitors to resolve inflammation and accelerate regeneration. Here, we evaluate interactions between macrophages in a neutral (M0) or pro-inflammatory (M1) state with mesenchymal stem cells (MSCs) in a basal or licensed state within a mineralized collagen scaffold. We reveal that MSC-macrophage crosstalk influences significant changes in osteoprogenitor cell differentiation and immune cell polarization. Notably, crosstalk between MSCs and macrophages drives an early-stage inflammatory response, which enhances the immunomodulatory activity of MSCs via secretion of IL-6, an effect that is heightened for already licensed MSCs. The presence of macrophages in the co-cultures upregulated osteogenic (ALPL, BMP2, COL1A2, and RUNX2) and angiogenic genes (ANGPT1) in basal MSC groups. Further, MSC-macrophage interactions subsequently drive increased M2-like macrophage polarization as early as 7 days of culture, as indicated by surface marker expression. These findings show that biomaterial scaffolds can be leveraged as mediators of MSC-mediated immunomodulation with an emphasis on achieving early-stage pro-inflammatory phenotypes that drive subsequent macrophage polarization and markers of increased regenerative potency.

Macrophages play a central role in early immune response after injury that can shape the success or failure of craniomaxillofacial (CMF) bone repair. While mineralized collagen glycosaminoglycan (GAG) scaffolds have been developed to support osteogenesis, here we define how scaffold pore size, pore alignment, and glycosaminoglycan (GAG) composition influence human monocyte-derived macrophage polarization. We establish flow cytometry, secretome, and gene expression benchmarks to assess primary macrophage polarization toward M1 versus M2 phenotypes in response to cytokine cocktails in 2D culture and 3D scaffolds. We then define the kinetics macrophage polarization in response to scaffold pore architecture and composition in the absence of exogenous cytokines. All scaffold variants support an early pro-inflammatory response followed by a shift toward M2-like phenotypes over seven days reflected by increased CD206 expression, secretion of pro-healing factors such as CCL18, and upregulation of M2a- and M2c-associated genes. Anisotropic scaffolds with smaller pores more robustly drove angiogenic and extracellular matrix related gene expression as well as earlier emergence of M2-like phenotypes. Scaffold GAG chemistry provided an additional tuning mechanism, with chondroitin-6-sulfate variants promoting the greatest late-stage M2 surface marker expression, heparin variants accelerating early M2 and pro-angiogenic phenotypes, and chondroitin-4-sulfate variants dampening both M1 and M2 phenotypes at early timepoints. These findings demonstrate that mineralized collagen scaffolds intrinsically guide macrophage polarization toward pro-regenerative states but that scaffold structure and composition can be used to shape the kinetics and intensity of these responses. These insights provide a critical foundation for immuno-instructive biomaterial designs that enhance CMF bone repair.

Human induced pluripotent stem cell (hiPSC) technologies offer human-relevant cardiac models for biomedical applications. However, workflows for differentiation of cardiac stromal cells and fabrication of engineered heart tissue (EHT) commonly rely on animal serum, contrary to growing policy demands to reduce use of these products. Applying marker analysis via COL1A, DDR2 and GATA4 for cardiac fibroblasts or CD31, CD34 and CD144 for endothelial cells, we tailored Panexin, a defined serum substitute, to support high efficiency differentiation of cardiac stromal lineages to 85% purity without additional purification steps. We evaluated fabrication of EHTs using hiPSC-cardiomyocytes only (monoculture) or further combined with cardiac fibroblasts and endothelial cells (triculture; 70%:15%:15%, respectively). Panexin poorly supported fabrication and contractility of EHTs, a finding unaltered by modulating spontaneous cardiac myofibroblast activation via TGF{beta} inhibition. In contrast, human serum enabled fabrication of mono- and tri-culture EHTs, wherein constructs made without TGF{beta} signalling inhibition delivered the strongest contractile forces (up to 0.25 mN) and exceeded comparator tissues engineered using animal serum. Our data show that iterative evaluation of serum substitutes, human serum, cell combinations and signalling pathway modulators can mitigate use of animal serum for functional EHT generation, aligning with the UK governments roadmap for alternative methods.

ObjectiveEvaluate the effects of bioabsorbable magnesium wires on dermal wound healing and tissue regeneration in a murine full-thickness wound model. Approach6 mm diameter stented dorsal skin wounds were created in C57BL/6J mice and treated with implanted WE43B magnesium alloy wires or PBS control. Wound healing was evaluated on days 7 and 28 by histology, immunohistochemistry, and micro-CT. Finite element analysis modeled mechanical strain distribution due to wire degradation during healing. ResultsAt day 7, magnesium wire-treated wounds showed 100% improved granulation tissue formation, reduced inflammation (37% fewer CD45+ leukocytes and 37% fewer F4/80+ macrophages), increased neovascularization (91% more CD31+ lumens), and 74% more nerve bundles. Improved wound closure (mean difference -1.48 mm) did not reach statistical significance (d = 1.06). By day 28, magnesium-treated wounds showed improved collagen organization and normalized epidermal thickness. The increase in dermal appendages (247%), and vascular density (41%) did not reach statistical significance. Micro-CT confirmed progressive wire degradation. Modeling revealed that degrading wires dynamically altered strain gradients in healing tissue, thereby modulating the spatial mechanical cues that govern fibroblast migration and extracellular matrix (ECM) remodeling. InnovationMagnesium is an essential trace element involved in cellular processes critical to wound repair, including angiogenesis, nerve growth, inflammation modulation, and ECM remodeling. Previous magnesium delivery systems incorporated polymers or other confounding materials that degrade rapidly. We directly applied bioabsorbable pure magnesium metal to provide sustained ion release and favorable mechanical properties to support regenerative healing. ConclusionBioabsorbable magnesium wires support regenerative wound healing by reducing inflammation, enhancing neovascularization, and promoting favorable ECM remodeling without adverse inflammatory reactions. These findings provide a strong rationale to harness magnesium metal use in wound healing applications.

PurposeFibrosis is the pathological remodeling of the extracellular matrix (ECM) that is largely orchestrated by activated fibroblasts. The mechanical properties of the ECM change drastically during fibrosis, and fibroblasts become increasingly activated by mechanical environments that mimic the properties of fibrotic tissues. While the effects of increased elastic modulus (stiffness) on fibroblast activation have been well-studied, the impact of changes in viscoelasticity are less clear. Here, we sought to determine how fibroblast activation is altered by changes in viscoelasticity in a three-dimensional, fibrillar microenvironment. MethodsWe employed 3D alginate collagen I hydrogels with independently tunable stiffness and stress relaxation rates. Dermal fibroblasts were encapsulated in hydrogels with four distinct mechanical profiles (soft: 3 kPa or stiff: 10 kPa, fast stress relaxing: {tau}1/2 {approx} 160 s or slow stress relaxing: {tau}1/2 {approx} 1600 s). We assessed fibroblast activation by changes in cell morphology, expression of key activation markers, and evidence of ECM remodeling. ResultsFibrillar alginate collagen networks enhanced fibroblast spreading, -smooth muscle actin stress fiber formation, and fibroblast activation protein- expression in matrices that were slow relaxing or stiff. The presence of the fibrillar network further enhanced fibroblast activation, independent of the changes driven by matrix viscoelasticity. ECM remodeling was also promoted by slow relaxing matrices, with increased fibronectin deposition and more remodeling of the local collagen fiber network. ConclusionsOur results demonstrate that fibroblast activation is highly responsive to matrix stress relaxation rate, and that models incorporating fibrillar, viscoelastic networks can provide new insights into the role of ECM mechanics driving fibroblast activation.

Mechanical homeostasis indicates the remarkable ability displayed by cells in tissues to maintain their mechanical properties near a stable homeostatic set-point. Experimental investigations and theoretical studies indicate that mechanical stress represents a key homeostatic target that stromal cells, such as fibroblasts, seek to maintain by tuning the intracellular structure and by remodeling the extracellular matrix. Much of what is known about mechanical homeostasis of tissues under tension, or tensional homeostasis, is based on experiments on tissue equivalents, that is fibroblast-populated collagen gels. However, existing platforms used to investigate tensional homeostasis cannot infer mechanical stress dynamically. Here we developed an integrated biomechanical bioreactor combining force sensing with confocal microscopy to dissect the mechanobiological mechanisms of tensional homeostasis. We used our novel platform to test the hypothesis that fibroblasts maintain a constant state of stress across varying collagen concentrations. Contrary to this assumption, synchronized force and imaging measurements revealed that stress is not constant but rather elevated at low collagen concentrations, where fibroblast contraction drives earlier collagen fiber alignment and greater tissue compaction. Conversely, force generation and -SMA expression increase with increasing collagen concentration, accompanied by modest transcriptional changes. However, at the highest collagen concentration, this homeostatic balance is disrupted, with lower force generation and -SMA expression, as gene expression shifts toward VEGFC-mediated autocrine survival signaling. These findings demonstrate that tensional homeostasis emerges from a dynamic balance between cellular contractility and extracellular matrix densification rather than stress maintenance, and reveal that excessive matrix density disrupts this balance by triggering a pro-survival response.

Spinal cord injury (SCI) results in loss of sensory and motor function below the level of damage, with chronic injuries presenting unique challenges for regenerative therapies. While multichannel biomaterial interventions have shown promise in promoting axonal regeneration, circuit restoration, and motor recovery in acute SCI, achieving similar outcomes in chronic injury models remains challenging due to a combination of intrinsic and extrinsic factors. These include the reduced capacity of the neuronal cell body to sustain a growth-activated state and the formation of a physical and chemical barrier at the injury site, preventing axonal growth. To address these challenges and promote motor recovery after chronic injury, we investigated the combinatorial effect of two regenerative approaches: 1) the implantation of poly (lactide-co-glycolide) (PLG) biomaterial bridge to guide axonal growth through the injury site, and 2) the delivery of Epothilone B (EpoB), a microtubule stabilizer that strengthens axons to promote regrowth. We used a transgenic mouse model that selectively expresses a red fluorescent protein variant (tdTomato) reporter throughout the corticospinal tract (CST) under control of the Crym promoter (Crym-tdTomato). We demonstrated that the combination of bridge implantation 60 days after surgical hemisection at C5 with EpoB improved locomotor function. At 12 weeks post-bridge implantation, immunohistology revealed axon regeneration in mice receiving implantation, but not EpoB or no-implant controls. The addition of EpoB significantly increased the volume of both total and CST axons regenerating through the biomaterial channels. Diffusion tensor magnetic resonance imaging (DTI) analysis identified enhanced fractional anisotropy (FA), axial diffusivity (AD), and mean diffusivity (MD) in the bridge region in the combination treatment group, consistent with new intact axons. Furthermore, EpoB enhanced the myelination of regenerated axons in the bridge. Finally, we investigated the proteomic profile of corticospinal neurons ipsilateral and contralateral to the SCI lesion and bridge, comparing the effect of EpoB treatment. Mass spectrometry-based analysis of laser-captured cells in this paradigm identified activation of a regeneration program by corticospinal neurons. These findings present a novel approach to enhance regenerative neural repair and locomotor recovery in chronic SCI.

In vitro reconstruction of human tissue microenvironments that integrate native biochemical and biomechanical cues is essential for disease modelling, regenerative medicine, and personalized therapeutic approaches. However, most currently available engineered matrices fail to recapitulate the complexity and tissue specificity of the human extracellular matrix (ECM). To address this limitation, we developed a novel hydrogel derived from decellularized human adipose tissue (atdECM) designed to support three-dimensional culture of human cells. The decellularization and delipidation processes were first validated, and the biochemical composition and biomechanical properties of atdECM were comprehensively characterized. Human pancreatic organoids were then cultured within atdECM hydrogel, and their structural organization and transcriptional profiles were analyzed and compared with those obtained in Matrigel, the current gold-standard matrix for organoid culture. Proteomic and cytokine analyses demonstrated efficient decellularization while preserving collagen-rich ECM architecture and a diverse repertoire of soluble bioactive factors. AtdECM exhibited physiological stiffness and retained tissue-specific extracellular cues. Pancreatic organoids cultured in atdECM displayed morphological similarities with those grown in Matrigel but exhibited transcriptional profiles more consistent with physiological epithelial homeostasis, with reduced activation of inflammatory and stress-related pathways. Altogether, these findings indicate that atdECM provides a human-derived, tissue-relevant, and permissive microenvironment for human organoid generation. This platform represents a promising alternative to Matrigel for studying human tissue biology and for developing physiologically relevant in vitro models.

Treating extensive full-thickness burn wounds remains difficult in clinical practice because available donor skin is often limited, the risk of infection is high, and many standard dressings do not perform well when defects are large or structurally complex. These limitations have shifted attention to decellularized extracellular matrix (dECM) scaffolds, which can provide physical coverage while preserving biochemical cues that may support tissue repair. Based on this rationale, we designed a decellularization method that improves reagent penetration to produce a full-thickness porcine decellularized small intestine (dSI) scaffold for use in burn wound coverage. The protocol removed most cellular material while leaving low levels of detergent residue, and it maintained the native three-layer structure of the intestinal wall. Most key ECM components, such as collagen and glycosaminoglycans, were also retained. In this study, the dSI showed several properties relevant to burn care, capacity to absorb large amounts of fluid, water vapor transmission rates similar to those reported for skin, and resisted microbial penetration in vitro. From a mechanical standpoint, the scaffold retained anisotropic behaviour and remained stable under cyclic loading. This pattern indicates that it could withstand repeated deformation instead of acting like a fragile membrane. Degradation tests under enzymatic and oxidative conditions indicate that the material breaks down in a controlled way over a period that appears consistent with typical wound-healing timelines. In vitro assays indicated that the scaffold was cytocompatible, as human dermal fibroblasts and keratinocytes both attached to its surface and continued to proliferate. Cell responses differed depending on surface orientation, suggesting that preserved intestinal layers may shape cell behaviour in ways that are often missing in thinner or more uniform matrices. Overall, full-thickness dSI appears to act as a biologically active scaffold and shows mechanical properties that exceed those of many currently used burn dressings.

Tissue engineering scaffolds such as collagen-based biomaterials have long been used to mimic native extracellular matrix in a wide range of regenerative applications. Their high porosity, tunable degradation and mechanics, and cell adhesion sites provide a structure upon which cells can grow and differentiate, while they also have the potential to act as carriers for loading and release of biomolecules to aid in healing. Here we describe the inclusion of a second lyophilization step in the fabrication process to enable improved loading efficiency of bone morphogenic protein 2 as well as increased ease of end-user handling. We report mineralized collagen scaffolds demonstrate maintained microarchitecture and mechanical properties post-relyophilization with reduced variability in biomolecule loading. Relyophilization allows consistent loading and release profiles and suggests the potential to improve the translational potential of collagen scaffold biomaterials for regenerative medicine applications.

Facial development is highly sensitive to genetic and environmental perturbation, with craniofacial malformation associated with over one-third of congenital birth defects. The face arises during an early and largely inaccessible window of embryonic development, with a large contribution from transient and multipotent cranial neural crest cells (CNCCs). Assessment of the molecular and cellular mechanisms driving normal and disordered human facial development therefore relies greatly on the use of in vitro cellular models. Here, we adapted a neurosphere-based CNCC differentiation protocol to facilitate robust quantification of early specification and migration events. Introduction of single-cell aggregation with arrayed plating enabled standardisation of neurosphere size, growth and patterning. Inclusion of fibronectin coating enhanced the efficiency of neurosphere attachment and synchronicity of CNCC migration timing. To demonstrate application of the Array-CNCC method, we developed a strategy for mosaic co-culture, which can facilitate differentiation of wildtype untreated cells directly alongside cells exposed to distinct drug treatments or genetic alterations. Finally, we present a screening approach which we use to test the impact of distinct extracellular matrix components on neurosphere morphology, CNCC migration and gene expression. Together, the Array-CNCC method is highly amenable to quantitative phenotyping and screening approaches, enabling enhanced craniofacial disease modelling with both cellular and molecular readouts.

Critical-sized craniomaxillofacial (CMF) defects affect the skull, face, and jaw, arising from conditions such as cleft palate, oncologic resections, and high energy impacts, and due to their large size and irregular geometry, cannot heal naturally by the body, thus requiring surgery. The field of biomedical research has long recognized the need to develop higher order biomaterial model systems for improved disease characterization and translational therapeutic/material progress. There is, however, difficulty in developing these workflows at the scale of conventional two-dimensional cell culture screening systems while simultaneously approaching a level of complexity necessary to consider translation to in vivo animal models. Here, we describe a three-dimensional (3D), in vitro model system to investigate the impact of stromal cell migration from one microenvironment to another at a medium-throughput scale. Importantly, we demonstrate the ability of this workflow to be utilized as a screening tool for collagen-based biomaterial motifs of interest in promoting craniomaxillofacial bone defect repair. Taken together we provide a strategy for interpreting cell-to-cell, cell-to-material, and material-to-material interactions across a multidimensional spatiotemporal scale.

Extracellular matrix (ECM) proteins assemble to form a heterogeneous connective scaffold that supports cells. Physical interactions between cells and the matrix regulate cellular behaviors and influence subsequent tissue construction. However, there is a lack of fundamental understanding regarding the contributions of individual native ECM proteins to the matrix. This gap arises from the need for nanoscopic characterization, which operates on a much smaller length scale than typical assessments in cell and tissue cultures, as well as in tissue reconstruction and clinical implantation. This study aims to systematically investigate how individual ECM proteins affect lipid membranes structurally and mechanically, and how these influences regulate cell migration. Results from Langmuir isotherm analysis, X-ray reflectivity measurements, and cell scratch assays demonstrate that strong collagen adsorption on the membrane surface disrupts lipid packing. However, its rigid network provides a sturdy scaffold for cell adhesion, thereby enhancing cell attachment and promoting cell migration. In contrast, elastin has a minimal structural or mechanical impact on the membrane during both adsorption and compression, but it benefits cells by facilitating migration and reducing the risk of infection. Fibronectin, on the other hand, exhibits complex mechanical responses to compression, characterized by significant structural rearrangements that occur during adsorption. This strong interaction with the membrane can result in excessively high adhesion forces, ultimately limiting cell motility. These findings lay the foundation for the design of artificial scaffolds that can manipulate cellular responses, a critical step toward advancing regenerative medicine and tissue engineering. SignificanceFabricating extracellular matrix (ECM) scaffolds from cells offers advantages over traditional approaches, such as decellularized tissues, which face donor limitations, and artificial scaffolds, which may hinder cellular communication. However, the slow harvesting process of cell-derived ECM has limited its clinical applications. This research is part of a larger mission to engineer ECM prescaffolds on lipid carriers tailored to cell requirements, enhancing ECM production and regulating cell behavior. The first step involves systematically analyzing the structural and mechanical effects of ECM on lipid membranes and how these effects regulate cellular behavior. This work confirms distinct characteristics of ECM proteins, advancing fundamental understanding of cell-matrix interactions and paving the way for scaffold engineering.

Articular cartilage repair is limited by the poor regenerative capacity of chondrocytes and their rapid dedifferentiation during in vitro expansion. This study investigated whether a decellularized and lyophilized cell-secreted matrix (CSM) could function as a bioactive material to regulate cell behavior, promote chondrogenic differentiation, and attenuate or reverse chondrocyte dedifferentiation without exogenous growth factor supplementation. CSM was generated from rabbit auricular perichondrial cells, decellularized, lyophilized, and characterized by histology, biochemical assays, and proteomic analysis. The resulting matrix was enriched in structurally and functionally relevant extracellular matrix proteins, including collagens, fibronectin, fibrillin, proteoglycans, and matricellular regulators, with minimal intracellular contamination and good batch-to-batch reproducibility. Functionally, CSM supported robust adhesion and proliferation of allogeneic and xenogeneic cells. Human articular chondrocytes cultured on CSM exhibited enhanced proliferation, sustained expression of cartilage-specific markers, and preserved type II collagen production over serial passages compared with standard plastic culture. CSM also promoted chondrogenic differentiation of human progenitor cells and partially reversed established chondrocyte dedifferentiation, as evidenced by increased expression of COL2A1, ACAN, SOX9, and COMP, with reduced COL1 expression and no induction of hypertrophic markers. These findings demonstrate that lyophilized CSM is a stable, off-the-shelf biomaterial capable of directing chondrocyte fate through intrinsic matrix-derived cues, highlighting its potential for cartilage tissue engineering and cell manufacturing applications.

Fibrotic responses at biomaterial-tissue interfaces limit implant integration and regenerative healing, yet how the interaction between biomaterials and the extracellular matrix (ECM) regulates fibroblast activation remains poorly understood. Granular hydrogels including microporous annealed particle scaffolds (MAP) reduce fibrosis, while chemically and mechanically matched hydrogels do not, suggesting a dominant role for scaffold architecture. In this model, MAP scaffolds allow collagen infiltration and form physically continuous composites, whereas hydrogels exclude collagen and generate interfacial slip planes. To isolate how biomaterial architecture influences extracellular matrix (ECM) integration and fibroblast activation, we developed a reductionist in vitro model that integrates collagen type I with either microporous annealed particle (MAP) scaffolds or chemically and mechanically matched bulk hydrogels. This physical integration stabilizes collagen architecture, limits fibroblast-mediated matrix compaction, suppresses contractility, and attenuates myofibroblast transition. Fibroblasts in mechanically integrated environments exhibit reduced expression and nuclear localization of NF-{kappa}B and are enriched for quiescent phenotypes. Together, these findings identify biomaterial-ECM physical continuity as a design principle for limiting fibrotic signaling.

Elastic and viscoelastic properties of extracellular matrices (ECM) are known to regulate cellular behavior and mechanosensation differently, with implications for morphogenesis, wound healing, and pathophysiology. Most in vitro cellular processes, including cell migration, are studied on linear-elastic substrates to mimic extracellular matrices. However, most tissues are viscoelastic and display a loss modulus (G) that may be 10-20% of their storage modulus (G) under biophysically relevant conditions. Recent research has shown that cells can distinguish between elastic and viscoelastic ECM, leading to alterations in their cellular morphology, migration rates, and contractility. Here, we present a protocol for creating PAH-based model ECMs that enables the fabrication of viscoelastic substrates with storage moduli similar to those of their elastic counterparts. To explore how G influences epithelial cell mechanobiology, we fabricated tunable viscoelastic model ECMs with G of 3 kPa, 8 kPa, and 12 kPa, and for each, independently tuned G values to approximately 300 Pa, 500 Pa, and 700 Pa, respectively. We found that A549 cells cultured on stiff elastic model ECMs migrated [~]30% slower and formed larger focal adhesions compared to their viscoelastic counterparts. Conversely, A549 cells on intermediate viscoelastic model ECMs exhibited a [~]54% reduction in migration speed, with no significant difference in focal adhesion size relative to their elastic counterparts. These findings highlight the complex interplay between substrate (ECM) elastic and viscoelastic properties in regulating epithelial cell mechanobiology and emphasize the importance of time-dependent matrix mechanics in governing epithelial responses.

Acellular Adipose Tissue (AAT) is an off-the-shelf, cadaveric adipose-derived ECM-based biomaterial for soft tissue reconstruction. AAT has been validated preclinically to promote angiogenesis and adipogenesis and demonstrated safety, biocompatibility, and tolerability in a Phase I study. In this study we report the findings for the first ten patients in the Phase II study for permanent reconstruction of modest soft tissue defects. AAT promoted macrophages, CD3+ T cells, and CD34+ progenitor activity. Multiplex immunofluorescence staining using the PhenoCycler (formerly CODEX) imaging platform found that AAT can induce tertiary lymphoid structures (TLS). Nanostring GEOMx spatial transcriptional data analysis found significant differential gene expression between neighboring tissues with EGR1, MCL1, and NR4A1 upregulated in AAT. These genes have roles in angiogenesis, anti-apoptotic processes, and promotion of anti-inflammatory genes, respectively. AAT promoted anti-fibrotic CD74+ adipose-derived stromal cells, confirmed by immunofluorescence staining. Our findings demonstrate that AAT promotes angiogenesis, adipogenesis, and anti-fibrotic remodeling.