Tissue Engineering Part A

A persistent challenge in cartilage tissue engineering is identifying cell sources capable of regenerating cartilage with native-like properties. Variability in chondrogenic potential among human- and animal-derived cells complicates graft development and disease modeling. Here we benchmarked ten chondrogenic cell sources under standardized culture conditions using hyaluronan-alginate biomaterials and implementing a functionality index combining histological and mechanical metrics referenced to native tissue. Mesenchymal stem cells and fetal chondroprogenitors demonstrated superior regenerative capacity, achieving 103% and 95% of native cartilage functionality, respectively. In contrast, adult articular chondrocytes from healthy and osteoarthritic donors produced fibrocartilaginous tissue with functionality scores below 50%. These findings provide a quantitative framework to guide cell source selection and optimize cartilage graft standardization for clinical applications and physiologically relevant disease models, addressing regulatory demands for product characterization.

Successful cartilage repair remains one of the most significant challenges in the musculoskeletal field. Microfracture (MFx), a form of marrow stimulation, remains the predominant repair technique, but it exhibits routine failure due to inadequate defect fill and inferior fibrotic tissue formation. Whereas current strategies focus on augmenting MFx with scaffolds and bioactive factors, the potential to target the MFx clot itself and use the capabilities of this dynamic environment to guide MFx repair remains largely unexplored. We verified that MFx contraction and fibrosis hinder repair success in minipigs and become evident as early as one week in multiple animal models. Therefore, our objective was to investigate and direct microenvironmental interactions in the MFx clot to promote volumetric maintenance and reprogram cells from a fibrotic to more chondrogenic phenotype. Extracellular control of cell-environment interactions, through fibrinogen augmentation or anti-fibrinolytic treatment, limited contraction but had no effect on or even exacerbated the fibrotic susceptibility of marrow-derived cells (MDCs). Intracellular control of microenvironmental interactions, through modulation of the Rho-ROCK pathway, drove TGF-{beta}3 activity of MDCs along a "chondro-fibro axis". In particular, treatment with the ROCK inhibitor Fasudil drove TGF-{beta}3-treated cells away from a myofibroblast phenotype and towards chondrogenesis. Short-term Fasudil treatment prevented TGF-{beta}3-driven macroscale clot contraction and enhanced cartilage-specific matrix deposition in vitro. In a pilot rat study, this combination treatment improved GAG deposition and better protected surrounding cartilage. These findings suggest that Rho-ROCK modulates TGF-{beta} signaling along this chondro-fibro axis and its precise control could be the key to promoting precise and volumetric cartilage repair through microenvironmental interactions.

Synthetic blood contacting devices frequently fail due to the lack of requisite biochemical and biomechanical cues needed to support transanastamotic endothelialization. During transanastomotic endothelialization, endothelial cells experience dynamic changes in extracellular mechanical cues as they migrate from compliant native vessels onto stiffer blood contacting device surfaces. However, how substrate stiffness and mechanical memory from prior mechanical environments influence temporal integrin remodeling and downstream endothelialization processes necessary to establish a stable endothelial layer remains poorly understood. In this study, human coronary artery endothelial cells (HCECs) were cultured on substrates spanning physiologically relevant stiffnesses to determine how substrate mechanics regulate collagen binding integrins and endothelialization. Increasing substrate stiffness promoted time dependent upregulation of 2{beta}1 integrin expression, whereas 1{beta}1 expression remained unchanged. Enhanced 2{beta}1 expression on stiff substrates was accompanied by increased vinculin associated focal adhesion maturation and accelerated endothelialization, characterized by increased proliferation, migration, and progression to confluence prior to reaching quiescence after 1 week. To better model transanastomotic migration and investigate mechanical history effects, cells initially expanded on compliant hydrogels were transferred to stiff substrates. Although these cells exhibited transient reductions in 2{beta}1 expression at early timepoints compared with tissue culture polystyrene expanded controls, no persistent differences in focal adhesion maturation, proliferation, migration, confluence, or quiescence were observed. Collectively, these findings demonstrate that substrate stiffness is a primary regulator of the early endothelialization processes required to establish a stable endothelial monolayer, whereas the influence of mechanical history is transient and ultimately superseded by the current mechanical environment. These findings also identify 2{beta}1 mediated mechanotransduction as a potential design target for blood contacting biomaterials that promote rapid endothelialization while supporting long-term endothelial cell quiescence.

Introduction Autologous and allogeneic chondrocyte cell sheet transplantation has shown safety and potential efficacy for cartilage defects associated with knee osteoarthritis. Although polydactyly-derived chondrocyte (PDC) cell sheets have been used clinically for allogeneic transplantation, a scalable and reliable alternative cell source is needed. This study aimed to establish a proof-of-concept protocol for generating chondrocyte cell sheets from clinical-grade human induced pluripotent stem cells (iPS cells). Methods Cartilaginous tissue was generated from human iPS cells, and isolated chondrocytes were used to fabricate iPS cell-derived chondrocyte sheets. In Phase 1, sheets were produced in high-serum medium under normoxic, hypoxic, or hypoxic/high-seeding-density conditions and compared with PDC sheets using a three-dimensional in vitro model and xenogeneic orthotopic transplantation in an immunodeficient rat model. In Phase 2, sheets were produced in low-serum medium under hypoxic/high-seeding-density conditions and evaluated for cartilage-related gene expression, secreted factors, cell surface marker expression, and in vivo cartilage repair. Results In Phase 1, hypoxic/high-seeding-density culture enhanced in vitro chondrogenic properties. However, after transplantation, iPS cell-derived chondrocyte sheets formed fibrotic repair tissue, whereas PDC sheets generated hyaline-like cartilage. In Phase 2, low-serum culture increased expression of cartilage-related genes, including collagen type II alpha 1 (COL2A1) and SRY-box transcription factor 9 (SOX9), enhanced secretion of transforming growth factor beta 1 and melanoma inhibitory activity, and upregulated CD9, CD26, CD56, CD201, CD227, and GD2. Low-serum iPS cell-derived chondrocyte sheets regenerated hyaline cartilage at 4 weeks, and the repair tissue was maintained at 12 weeks without bone formation. Conclusions A robust fabrication protocol enabled human iPS cell-derived chondrocyte sheets to regenerate hyaline-like cartilage in vivo. These findings support their potential as a scalable cell source for clinical cartilage repair.

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

Osteochondral repair requires restoration of zonally organised articular cartilage and subchondral bone, yet translatable implants rarely reproduce this spatial complexity. Here, we developed an off-the-shelf, cell-free multilayer scaffold comprising a superficial 2% (w/v) articular cartilage extracellular matrix (AC-ECM) phase, an intermediate 5% AC-ECM phase and a basal 6% bone ECM (BN-ECM) phase. The scaffold formed continuous interfaces, displayed regionally distinct pore sizes and resisted permanent deformation during cyclic compression. In vitro, constructs seeded with caprine mesenchymal stromal and articular cartilage progenitor cells supported cell expansion and the accumulation of sulfated glycosaminoglycan- and collagen-rich matrix, with regional differences in collagen I, II and X deposition. Following eight weeks of subcutaneous implantation, cell-seeded scaffolds contained more collagenous matrix than unseeded controls, while vascularisation preferentially localised to the BN-ECM phase. The scaffold was then evaluated against empty defects in a caprine osteochondral model for six months. Scaffold treatment significantly improved macroscopic and histological repair, increased chondral tissue fill (~60% versus ~40%), limited cartilage-like tissue extension into the subchondral region and generated a more native-like superficial collagen organisation. Repair tissue further exhibited greater collagen II immunoreactivity, increased ACAN and COL2A1 expression and reduced COL1A2 expression relative to empty defects, although deeper bone repair was not significantly improved. These findings demonstrate that tissue-specific ECM layering can spatially guide endogenous repair and substantially improve cartilage restoration without exogenous cells or growth factors in a clinically relevant large-animal model, while identifying subchondral bone regeneration as the remaining design challenge for complete osteochondral repair.

Current treatment of orthopedic ligament injury relies principally on non-regenerative materials and surgical repair rather than strategies that promote intrinsic tissue regeneration. This study evaluated whether biologic augmentation with XWRAP, a human amniotic membrane allograft, combined with a cryopreserved amniotic-fluid-derived biologic containing micronized human amnion membrane, promotes regeneration of ligament architecture in a rabbit medial collateral ligament (MCL) gap-defect model. Standardized 4-mm mid-substance MCL gap defects were created in New Zealand White rabbits (n=3) and either treated with the amniotic membrane/amniotic-fluid biologic combination or left untreated, with intact contralateral or adjacent MCL serving as native controls; ligaments were evaluated by transmission electron microscopy at 2 and 6 weeks post-procedure, with collagen fibril diameter quantified from calibrated high-magnification micrographs. Treated defects developed significantly larger mean collagen fibril diameters than untreated defects at both time points (75 vs. 50 nm at 2 weeks and 88 vs. 62 nm at 6 weeks; p<0.0001 for each comparison), reaching approximately 90% of native, uninjured ligament fibril diameter (98 nm) by 6 weeks. Treated defects also developed a broader, bimodal fibril-diameter distribution resembling native ligament architecture, whereas untreated defects remained characterized by a narrow, unimodal population of small-diameter fibrils typical of scar-associated repair. These findings indicate that biologic augmentation with XWRAP combined with an amniotic-fluid-derived biologic promotes a regenerative, rather than purely reparative, healing response following ligament injury, progressively restoring collagen fibril architecture toward that of native tissue in this preclinical model.

The ability to generate vascularized tissue constructs supplied by surgically accessible vessels remains a major limitation in reconstructive tissue engineering. Previous work demonstrated that positioning porous biomaterials around a vein can induce extensive vascular sprouting and generate a vascularized tissue volume without creation of an arteriovenous loop. However, this phenomenon has principally been demonstrated using rigid calcium-phosphate scaffolds, limiting its direct relevance to soft-tissue reconstruction. Here, we investigated whether a flexible decellularized dermal matrix could similarly be vascularized around a single unmodified vein and whether autologous dermal fibroblasts could further enhance this response. Decellularized rat dermis, with or without autologous dermal fibroblast seeding, was implanted either subcutaneously in the dorsal region or around the femoral vein for 8 weeks. Following Microfil perfusion, three-dimensional micro-CT demonstrated an extensive branching vascular network within perivenous constructs. Vessel volume relative to tissue volume was 0.2 +/- 0.1% in subcutaneous constructs without fibroblasts (n = 11) and 0.6 +/- 0.2% with fibroblasts (n = 9), compared with 6.+/- 1.4 % in perivenous constructs without fibroblasts (n = 10) and 7.3+/- 1.2% with fibroblasts (n = 10). Histology and immunohistochemistry similarly showed greater cellular repopulation and substantially higher CD31- and -SMA-positive vessel densities following perivenous implantation. Integrated two-way ANOVA of the same 8-week data confirmed a dominant effect of implantation site across the vascular outcomes, while fibroblast seeding significantly affected vascular volume, dermal remodeling, and nuclear density. The effect of fibroblast seeding on vascular volume was site dependent, with a significant implantation site fibroblast interaction. At 8 weeks, the vascularized dermal construct was completely dissected free from the surrounding tissues while maintaining its connection to the femoral vein. A small superficial incision into the isolated dermis produced visible bleeding, supporting an acute functional connection of the intradermal vascular network to the host circulation while the femoral venous connection was preserved. After flap elevation, perivenous constructs retained substantially greater vascular volume and showed more complete surface healing and organized skin architecture than subcutaneous constructs. Fibroblast seeding accelerated wound closure, while its effect on post-elevation vascular volume depended on implantation site: VV/TV decreased in subcutaneous constructs but increased in perivenous constructs. These findings show that single-vein vascularization can extend from rigid calcium-phosphate biomaterials to a compliant biological extracellular-matrix scaffold and may provide a simplified axial vascularization strategy without arterialization or incorporation of an arterial vessel.

Neural progenitor-stem cells (NPSCs) reside in mechanically dynamic brain microenvironments and give rise to neurons, astrocytes, and oligodendrocytes. Although recent studies have shown that matrix viscoelasticity can influence neural maturation and neurogenic differentiation, its role in primary NPSCs beyond neurogenesis remains less defined. How matrix viscoelasticity or stress relaxation regulates primary NPSC stemness, neuronal and glial differentiation, and the associated matrix-cell mechanotransduction pathways are not well understood. Here, we use alginate hydrogels with independently tunable stiffness and stress relaxation properties to investigate how matrix stress relaxation regulates the fate of primary subventricular zone (SVZ)-derived NPSCs in 3D. The results suggested that matrices with faster stress relaxation enhances stemness maintenance, radial glial-like marker expression, and differentiation of NPSCs toward neuronal, astrocytic, and oligodendrocytic lineages in the corresponding biochemical environments. In mixed neuronal/astrocytic differentiation conditions, fast-relaxing matrices preferentially promote neuronal differentiation. Mechanistically, NPSC responses to matrix stress relaxation involve integrin-mediated adhesion, actomyosin contractility, actin polymerization, and Piezo1 activity, with distinct contributions across differentiation lineages. Together, these findings reveal how matrix stress relaxation regulates primary NPSC stemness and multilineage differentiation through multiple mechanotransduction pathways in 3D.

Synthetic grafts remain ineffective for small-caliber vascular applications due to thrombosis and intimal hyperplasia. To address these limitations, our lab designed a multilayer graft consisting of a hydrogel coating that promotes post-implantation endothelialization and an electrospun mesh that matches arterial mechanical properties. Damage-resistant hydrogels were engineered using a double-network system composed of polyether urethane diacrylamide and N-acryloyl glycinamide to enhance fracture resistance through hydrogen bonding. In this study, we utilized redox initiation to apply conformal, durable hydrogels to electrospun grafts. Bioactivity wa introduced using streptococcal collagen-like proteins containing 1{beta}1 and 2{beta}1 integrin-binding motifs, enabling selective cell-material interactions that support endothelialization while preserving acute thromboresistance. To establish the feasibility of these grafts as off-the-shelf devices, we evaluated coating integrity and bioactivity retention following sterilization and dynamic physiological loading. Sterilized composites exhibited surgically-associated damage resistance, indicating that sterilization did not compromise hydrogel durability. Coating integrity and bioactivity were also preserved after six weeks of physiological loading. Acut thromboresistance was supported by both static platelet adhesion assays and dynamic whole-blood bioreactor studies using heparinized blood, with low platelet adhesion observed relative to ePTFE. Finally, a pilot ovine carotid model demonstrated successful surgical handling and sustained graft patency. Collectively, these results highlight the promise of multilayer vascular grafts as durable, thromboresistant conduits for small-diameter vascular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/741222v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14ec910org.highwire.dtl.DTLVardef@17259e8org.highwire.dtl.DTLVardef@6cb611org.highwire.dtl.DTLVardef@1251d7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.

Abstract Chondrocytes in native articular cartilage are enclosed within a collagen VI (COL VI)-rich pericellular matrix (PCM), forming functional units known as chondrons. However, enzymatic isolation disrupts the PCM, and the functional consequences of its loss and restoration remain poorly understood. In this study, primary human chondrocytes were cultured in alginate beads to promote PCM reconstitution and subsequently recovered as reconstituted chondrons. Chondrocytes and reconstituted chondrons were encapsulated in gelatin methacryloyl hydrogels and compared under inflammatory and mechanical stimulation. Alginate preconditioning generated chondron-like units with a distinct COL VI-positive PCM that was retained after transfer to three-dimensional culture. Under inflammatory conditions, reconstituted chondrons exhibited reduced inflammatory, catabolic, neuroinflammatory, and angiogenic responses compared with isolated chondrocytes at both gene and protein levels. Under interleukin-1{beta} stimulation, mechanical loading further increased inflammatory gene expression in chondrocytes in a donor-dependent manner, whereas responses remained comparatively limited in reconstituted chondrons. Conditioned medium from reconstituted chondrons was also associated with lower capsaicin- and potassium chloride-evoked calcium responses in human induced pluripotent stem cell-derived sensory neurons than corresponding chondrocyte-conditioned medium. These findings demonstrate that reconstitution of a COL VI-rich PCM restores a chondron-like pericellular microenvironment that attenuates inflammatory activation and buffers load-associated inflammatory amplification, while exploratory sensory neuron experiments suggest downstream modulation of neuronal responsiveness. PCM restoration may therefore provide a promising strategy for cartilage tissue engineering and regenerative applications.

Three groups of touch-spun 3D scaffolds with uniform fiber diameters and varying interfiber spacing were studied to identify optimal conditions for NIH/3T3-GFP fibroblast growth. All scaffold groups exhibited higher cell viability relative to control levels after 14 days of culture, with the highest viability observed for scaffolds with a 50.61 {+/-} 15.35 m interfiber spacing. Although cell counts indicated comparable cell growth across groups, scaffolds with medium and large interfiber spacing demonstrated an improved capacity to sustain viable cells without necrotic core formation. These findings provide insight into the optimal structural design of touch-spun 3D scaffolds for tissue engineering applications.

Introduction: Placental tissue allografts are widely utilized in the management of chronic wounds, including diabetic foot ulcers. In addition to serving as wound coverings, these products may influence wound repair through extracellular matrix architecture and biologically active signaling molecules native to human amniotic tissue. Manufacturing and processing methodologies vary considerably across commercially available products and may substantially impact biologic activity, tissue remodeling, and wound-healing trajectories. In this study, a rodent wound-healing model was utilized to compare a proprietary processed placental tissue allograft with a conventionally processed placental tissue comparator. Methods: Two arms of a five-arm pilot rodent wound-healing study were evaluated to compare a proprietary processed human amniotic tissue allograft against a conventionally processed comparator; the remaining arms evaluated test articles under development and will be reported separately. Male rats (n=8 per arm), matched for weight, age, and diet, each received two full-thickness dorsal excisional wounds approximately 10 mm in diameter and 2 mm deep. Test articles were reapplied upon resorption over a 14-day period. At Day 14, sections were scored semi-quantitatively (0-4 Severity Injury Score) across histologic and immunohistochemical parameters spanning epidermal repair, inflammation, angiogenesis, proliferation, and remodeling. Cranial and caudal wounds were averaged within each animal, retaining the animal as the experimental unit. A Day 14 Histology Resolution Score was derived as a signed composite of pro- and anti-regenerative markers and evaluated alongside percent wound closure. Inferential statistics (Welch ANOVA with Games-Howell post hoc) were derived from the full five-arm cohort (n=40). Results: Animals treated with the proprietary processed allograft showed more advanced progression toward wound resolution than those receiving the conventionally processed comparator, across both surface and tissue-level endpoints. Mean animal-level wound closure at Day 14 was 95.2% with the proprietary processed allograft versus 89.9% with the conventional comparator. The integrated Day 14 Histology Resolution Score, a composite of epidermal repair, inflammation, angiogenesis, proliferation, and remodeling endpoints was 8.94 versus 5.13, corresponding to a shift from the poor-resolution range into the partial-resolution range on the pre-defined interpretive scale. Welch ANOVA across the full five-arm cohort (n=40) detected a significant overall effect of treatment on this composite (p = 0.010). The pairwise contrast between these two arms did not reach significance under Games-Howell correction across ten comparisons (p = 0.337); at n=8 per arm the study was not powered to resolve individual pairwise differences, and the observed standardized difference was large (Hedges' g = 0.93). Injury endpoints were consistently lower in the proprietary processed allograft: 69% lower acute inflammation, 60% lower dermal necrosis, approximately 57% lower epithelial erosion, 46% lower ulceration, and 42% less hemorrhage. Importantly, these reductions in residual tissue injury occurred while maintaining robust granulation tissue formation, angiogenesis, collagen remodeling, cellular proliferation, and macrophage recruitment. Discussion: These findings suggest that processing methodologies can materially influence the biologic performance of placental tissue allografts. The proprietary processed allograft was associated with lower residual tissue injury and greater regenerative and reparative activity relative to a conventionally processed comparator. These observations support the concept that placental tissue allografts should not necessarily be viewed as biologically equivalent and may have important implications for clinical outcomes and product selection in wound care.

Oncofetal fibronectin (oFN) is a known biomarker of cancer progression and is involved in events such as epithelial mesenchymal transition and multidrug resistance. It is also understood that fibronectin (FN) and its splice variants are differentially arranged by cancer associated fibroblasts (CAFs) to support cancer cell proliferation and migration. Contrastingly, little is understood about the mechanobiology of oFN, a post-translationally modified FN isoform defined by O-glycosylation of a specific threonine residue. To investigate the architectural features of the oFN matrix, CAFs were simulated by culturing human mammary fibroblasts (HMFs) on stiff polyacrylamide gels (20 kPa) in media collected from HCC-1937 (primary site breast adenocarcinoma) and MDA-MB-231 (metastatic breast adenocarcinoma) cells. As a control, HMFs were also cultured on softer gels representing healthy adipose tissue (2 kPa) in media not exposed to cancer cells. After immunocytochemical analysis, tumor conditioned HMFs displayed an expected CAF-like phenotype that was most prominent with MDA-MB-231 conditioned media. Matrices produced by tumor-conditioned HMFs contained more oFN with structural features mostly following that of the overall dense, aligned FN matrix. Cancer cells reseeded on their corresponding decellularized tumor-associated matrices had greater F-actin/oFN colocalization than control matrix. The lack of distinct oFN topographical cues may indicate that oFN O-glycosylation is more directly involved in biochemical signaling than mechanosignaling through matrix architecture. Together, this study established a platform for investigating stromal oFN which revealed that tumor-conditioned media sourced from cancer cells with different metastatic potential influences HMF activation, leading to distinct total FN and thus oFN matrix microarchitecture features.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, affecting 50-60 million people annually. Cascading secondary injury mechanisms, including oxidative stress and neuroinflammation, impair motor and cognitive function, while current therapies often manage symptoms rather than restore lost neurological function. This study investigated an ultrasound-activated piezoelectric hydrogel scaffold composed of barium titanate nanoparticles (BTNPs), sodium alginate (SA), and polyethylene oxide (PEO). The hypothesis was that TBI flies receiving the scaffold and ultrasound would display a higher climbing assay pass rate than TBI flies receiving no treatment or BTNPs alone. TBI was induced in Drosophila melanogaster with a high-impact trauma (HIT) apparatus. Treatments were applied by opening and resealing the fly head cuticle, followed by ultrasound stimulation. Motor recovery was quantified with a climbing assay across four groups (N=10 flies per group), which measured the percentage of flies that could cross a 5 cm mark within 30 seconds. Assay validity was supported by a statistically significant difference between flies without TBI and those with TBI using Mann-Whitney U testing (p=0.0035). Although TBI treatment groups did not differ significantly (p>0.05), median pass rates increased from injury control (35%) to BTNPs (45%) to scaffold (50%). However, high inter-trial variation, a 45.2% procedural mortality rate, and small sample size limited statistical power. These results suggest that piezoelectric electrospun hydrogel scaffolds may be a pathway for safer and more biocompatible restoration after TBI, but provide insufficient evidence to conclude that the scaffold significantly improved motor recovery.

Background: Post-operative uterine adhesions remain a major clinical challenge, often leading to infertility and severe pelvic pain. Current anti-adhesion barriers lack the mechanical strength, tissue specificity, or bioactivity required for optimal regeneration of uterine tissue. To address these limitations, we developed a biomimetic, multilayer scaffold that replicates the structural and functional complexity of the myometrial and serosal layers of the uterus by combining a tunable polycaprolactone (PCL) mesh with a bioactive hydrogel derived from decellularized uterine extracellular matrix (dUECM) reinforced with sodium alginate (Alg). Materials and Methods: Composite scaffolds were fabricated using near-field melt electrowriting (MEW) to create precision-engineered PCL meshes with various infill angles (90{degrees}, 60{degrees}, 45{degrees}, and 30{degrees}), followed by impregnation with an alginate-dUECM hydrogel. A comprehensive physicochemical characterization was conducted using Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), and contact angle measurements. Mechanical properties were evaluated in both dry and wet states via uniaxial tensile testing. Swelling and degradation were assessed over 10 days. hTERT-HM cells were used for in vitro evaluation through Live/Dead staining, MTT, SEM, and immunocytochemistry for -SMA and DAPI. Results: The successful incorporation of the hydrogel into the PCL mesh was confirmed by the results obtained from FTIR and TGA analyses; these findings were further supported by contact angle studies, which showed a marked increase in hydrophilicity, resulting in a reduction of the static contact angle from 113 {+/-} 4{degrees} in the pure PCL to 60 {+/-} 12{degrees} in the hydrogel-infiltrated samples in the 30{degrees} group. Mechanical testing showed that the wet composite scaffolds maintained higher stiffness than hydrated native uterine tissue. Cell culture assays confirmed excellent cytocompatibility and proliferation, especially in 30 infill architecture. Immunofluorescence revealed strong -SMA expression and cytoskeletal organization, indicating phenotypic maturation of uterine smooth muscle cells for all groups. Conclusion: Among all groups, the 30 MEW mesh impregnated with Alg-dUECM hydrogel demonstrated the most balanced combination of mechanical resilience, degradation profile, wettability, and cellular compatibility. This structure most closely recapitulates the biological characteristics of uterine outer myometrium tissue, positioning it as a highly promising scaffold for regeneration and biofunctional anti-adhesion barrier applications.

Decellularized extracellular matrix (dECM) scaffolds are increasingly used in regenerative medicine, yet the extent to which processed placental dECM retains properties capable of influencing cellular responses remains unclear. This study combines functional cell assays with deep learning-enabled quantitative imaging to determine how dehydrated placental ECM regulates cellular behavior across multiple human cell lineages. Human dermal fibroblasts, cardiac fibroblasts, and osteoblasts were cultured on dehydrated placental ECM or standard cell culture surfaces and assessed for cell attachment, viability, extracellular matrix production, and nuclear morphology. Placental dECM supported attachment and survival across all three cell types, while Pro-Collagen I Alpha 1 secretion varied by cell lineage relative to negative controls. To identify structural responses associated with scaffold culture, an automated imaging pipeline combining Cellpose-based nuclear segmentation with nuclear morphometric analysis was used to quantify nuclear area, eccentricity, and circularity. Quantitative profiling of hundreds of nuclei revealed scaffold-dependent remodeling of nuclear morphology that was not apparent by conventional microscopy. Cells cultured on placental dECM exhibited reduced nuclear area and increased nuclear eccentricity, while cardiac fibroblasts and osteoblasts showed alterations in nuclear circularity. These lineage-dependent morphological responses demonstrate that placental dECM provides more than a permissive substrate for cell attachment and is associated with measurable changes in cellular architecture following processing. Together, these findings support the biological relevance of processed placental dECM as a regenerative biomaterial and demonstrate the utility of quantitative single-cell morphometric analysis for detecting cell-material interactions that may not be apparent through qualitative imaging alone, guiding the rational design of regenerative therapies.

Mechanical characterization of biomaterial scaffolds is essential to evaluate their capacity to meet the functional demands of target tissues in tissue engineering and regenerative medicine applications. Scaffolds designed to interface with living tissues must support the transmission of mechanical cues to resident cells and stimulate mechanosignaling pathways that are essential to their function. In joints, bone and cartilage cells act as primary mechanosensors, converting mechanical stimuli into biochemical signals that regulate tissue homeostasis and remodelling. Therefore, evaluating cellular mechanoresponses to scaffold-transmitted compression in vitro can inform the development of functional tissue-engineered constructs. For example, poly({epsilon}-caprolactone) (PCL) scaffolds are highly relevant for bone and cartilage tissue engineering due to their biocompatibility, stable mechanical properties and slow degradation. Here, we applied a custom-built device to study compression-induced mechanosignaling in MC3T3-E1 pre-osteoblast cells. The device is composed of a polydimethylsiloxane (PDMS) pillar, a force-sensing load cell, and a piezoelectric linear track. A protocol is described in which MC3T3-E1 cells are repeatedly compressed, while in parallel live tracking of force measurements and live imaging of intracellular calcium dynamics in MC3T3-E1 cells are recorded. PCL scaffolds fabricated by melt electrowriting (MEW) were subsequently integrated into the platform. Scaffold-transmitted compression triggered dynamic increases in cytosolic calcium; in MC3T3-E1 cells located directly under the PCL microfibers, but also in cells located in the interfiber spaces. This device and workflow facilitate in vitro investigations of real-time cellular mechanoresponses to dynamic compression applied with biomaterial scaffolds, and provides a testing platform for evaluating the mechanotransductive properties of scaffolds intended for tissue engineering applications.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.