Fibrosis causes altered tissue structure and function in multiple organs due to a complex interplay between inflammatory cells, myofibroblasts, and extracellular matrix (ECM) components. While it is known that T cells play a role in tissue fibrosis, it remains unclear how they modulate cellular interactions to activate fibrogenesis. Since conventional monolayer cell cultures do not mimic the tissue complexity and cellular heterogeneity in the fibrotic tissue environment, there is a need to bridge the gap between monolayer cultures and in vivo animal studies of fibrosis by providing a more predictive 3D model for preclinical drug screening and mechanistic studies of fibrotic diseases. We have developed 3D skin-like tissues harboring blood-derived human T cells that offer a model to better understand the role these cells play in the pathogenesis of tissue fibrosis. In the current study, we constructed skin-like tissues harboring T cells, fibroblasts, macrophages, and keratinocytes and analyzed them using tissue analysis and single-cell RNA sequencing (scRNA-seq). Skin-like tissues constructed with fully autologous cells (donor-matched fibroblasts and T cells) or nonautologous cells (mismatched fibroblasts and T cells) derived from patients with scleroderma (SSc) demonstrated normal distribution of tissue markers of epithelial differentiation and proliferation. T cells in these tissues were viable and functional as seen by elevated IL-6 production by enzyme-linked immunosorbent assay, expression of alpha smooth muscle actin in fibroblasts, and scRNA-seq. We used scRNA-seq to identify five distinct T cell subpopulations: CD8 T cells (identified by KLRK1 and CD8A), proliferating CD4 T cells (identified by PCNA, MKI67, and CD4), activated CD4 T cells (identified by IL2RA, RORA, and CD4), naïve CD4 T cells (identified by CCR7 and CD4), and Th17 CD4 T cells (identified by KLRB1, RORA, IL2RA, and CD4). Fabrication of complex 3D tissues are an important step toward establishing tissue engineering approaches to study fibrosis in multiple diseases, including SSc, idiopathic pulmonary fibrosis, as well as liver and kidney fibrosis. Understanding the roles of T cells in the ECM environment and their interactions with fibroblasts will support the development of novel treatments to reverse fibrosis and restore normal tissue and organ function.
Owing to the high occurrence of tissue detachment during the sample preparation process, the application of multiplex immunohistochemistry (mIHC) technology is limited in the field of fragile tissue samples, such as tendons, ligaments, and bones. To optimize a method for preparing sections for mIHC on fragile tissue samples, taking the human anterior cruciate ligament as an example, paraffin-embedded continuous sections with a thickness of 4 μm were divided into two groups: baking groups underwent routine section processing, and after being mounted on glass slides, they were baked at 65°C for 4 h, 8 h, or 24 h; ultraviolet (UV) photosensitive cross-linking groups used adhesive-coated slides for mounting and were directly subjected to UV light-induced cross-linking, with the cross-linking time set at 0 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min, respectively. After deparaffinization and rehydration, we simulated the microwave step, which was most likely to cause tissue detachment during the mIHC experimental procedure, and then, the sections were stained with eosin. Finally, using the optimal cross-linking time selected from the UV cross-linking groups, mIHC staining of tendon and bone tissues was performed. After deparaffinization and rehydration, both groups were able to maintain the integrity of the tissue structure, except for the slides from the UV-sensitive cross-linking 0 s group, which showed complete tissue detachment. Following the seventh microwave treatment, the baking groups presented significant tissue detachment. The UV cross-linking groups were affected by the cross-linking time, and severe tissue detachment occurred with cross-linking times of 20 s, 40 s, and 5 min, whereas the tissues cross-linked for 1 min, 2 min, 3 min, and 4 min all maintained complete tissue morphology and structure. Finally, after 2 min of cross-linking, the results of spectral imaging revealed that the tissue morphology and structure were intact. During the process of mIHC staining, photocrosslinking with UV irradiation for 1-4 min effectively preserves the integrity of the tissue morphological structure.
The demand for effective vascular grafts continues to increase with the increasing prevalence of cardiovascular disease. The decellularized human umbilical vein (HUV) offers a promising scaffold for cellular removal, extracellular matrix (ECM) preservation, and balanced biocompatibility. To evaluate the efficacy of a sodium dodecyl sulfate (SDS)-based decellularization protocol for HUV preparation intended for future stem cell seeding. HUVs were obtained from four full-term cesarean deliveries (37-39 weeks; total length 210 cm) and assigned to a control group or five SDS-treated groups (0.5% SDS for 6, 12, and 24 h; 1% SDS for 12 and 24 h). Following decellularization, scaffolds were freeze-dried, gamma-sterilized, and analyzed for DNA content, cell viability (MTT assay), collagen/elastin retention, and histological and ultrastructural changes. The 0.5% SDS for 6 h group achieved optimal results, with significant cell clearance (1.82 ± 1.75 vs. 5.55 ± 2.49 cells/field; p = 0.009) and DNA reduction (59.6 ± 22.62 vs. 258.09 ± 107.63 ng/µL), while maintaining cell viability (87.05 ± 17.14% vs. 84.79 ± 14.3%; p = 0.781). Collagen (20.80 ± 5.26% vs. 27.34 ± 5.34%; p = 0.152) and elastin (21.13 ± 5.02% vs. 24.47 ± 8.01%; p = 0.437) retention were comparable to controls. A 0.5% SDS 6 h decellularization protocol effectively removed cellular components while preserving ECM integrity and biocompatibility, offering a balanced and feasible approach for preparing HUV scaffolds for vascular tissue engineering applications.
Scaffold-free tissue engineering strategies using cellular aggregates, microtissues, or organoids as "biological building blocks" could potentially be used for the engineering of scaled-up articular cartilage or endochondral bone-forming grafts. Such approaches require large numbers of cells; however, little is known about how different chondrogenic growth factor stimulation regimes during cellular expansion and differentiation influence the capacity of cellular aggregates or microtissues to fuse and generate hyaline cartilage. In this study, human bone marrow mesenchymal stem/stromal cells (MSCs) were additionally stimulated with bone morphogenetic protein 2 (BMP-2) and/or transforming growth factor (TGF)-β1 during both monolayer expansion and subsequent chondrogenic differentiation in a microtissue format. MSCs displayed a higher proliferative potential when expanded in the presence of TGF-β1 or TGF-β1 and BMP-2. Next, the chondrogenic potential of these human MSCs was explored in a medium-high throughput microtissue system. After 3 weeks of culture, MSCs stimulated with BMP-2 during expansion and differentiation deposited higher levels of glycosaminoglycans (GAGs) and collagen, while staining negative for calcium deposits. The fusion capacity of the microtissues was not impacted by these different growth factor stimulation regimes. After 3 weeks of fusion, it was observed that MSCs stimulated with TGF-β1 during expansion and additionally with BMP-2 during chondrogenic differentiation deposited the highest levels of sulfated GAGs. No increase in type X collagen deposition was observed with additional growth factor stimulation. This study demonstrates the importance of carefully optimizing MSC expansion and differentiation conditions when developing modular tissue engineering strategies (e.g., cellular aggregates and microtissues) for cartilage tissue engineering applications.
Bioengineering aims to develop biomaterials that closely mimic the native extracellular matrix (ECM) to support tissue regeneration. This study presents a detailed protocol for producing hydrogels derived from decellularized bovine placental cotyledons. Bovine placentas at 4-5 months of gestation (n = 10) were subjected to vascular perfusion with increasing concentrations of sodium dodecyl sulfate (0.01-1%) and Triton X-100 (1%), which effectively removed cellular components. Decellularization efficacy was confirmed by histological (hematoxylin and eosin and 4',6-diamidino-2-phenylindole [DAPI] staining), molecular, and structural analyses, including residual genomic DNA quantification averaging 9.1 ng/mg of dry tissue. The ECM scaffolds were enzymatically digested using 0.1% (w/v) pepsin in 0.01 M HCl and reconstituted with sodium alginate at concentrations of 5%, 8%, 10%, and 12% (w/v). Crosslinking was achieved with 1% calcium chloride. Among the tested formulations, hydrogels containing 12% alginate demonstrated greater mechanical stability and preserved three-dimensional architecture, including interconnected porosity, as evidenced by scanning electron microscopy. Cytocompatibility was evaluated by culturing canine adipose-derived mesenchymal stem cells on both decellularized biomaterials and hydrogels. DAPI staining revealed nuclei after 7 and 25 days of culture, indicating cell presence and distribution throughout the constructs. These results indicate that bovine cotyledon-derived ECM hydrogels maintain structural and biochemical features favorable for cell interaction and may serve as adaptable platforms for tissue engineering, dermal repair, and three-dimensional cell culture.
In tissue engineering and regenerative medicine, biomaterials have transitioned from passive structural supports to dynamic platforms capable of actively modulating regenerative microenvironments. Their tunable physical and chemical properties, combined with the capacity for controlled release of bioactive signals or extrinsic stimuli, allow precise regulation of stem, immune, and tissue-specific cell behaviors. However, single-modality of signal remains insufficient to recapitulate the multifactorial and spatiotemporally coordinated processes underlying complex tissue regeneration. From a biomaterial perspective, we proposed biomaterial-based multimodal tissue engineering strategy, focusing on the synergy of multimodal cell-regulatory signals for enhanced tissue regeneration. These multifunctional biomaterials serve as advanced artificial regenerative niches, capable of delivering coordinated multimodal signals to precisely guide cellular behavior and tissue formation. Inspired by bionic design principles, decoding the compositional, structural, mechanical, and biological parameters of the native extracellular matrix-and elucidating their regulatory effects and molecular mechanisms on cellular activities-has informed the development of multifunctional biomaterials for tissue regeneration. Key material properties-spanning mechanical, topological, biochemical, and dynamic characteristics-can be strategically engineered to function as distinct yet complementary regulatory signals in this multimodal approach.
Tissue-engineered vascular grafts (TEVGs) are emerging as promising alternatives to synthetic grafts, particularly in pediatric cardiovascular surgery. While TEVGs have demonstrated growth potential, compliance, and resistance to calcification, their functional integration into the circulation, especially their ability to respond to physiological stimuli, remains underexplored. Vasoreactivity, the dynamic contraction or dilation of blood vessels in response to vasoactive agents, is a key property of native vessels that affects systemic hemodynamics and long-term vascular function. This study aimed to develop and validate an in vivo protocol to assess the vasoreactive capacity of TEVGs implanted as inferior vena cava (IVC) interposition grafts in a large animal model. Bone marrow-seeded TEVGs were implanted in the thoracic IVC of Dorset sheep. A combination of intravascular ultrasound (IVUS) imaging and invasive hemodynamic monitoring was used to evaluate vessel response to norepinephrine (NE) and sodium nitroprusside (SNP). Cross-sectional luminal area changes were measured using a custom Python-based software package (VIVUS) that leverages deep learning for IVUS image segmentation. Physiological parameters including blood pressure, heart rate, and cardiac output were continuously recorded. NE injections induced significant, dose-dependent vasoconstriction of TEVGs, with peak reductions in luminal area averaging ∼15% and corresponding increases in heart rate and mean arterial pressure. Conversely, SNP did not elicit measurable vasodilation in TEVGs, likely due to structural differences in venous tissue, the low-pressure environment of the thoracic IVC, and systemic confounders. Overall, the TEVGs demonstrated active, rapid, and reversible vasoconstrictive behavior in response to pharmacologic stimuli. This study presents a novel in vivo method for assessing TEVG vasoreactivity using real-time imaging and hemodynamic data. TEVGs possess functional vasoactivity, suggesting they may play an active role in modulating venous return and systemic hemodynamics. These findings are particularly relevant for Fontan patients and other scenarios where dynamic venous regulation is critical. Future work will compare TEVG vasoreactivity with native veins and synthetic grafts to further characterize their physiological integration and potential clinical benefits.
Evaluating the complex, three-dimensional (3D) architecture of de novo angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for complete visualization of the microcirculatory system is needed. We present an integrated workflow for high-resolution 3D visualization of neovascularization within arteriovenous (AV) loop-based tissue constructs in a rat model. An intravascular perfusion with a cationic near-infrared fluorescent dye, MHI148-polyethylenimine, was used to 3D label the patent vasculature. Following perfusion-fixation and explantation, the construct was rendered optically transparent using an ethyl cinnamate-based clearing protocol. The fluorescent signal was then imaged using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation. Our workflow successfully achieved high-contrast, 3D visualization of the microvascular network, allowing for whole-mount and segmental analysis of the vascular tree. At day 7, imaging delineated solely the AV loop axis while by day 28, a dense and complex, interconnected capillary plexus from the central axis demonstrated a progressive neovascularization. Downstream processing compatibility was confirmed through successful rehydration and 3D nuclear counterstaining. This workflow offers a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs, overcoming key limitations of existing techniques.
Decellularization does not completely remove the matrix-bound α-Gal epitopes in porcine acellular dermal matrix (pADM), and the presence of residual α-Gal epitopes could elicit adverse immunological reactions and cause potential early failure of xenografts. The present study had evaluated the effectiveness of decellularization and α-galactosidase treatment to eliminate the matrix-bound α-Gal epitopes in pADM, as well as the effect of tissue form (intact pADM vs. microparticle). Decellularization eliminated ∼80% of α-Gal epitopes in porcine dermis, and pADM retained ∼20% of the matrix-bound α-Gal epitopes. While Aspergillus α-galactosidase and Coffea α-galactosidase both hydrolyzed the terminal alpha-galactosyl moiety from oligosaccharides, only Coffea α-galactosidase was effective in eliminating the matrix-bound α-Gal epitopes in intact pADM. Aspergillus α-galactosidase did not work for intact pADM, even at an enzyme activity more than an order of magnitude higher than that of Coffea α-galactosidase used. The different efficacy between Aspergillus α-galactosidase and Coffea α-galactosidase was associated to the accessibility to the matrix-bound α-Gal epitopes in intact pADM. When intact pADM was micronized into fine microparticles, Aspergillus α-galactosidase and Coffea α-galactosidase eliminated the matrix-bound α-Gal epitopes equally well. Thus, the tissue form had significant influence on the efficacy of enzymic cleavage. The findings of the study offer valuable insight for enzyme selection and process development for efficient α-Gal antigen reduction in xenogeneic grafts or tissue scaffolds.
Advanced tissue-engineered respiratory models are essential for studying drug or cosmetic toxicity, infection biology and xenobiotic metabolism. Here, we investigated a polyamide 6 (PA6)-based electrospun stromal scaffold as a substitute for porcine-derived small intestinal submucosa (SIS) to build human airway mucosa tissue models at the air-liquid interface. We demonstrate that the porous PA6 scaffold supports extracellular matrix production by human nasal fibroblasts and facilitates the complete differentiation of respiratory epithelial cells to the mucociliary phenotype. These models reduce reliance on animal-derived materials, improve reproducibility, and minimize potential interference from animal-derived antigens and pathogens. Both PA6- and SIS-based models promote fibroblast migration, epithelial differentiation, and the expression of key xenobiotic metabolizing enzymes. They exhibit comparable epithelial barrier integrity and susceptibility to influenza A virus infections. These findings establish PA6 scaffolds as a suitable, animal-free alternative to the SIS to build human airway mucosa tissue models.
Vascular tissue engineering technology uses tubular viscoelastic materials as intermediaries to transmit the mechanical stimuli required for the construction of vascular grafts. However, most existing studies rely on elastic models, which fail to capture the time-dependent nature of viscoelastic materials. Moreover, the long fabrication cycles, high costs, and complex parameter measurements in tissue engineering pose significant challenges to experimental approaches. There is thus an urgent need to develop a viscoelastic mechanical model that combines physical interpretability, computational efficiency, and predictive accuracy, enabling precise characterization of material responses and unified quantification across experimental platforms. Here, we propose an error-corrected linear solid (ECLS) model with an embedded correction term to address the predictive deviations of conventional models in nonlinear viscoelastic scenarios. Instead of expanding the traditional model structure, the ECLS incorporates an error correction method that improves predictive performance while maintaining structural simplicity. Experiments were conducted on three representative viscoelastic materials-silicone rubber, polyurethane, and polytetrafluoroethylene-to acquire time-resolved response data through stress relaxation and creep tests. The fitting performance was quantitatively evaluated using the Euclidean norm and the Akaike information criterion, enabling a systematic comparison between the ECLS model and three classical models (Kelvin-Voigt, Maxwell, and standard linear solid [SLS]). The results show that the ECLS model exhibits higher predictive accuracy over a wide time range, with an average goodness of fit (R2) of 0.99, representing an improvement of ∼6% compared to the SLS model. Furthermore, the Root Mean Square Error (RMSE) and Mean Absolute Error (MAE) of the ECLS model are at least one order of magnitude lower than those of the traditional models, significantly improving the description of nonlinear viscoelastic behavior and providing more accurate predictions of material viscoelastic mechanical behavior. Therefore, the ECLS model not only improves the modeling accuracy of viscoelastic behavior but also establishes a unified and scalable framework for predicting and optimizing the mechanical performance of tissue-engineered vessels, expanding the application potential of mechanical modeling in bioreactor design and biomaterials development.
Colorectal organoids, which accurately replicate the structure and function of the human colorectal epithelium, have become a valuable platform in a broad spectrum of fundamental biological research and clinical applications. This study employs bibliometric analysis to develop a knowledge domain map specifically focusing on colorectal organoid research. Articles were sourced from the Web of Science Core Collection, and CiteSpace 6.3.R1 was utilized to analyze the literature, including outputs, journals, countries, institutions, authors, cocited authors, references, co-occurring terms, and burst terms. Subsequently, we examined prevailing research themes and focal points and identified potential future research directions within this domain. Between 2010 and 2025, a total of 719 articles related to colorectal organoid research were published. Among these, the journal Nature Communications published the highest number of papers. The United States and Utrecht University were identified as the most prolific country and institution, respectively. Hans Clevers emerged as the most prolific author, while Toshiro Sato had the highest number of cocitations, indicating that both are ideal candidates for academic collaboration. The research focus on colorectal organoids has evolved from basic biological characteristics to disease modeling and clinical applications, and further towards an in-depth exploration of functional mechanisms and precision medicines. The terms "patient-derived organoids", "disease modeling", "epithelial barrier", and "personalized medicine" have garnered significant attention between 2020 and 2025, highlighting them as promising areas for future research. Research on colorectal organoids has achieved substantial progress, positioning itself as a vital interdisciplinary field that integrates fundamental biology with clinical medicine. Future studies should focus on optimizing organoid culture methodologies, exploring functional mechanisms, and expanding clinical applications-especially in disease modeling and personalized medicine.
Adipose tissue is an abundant and clinically accessible source of stromal cells. Stromal vascular fraction (SVF) and nanofat have been widely investigated for their regenerative potential; however, commercial systems vary considerably in yield, viability, and regulatory oversight. Most devices report fresh results only, with limited validation following cryopreservation. Mesenchymal stromal cells derived from adipose tissue have also attracted attention due to their accessibility, immunomodulatory effects, and multipotent differentiation capacity. Uvence has developed a proprietary workflow for adipose tissue processing that integrates washing, cryopreservation, thawing, and emulsification within a Human Tissue Authority-regulated laboratory. The process includes Good Manufacturing Practices (GMP) Annex 1-aligned environmental monitoring and independent quality control (QC) testing. Critically, this workflow validates postthaw cell viability, addressing a gap in current SVF/nanofat approaches. Three cryopreserved donor samples demonstrated a mean postthaw viability of ∼91% (range 90.5-92%), consistently exceeding the International Federation for Adipose Therapeutics and Science (IFATS)/ International Society for Cell and Gene Therapy (ISCT) 70% threshold. Benchmarking against global systems showed Uvence postthaw viability to be equivalent to or higher than fresh outcomes reported for enzymatic platforms (Celution, 85-91%; InGeneron, 86%) and mechanical platforms (Lipocube, Tulip, ∼96%). Unlike competitor devices, Uvence has validated freeze-thaw performance, providing a stable and compliant platform. This study also presents in vitro culture and characterization of stromal cells expanded from Uvence nanofat-derived SVF samples, including flow cytometry, morphology, and trilineage differentiation. Flow cytometry confirmed high expression of CD73, CD90, and CD105, with minimal expression of CD34/CD45, consistent with the ISCT criteria. While these findings are limited to research characterization and do not constitute approval for therapeutic use, they demonstrate that the Uvence workflow delivers a quality-focused approach to adipose tissue processing.
Patients with breast or prostate cancer have a high chance of developing bone metastasis, which is associated with many skeletal-related events. The development of novel bone metastasis treatments is lagging behind due to the lack of reliable models. We aimed to develop a humanized bone metastasis model comprising vital human bone discs and human metastatic cancer cells (bone metastasis discs), which were subsequently cultured ex vivo or subcutaneously implanted into nude mice. Ex vivo culture experiments confirmed that cells within the bone metastasis discs remained metabolically active, while the presence of metastatic cancer cells could be monitored using bioluminescence. Although histological analyses confirmed the presence of relevant bone cells in the human bone tissue, no apparent formation of metastatic lesions was detected over the 2-week ex vivo culture period. In contrast, subcutaneously implanted bone metastasis discs demonstrated clear metastatic lesion formation, with osteolytic characteristics, that progressed from 3 to 6 weeks after implantation for both breast and prostate cancer bone metastasis discs. Histologically, healthy bone tissue with bone marrow compartments as well as anastomosis was observed. Cisplatin treatment of ex vivo cultured bone metastasis discs significantly decreased the bioluminescent signal from (prostate) cancer cells, while no effects of cisplatin treatment were observed for in vivo implanted bone metastasis discs. Our data provide a proof of concept for an ex vivo/in vivo bone metastasis model with vital human bone and human metastatic cancer cells but require further fine-tuning to improve robustness, relevance, and quantification methods. Future research could potentially use these models for the evaluation of novel bone metastasis treatments, accelerating their potential clinical application.
Biological research groups may face a high barrier to entry when constructing custom 3D cell culture devices to investigate multi-tissue interactions in vitro. Standard fabrication methods such as lithography, etching, or molding are expensive and require specialized equipment and expertise. To address this, we developed an accessible approach for producing polyethylene glycol (PEG)-based cell culture devices using stereolithography 3D printing with a polydimethylsiloxane intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, and the final device swells to predictable dimensions and retains its shape for at least 10 days. We used this approach to develop a human pluripotent stem cell-derived neural spheroid outgrowth model that supports directed neurite extension over 14 days. Together, this method provides a highly customizable, affordable platform for rapid fabrication of PEG-based microphysiological systems for diverse tissue engineering applications.
Bone and cartilage injuries are highly prevalent and arise from diverse pathogenic mechanisms, placing a substantial burden on patients' health, quality of life, and on families and society. Piezoelectric materials, inspired by tissue engineering concepts and the intrinsic piezoelectricity of human tissues, can harness the physiological electrical microenvironment to enhance tissue regeneration. To better understand the development of this field, we employed data from the Web of Science Core Citation (WoSCC) database as the core and primary focus for conducting bibliometric research and applied tools including Bibliometrix, Origin, Python, CiteSpace, and VOSviewer. A total of 388 publications from 46 countries were identified, with China, the United States, and Iran being the leading contributors. Fangwei Qi had the highest publication output, while C. Ribeiro had the highest cocitation frequency. The most productive institutions were Shanghai Jiao Tong University, the Fourth Military Medical University, and the University of Chinese Academy of Sciences. ACS Applied Materials & Interfaces published the largest number of articles. The most frequent keywords included "bone regeneration," "osteogenic differentiation," "piezoelectric," "scaffolds," and "hydroxyapatite." Furthermore, we employed Scopus as a validation database to cross-verify the publication trends and keyword hotspots derived from WoSCC, with the results demonstrating a high degree of consistency. These findings reveal that research on the role of piezoelectric materials in bone and cartilage regeneration is expanding rapidly, highlighting the current hotspots and emerging trends and providing valuable insights to guide future studies in this area.
Orofacial bone tissue engineering addresses bone loss caused by trauma, malformations, or tumors, enabling restoration and implant rehabilitation. Angiogenesis plays a crucial role in osteogenesis by ensuring nutrient and oxygen transport essential for bone regeneration. Preclinical large animal models are vital for translational research and require noninvasive, nondestructive methods aligned with 3Rs principles (Replacement, Reduction, and Refinement) to assess angiogenesis. This study proposes high-resolution cone-beam computed tomography subtraction angiography (HR-CBCT-SA) adapted for the orofacial region as an innovative method for monitoring angiogenesis during jawbone regeneration. Three Yucatan minipigs with a surgically created buccal wall jawbone defect per hemimandible were followed for 90 days by CBCT-SA to assess vascular remodeling. Morphometric parameters, including vessel number, node count, radius, and length, were analyzed and validated against histological morphometry. CBCT-SA revealed vascular dynamics during healing. By day 10, increased vessel and node counts along with reduced vessel radius and length indicated neoangiogenesis. At day 30, vessel maturation was aligned with transition of fibrous tissue to osteoid matrix deposition. By day 90, vascular metrics stabilized, reflecting bone remodeling phases characterized by replacement of lamellar and medullary bone replacement. Extrabony vascular networks underwent more pronounced changes than intrabony vessels, underscoring the leading role of periosteum in regeneration. Histology validated CBCT-SA findings, although resolution limitations prevented detection of vessels smaller than 500 µm. Nevertheless, CBCT-SA captured angiogenic changes over time and supported nondestructive monitoring without compromising tissue integrity. This study establishes HR-CBCT-SA as a reliable, nondestructive imaging technique for assessing vascular changes during jawbone regeneration in preclinical models. It demonstrates significant translational potential because of the clinically validated use of CBCT-angiography. Advances in artificial intelligence (AI)-driven image analysis are expected to enhance sensitivity and accuracy, improving vascular assessment. Moreover, this approach can be extended for investigating vascular-related oral pathologies (e.g., radiochemical osteonecrosis of the jaws), offering valuable tool to advance research in jawbone regeneration.
During development and regeneration, bone is formed by endochondral ossification (EO) through the remodeling of a cartilage template. This complex process involves multiple cell types and interactions that cannot currently be modeled in vitro. This study aimed to develop a novel tissue-engineered human in vitro model of certain aspects of the early stages of EO by integrating cartilage which undergoes mineralization, self-assembled vascular networks, and osteoclasts into a single system. We first studied the dynamics of osteoclastogenesis and vascularization in an in vivo model of stromal cell-mediated EO, to inform our in vitro system. Next, we aimed to develop a fully human cell-based three-dimensional model of EO by combining pediatric bone marrow stromal cells differentiating into chondrocytes, osteoclasts derived from human CD14+ monocytes, and human umbilical vein endothelial cells and adipose-derived stromal cells as vessel-forming cells. We investigated how mineralizing cartilage affects osteoclast and vessel formation in vitro through separate cartilage-osteoclasts and cartilage-vessels cocultures. Finally, we combined these elements and established a complex in vitro model that supports the functionality of all these cell types and recapitulates chondrogenesis, cartilage mineralization, vessel formation and osteoclastogenesis. This integrated approach reaches unprecedented complexity and will enable new tissue engineering strategies to model skeletal diseases or cancer metastasis to the bone.
Endoscopic Submucosal Dissection (ESD) effectively treats early gastric cancer, but postoperative complications limit its clinical use. Therefore, this study examines how esophageal mucosal wound protective gels improve wound healing and reduce post-ESD complications. The gels were characterized for physical properties and stability using rheological behavior, injectability, swelling capacity, and enzymatic degradation resistance. Biocompatibility was assessed via hemolysis testing, cytotoxicity assays, and oral mucosal irritation tests. Furthermore, wound repair potential was evaluated through cell proliferation, migration, and cell cycle analysis in Het-1A cells. Finally, in vivo recovery experiments were conducted to assess post-ESD wound healing efficacy. The gels exhibited favorable physical properties, chemical stability, and biocompatibility. Specifically, they maintained stability in the digestive tract, underwent rapid gelation at 37°C, and promoted cell proliferation. Post-ESD evaluation further revealed improved mucosal healing with no significant bleeding events. The developed esophageal mucosal wound-protective gels fulfill the requirements for submucosal interventions and show promising potential for ESD wound repair via rapid in situ gelation. This platform could be adapted for various endoscopic procedures and provides new insights for digestive tract tissue engineering applications.
Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surrounding brain microenvironment and drug resistance. Multicellular spheroids are an increasingly common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogeneous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild-type (WT) and temozolomide (TMZ)-resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 1,000 to 10,000 cells in size were embedded into a methacrylamide-functionalized gelatin hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid-size-dependent effects on TMZ efficacy for both TMZ-resistant and WT cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing, while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.