Although embryonic chondrogenesis follows conserved developmental stages across mammalian species, the rate of this process varies significantly. We hypothesized that these varying developmental rates could be normalized and modeled by aligning key developmental milestones of articular cartilage: mesenchymal condensation, chondrogenic differentiation, joint interzone formation, cavitation, and primary ossification center formation. A mathematical model was developed in MATLAB by fitting the developmental curves of these key stages in mice, rats, pigs, horses, and humans. Among various mathematical models, a sigmoid curve model demonstrated the best fit, with the highest R[2] (0.98 ± 0.02) and lowest residual norm (0.36 ± 0.21) after normalizing for gestation period and femoral condylar width to account for species-specific differences. The model's high predictive accuracy (R[2] = 0.98) was further validated using data from rabbits, canines, and sheep. By differentiating the developmental curve equation, we determined the developmental rate for each species. Our results indicate that embryonic chondrogenesis occurs within a relatively narrow window of gestation (11-32%), with rodents exhibiting faster rates than larger animals. This study presents the first mathematical model to quantitatively align embryonic chondrogenesis across mammals. This model has the potential to fill gaps in developmental timelines through rational calculation and by improving the precision of developmental timing, factors that have often been overlooked in the field. As a pioneering effort, this work opens new avenues for cross-species alignment and enhances the accuracy of data related to embryonic chondrogenesis.Impact Statement As the first mathematical model to align chondrogenesis across different mammals, this work has the potential to enhance the consistency and accuracy of data from various species by correcting inaccuracies and supplementing missing information.

Normothermic machine perfusion (NMP) has emerged as a new technique for organ storage before liver transplantation, making viability testing of the organ feasible and possibly allowing for reconditioning of marginal liver grafts. Erythrocytes form a substantial component in the composition of NMP perfusates. The optimal hematocrit (HCT) for preserving organ function during NMP and the effects of the HCT on metabolic graft function and parameters commonly used to determine organ viability are still unclear. We retrospectively analyzed our rat NMP database of 123 perfusions and identified a possible link between HCT and liver damage parameters in 34 cases following a dual perfusion protocol. To further investigate this relationship, we perfused a total of 16 rat livers for 6 h each in our rat liver NMP setup with four different HCTs at 10%, 16%, 22%, and 28%. We performed blood gas analysis and measured common viability and damage parameters such as bile production, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, histological analysis, as well as enzyme-linked immunosorbent assay and colorimetric assay analysis for reactive oxygen species, glycogen content, and hypoxia-inducible factor (HIF), was performed. The perfusate HCT significantly impacted ALT and LDH levels during NMP of rat liver grafts, with lower HCT values corresponding to decreased damage parameters. We investigated differentiating oxygenation levels and identified a possible cause for our observations in HIF-1α stabilization in lower HCT groups. Furthermore, we demonstrate that HCT affects key viability parameters such as lactate, pH levels, and bile production. Our findings illustrate the importance of adequate HCT levels during NMP and propose mechanisms influencing the perfusion outcome.Impact StatementNormothermic machine perfusion (NMP) is becoming an increasingly important tool in liver transplantation. We here show in an exploratory setting the importance of an adequate hematocrit for the outcome of liver graft perfusion and its importance in defining hepatic viability parameters during NMP.

Urethral strictures can cause significant discomfort and progressive urinary tract damage if left untreated. Current reconstructive options, including urethral resection and buccal mucosa grafting, are associated with several limitations such as donor-site morbidity, limited tissue availability, and variable long-term outcomes. To address these challenges, we developed a novel multilayered 3D-bioprinted urethral construct designed to closely mimic the native urethral architecture. Using an Integrated Tissue and Organ Printing System (ITOP) equipped with a rotating mandrel, tubular urethral constructs were fabricated with distinct layers consisting of urothelial cells (UC), basement membrane (BM), smooth muscle cells (SMC), and supportive polycaprolactone (PCL). Autologous UC and SMC isolated from urinary bladder tissue were incorporated into a fibrinogen-based hydrogel bioink. Following in vitro maturation, the constructs were evaluated using viability assays, immunohistochemistry, and biomechanical testing. Live/Dead staining demonstrated an average cell viability of 75% for both UC and SMC populations. Immunostaining confirmed appropriate localization of the different cell types within their respective layers. Tensile testing showed that constructs matured for 14 days developed stable and elastic tissue-like mechanical properties. To further improve construct handling and structural integrity, horizontal reinforcement bands were incorporated into the PCL layer. Using this approach, 4 cm-long and 0.5 cm-diameter urethral constructs with native-like multilayered organization were successfully fabricated. This novel 3D-bioprinting strategy demonstrates strong potential for generating customizable and biologically relevant urethral grafts for future reconstructive applications. Ongoing in vitro optimization and planned in vivo evaluation in a porcine model aim to further validate the structural and functional performance of the construct and support future clinical translation for urethral and other tubular tissue reconstruction.

Severe burn injuries pose significant clinical challenges, with high risks of infection, excessive inflammation, and impaired wound healing. Mesenchymal stem cells (MSCs) have shown regenerative and antimicrobial potential; however, their therapeutic efficacy is constrained by poor survival and engraftment. Here, we demonstrate that priming MSCs with insulin-secreting cells (ISCs) and encapsulating them in hydrogels (HEMI) enhances their regenerative function, leading to accelerated healing of full-thickness burns in a porcine model. By 5 weeks, 80% of HEMI-treated wounds achieved complete closure, compared with 50% of MSC-only wounds and 0% of standard-of-care control wounds. All groups exhibited partial wound closure over time, but no control wounds reached complete closure within the study period. Histological analysis revealed complete epidermal and dermal regeneration with minimal fibrosis in HEMI-treated wounds. Single-cell RNA sequencing and differentially abundant sequencing analysis identified distinct MSC subpopulations whose relative abundance and transcriptional profiles differed between insulin-primed and control MSCs, with insulin priming promoting pathways involved in extracellular matrix stabilization, immune modulation, and oxidative stress resistance. Our findings suggest that insulin priming enhances MSC-mediated tissue repair via paracrine mechanisms, providing a clinically translatable strategy for improving burn treatment and regenerative medicine applications.

To develop bone grafts with superior osteoconductivity, we have fabricated unidirectionally connected porous carbonate apatite (CAp) via gelatin gelation combined with freeze-drying. A composite of calcium carbonate (CaCO3) and gelatin was first gelled at low temperature and then subjected to directional freeze-drying to create well-aligned macroporous channels. The samples were then sintered to remove the organic components and subsequently phosphatized to convert the composition into CAp. The resulting material exhibited a porosity exceeding 80% with fully interconnected unidirectional pores favorable for cell migration and vascular infiltration. Bone regeneration was evaluated by implanting the material into rabbit femoral bone defects; a comparison was also made with nonoriented porous controls. Histological and radiographical analyses demonstrated that the unidirectional porous structure significantly enhanced directional ingrowth of new bone tissue and accelerated early-stage bone formation relative to the control. This study demonstrates that unidirectional porous CAp fabricated via a gelatin-based freeze-casting method is promising for bone regeneration, particularly in clinical applications requiring guided bone formation.

Chronic tympanic membrane (TM) perforation remains difficult to resolve without surgery because of poor intrinsic healing capacity and limitations of passive, nonsurgical materials. Although patch-based approaches are common, most merely provide passive coverage and lack intrinsic therapeutic capability to drive tissue regeneration. We developed and clinically evaluated an extracellular matrix (ECM)-mimetic, drug-free nanopattern guidance (NG) patch for TM repair, marking a paradigm shift from passive coverage to active, biophysically driven regeneration. The NG patch is a biocompatible, implantable scaffold with aligned nanotopography that recapitulates native ECM architecture, coupled with a hydrocolloid adhesive layer to ensure secure placement. In a prospective study of 18 patients, the NG patch achieved a 61% overall healing rate with 50% complete closure and no serious adverse events. In rat models, a single application induced complete healing within 2 weeks. Mechanistic in vitro assays demonstrated that the nanopatterned surface enhances fibroblast adhesion, alignment, and directional migration, supporting organized tissue closure. Collectively, the NG patch offers a minimally invasive approach that activates TM repair through physical cues without exogenous drugs. This first clinical evaluation of an ECM-mimetic nanotopographical scaffold introduces a strategy to redefine the standard of care for chronic TM perforation and related defects.

Pelvic organ prolapse (POP) is an urgent clinical challenge, and traditional surgical treatment is limited by high recurrence rate, erosion, mesh exposure, pain, and other complications. Recently, umbilical cord mesenchymal stem cells (UMSCs) have emerged as a promising modality for tissue regeneration and Pelvic floor repair. This study aimed to assess the effectiveness of polypropylene mesh (Gynemesh[TM] PS) loaded with human UMSCs (HUMSCs) primed with IFN-γ for the treatment of POP. In this study, an ideal IFN-γ concentration was chosen to stimulate HUMSCs and create an enhanced version by incorporating the activated HUMSCs onto the mesh. Balloon distension was used to create a rat POP model, and the mesh was implanted into the vaginal wall of rats. The meshes were removed at week four after implantation in order to assess the treatment effect. The improved mesh displays good biocompatibility. In vitro results indicate that IFN-γ stimulates HUMSC proliferation and enhances paracrine effects. In vitro results also demonstrate that it effectively reduces inflammation and promotes angiogenesis, collagen deposition, and cell proliferation, thereby accelerating tissue repair. Overall, this innovative therapeutic approach offers a new avenue for POP.

In tendons, ligaments, and menisci, collagen fibers running the length of the tissue are the primary source of strength and function. Cells assemble these fibers hierarchically from nanometer-wide fibrils into larger fibers and fascicles, increasing in size throughout development and with mechanical loading. These fibers largely do not regenerate after injury or with repair, limiting recovery options. Engineered replacements are a promising treatment option; however, it remains a challenge to produce the hierarchical collagen fibers essential to tissue strength, limiting their applications. To better repair, regenerate, and engineer these tissues, we must better understand how cells regulate hierarchical fiber formation and maintenance. It is well established that mechanical cues are critical for cell-driven hierarchical fiber formation, which cells sense through several mechanisms, such as integrin-mediated adhesions, cell-to-cell connections, mechanosensitive ion channels, primary cilia, and caveolae. These mechanisms of mechanosensation have been well studied at the fibril scale of collagen organization, but as tissues mature, the loading environment becomes more complex, with cells experiencing increasing secondary shear and compressive loads generated by the developing hierarchical structure. Mechanical cues in this environment are likely sensed through several pathways, each likely playing a role in tissue maturation and injury. There remains a clear gap in our understanding of the later stages of hierarchical fiber formation, which are crucial to better understand since large hierarchical fibers dominate the human musculoskeletal system. Here, we review the role of mechanobiology in hierarchical fiber development and maintenance, and highlight what still needs further research to better regenerate fibers in engineered replacements or in vivo after injury. A better understanding of the mechanisms by which cells form hierarchically organized collagen fibers could help to overcome the limitations of current tissue engineering techniques and help to create functional repairs and replacements.

Because articular cartilage (AC) lacks inherent repair capacity, research has focused on translating tissue-engineered cartilage to the clinic. Toward this, rapid and nondestructive methods would be useful for determining in-process and release characteristics during the manufacture of tissue-engineered products. The current work aims to introduce a Raman-based methodology for nondestructive qualitative and quantitative characterization of tissue development using AC. First, Raman shifts associated with critical biochemical components of AC, with particular emphasis on DNA, glycosaminoglycans (long chains of sugar molecules and a key component of cartilage), total collagen, as well as pyridinoline (a marker of collagen crosslinking and maturation), were collated. Next, verification of the molecular spectroscopic biomarkers was conducted by temporally tracking tissue maturation/development of nascent and mature AC, establishing a temporal reference dataset. Finally, validation was performed by correlating the spectroscopic biomarkers with traditional photometric biochemical assays and mass spectrometry. The results presented here include a Maturity Index for quantification of tissue development/maturation. Strong correlations were found between nondestructive spectroscopic-based measurements and destructive (photometric and mass spectrometric) measurements with high linearity for both nondestructive Raman (R[2] > 0.96) and destructive biochemical (R[2] > 0.97) assays, respectively. Uses of the proposed rapid and nondestructive method include in-line quality assessment (in which the sample is not removed from the process stream) to monitor the manufacturing of tissue-engineered medical products. This study shows that Raman spectroscopy has the capacity of being a powerful tool for nondestructive quality control and assurance in traditional biomanufacturing workflows, and the approach taken here may also be utilized as a template and research tool for studies on the development of other native and engineered tissues.

Volumetric muscle loss (VML) injuries result in an irrecoverable loss of muscle mass and function. VML injury causes loss of both contractile tissue and associated neuromuscular junctions (NMJs). Biosponge (BSG) scaffolds, composed of gelatin, collagen, and laminin-111, have improved recovery following VML. However, improvements in NMJ quantity were not observed. Glial cell line-derived neurotrophic factor (GDNF) is known to promote motor unit survival and stimulate neurite outgrowth. In this work, BSG scaffolds were electrostatically coupled with GDNF via gelatin nanoparticles (GNPs) to support myofiber regeneration and preserve NMJs post-VML in a rodent model. In vitro determination of release kinetics revealed an initial burst release of surface-bound GDNF with almost an equivalent amount of electrostatically bound GDNF retained within the BSG post 1 week of incubation at 37°C in phosphate-buffered saline. To create the VML injury in male Lewis rats (10-12 weeks old), ∼20% of the muscle mass was removed from the tibialis anterior (TA) muscle of both hindlimbs. Relative to BSG + GNP alone, treatment with BSG + GNP + GDNF showed a significant increase (∼25%) in peak isometric torque at 6 weeks postinjury. Qualitative and quantitative histological analysis of NMJs revealed an enhanced overlap between pre and postsynaptic structures in the BSG + GNP + GDNF group. Additionally, the incorporation of GDNF slowed BSG remodeling and degradation. Overall, these results suggest that BSG-mediated GDNF delivery is an effective strategy for mitigating NMJ loss and enhancing muscle recovery following VML.

Bone is a common site of breast cancer metastasis, which dramatically increases fracture risk. Recent in vivo bone metastasis studies show that mechanical loading is osteoprotective; however, little is known about how loading regulates breast cancer cell function in the unique bone mechanical environment, especially in combination with radiotherapy, one of the first-line treatments for advanced breast cancer patients. Here, we characterize the breast cancer cell response to a range of bone-mimicking fluid shears and determine how irradiation further modulates one candidate gene: SERPINE1. We found that irradiation, regardless of dosage, modulates SERPINE1 expression and is sensitive to the timing of administration. Additionally, protein expression of SERPINE1 accompanies a protumorigenic gene expression profile, which is elevated with higher-magnitude fluid shear stresses in a bone-mimicking 3D environment. Thus, we postulate that plasminogen activator inhibitor-1 (PAI-1) (encoded by SERPINE1) is a critical growth factor contributing to osteolytic lesion development in the bone metastatic vicious cycle of breast cancer.

Cartilage and osteochondral disorders pose an increasing clinical, economic, and social challenge. With an aging population, there is a need to develop innovative, nonsurgical strategies for treating defects, fractures, and other osteochondral disorders. Bone implants require surgical intervention, carry a risk of complications, are expensive, and do not always provide comfort to the patient. Alternatively, stimulation of bone regeneration using synthetic peptides is a promising and less invasive option in the treatment of trauma, orthopedics, craniofacial surgery, and dentistry. In the present study, we evaluated the biological effects of two peptides: the novel peptide UG27 and CDP4 derived from the protein Cpne7 (Copine 7), on the activity of adipose tissue-derived mesenchymal stem cells. Using labeling, differentiation, and imaging methods, we demonstrated the effect of UG27 on viability, biomineralization, extracellular matrix, and calcium salt growth in osteocytes. The peptides were immunologically safe and stimulated cell migration without showing any cytotoxic effects. The peptide, UG27, has an active connection with the biomaterial and is a promising compound in bone injury therapies.

Critical-sized bone defects remain a significant clinical challenge, as their size prevents spontaneous healing and necessitates surgical intervention. Although autografts are considered the clinical gold standard, their use is limited by donor site morbidity and tissue availability, while allografts carry risks of disease transmission and long-term failure. In this study, we developed a composite polymer ink for high-resolution digital light processing (DLP) 3D printing of bone tissue engineering scaffolds intended for load-bearing applications. A combination of poly(propylene fumarate) (PPF), poly(caprolactone fumarate) (PCLF), and hydroxyapatite (HA) was formulated to achieve tunable mechanical properties and controlled scaffold architecture. Scaffolds with varying porosities and material compositions were fabricated and evaluated using compressive mechanical testing and finite element modeling to assess structural integrity and stress distributions. In vitro studies using preosteoblast cells demonstrated consistently high cell viability (>75%) across all scaffold designs, with sustained proliferation over 7 days. Notably, scaffold porosity and material composition influenced proliferative responses, with significant increases observed in select formulations. Collectively, these results demonstrate that DLP-printed PPF/PCLF/HA composite scaffolds provide a mechanically viable and cytocompatible platform with tunable properties, supporting their potential utility in bone tissue engineering applications.Impact StatementCritical-sized bone defects lack effective, widely accessible treatment options due to the limitations of current grafting strategies. This work introduces a digitally light processed (DLP) 3D-printable composite scaffold with tunable mechanical properties and architecture suitable for load-bearing applications. By integrating poly(propylene fumarate), poly(caprolactone fumarate), and hydroxyapatite, the platform enables control over structural and biological performance while maintaining high cytocompatibility. These findings highlight a scalable and customizable approach to bone tissue engineering that may reduce reliance on traditional grafts and improve outcomes in complex bone repair.

The Coronavirus Disease of 2019 (COVID-19) pandemic caused by SARS-CoV-2 resulted in widespread mortality, economic strain, and healthcare system disruption, highlighting the need for effective strategies to address viral threats. Tissue engineering and biomaterials can contribute significantly in advancing our understanding and treatment of respiratory viral infections by developing physiologically relevant in vitro models, controlled and targeted drug delivery systems, and effective next-generation vaccine platforms. Over the last 5 years, tissue-engineered cell-based models, including organ-on-a-chip systems, have improved our understanding of viral entry, immune response, and therapeutic efficacy in pulmonary, cardiac, neurological, and vascular tissue. Biomaterial-based delivery systems have enhanced the targeting, bioavailability, and sustained delivery of therapeutics such as antivirals, monoclonal antibodies, nucleic-acid-based vaccines, and anti-inflammatory drugs, while also reducing or eliminating system toxicity and our reliance on intravenous administration. Advancements in vaccine platforms using lipid nanoparticles, protein scaffolds, and vesicular or cell-based platforms are in different phases of development to stimulate more robust responses against emerging SARS-CoV-2 variants. Collectively, these advancements highlight the influence of tissue engineering and biomaterials in SARS-CoV-2 research and treatment. This review provides an overview of recent developments in in vitro modeling, drug delivery systems, and vaccine platforms, highlighting their future role in improving clinical outcomes, managing variants, and preparing for potential future pandemics, including addressing challenges in infrastructure such as limited access to high-containment biosafety facilities used to study emerging infectious pathogens.Impact StatementDuring the COVID-19 pandemic, we introduced basic tenets of virology to a tissue engineering audience and proposed different areas in which the field could contribute to developing diagnostics and therapeutics for respiratory viral infections. In this 5-year update, we highlight how tissue engineers and biomaterial scientists contributed tools to dissect virus pathophysiology and deliver therapies such as mRNA vaccines to prevent mortality during the pandemic. With the emergence of new variants and the threat of new respiratory viral pandemics, tissue engineers can continue to play important roles in virology.