Tissue-engineered Model Research Articles

Merging tissue engineering models with systems biology analysis for more collaborative understanding

The two fields have much to gain by working together and taking advantage of each other’s recent developments.

Multi‐Material Digital Light Processing (DLP) Bioprinting of Heterogeneous Hydrogel Constructs with Perfusable Networks

AbstractThe challenges of replicating the complex mechanical and structural diversity of natural tissues in vitro by leveraging multi‐material digital light processing (DLP) bioprinting are addressed. This technique utilizes PEGDA‐AAm bio‐ink to develop multi‐component, cell‐laden hydrogel constructs with varying moduli. These constructs not only possess heterogeneous mechanical properties but also feature complex architectures and precisely engineered surface microstructures. The hydrogel microfluidic chips are successfully fabricated with perfusable microchannels, embedding various cell types within the matrix. This approach enables the bioprinting of intricate cell‐laden structures with unique surface topologies, such as spiral grooves and triply periodic minimal surfaces (TPMS), which effectively influence cell alignment, spreading, and migration. By integrating various cell‐laden PA hydrogels, the diverse mechanical moduli of biological tissues, including bone, liver lobules, and vascular networks are replicated. This technique ensures high‐fidelity differentiation between cell types and regions. These findings provide valuable insights into the impact of substrate modulus and structure on cell behavior, underscoring the potential of multi‐component, multi‐modulus hydrogel constructs in creating sophisticated structures with custom‐tailored mechanical properties. This study significantly advances the field by demonstrating the feasibility and effectiveness of multi‐material DLP bioprinting in developing complex, functionally relevant tissue models for tissue engineering and regenerative medicine.

Abstract 1501: Engineering tissue models to elucidate prostate cancer bone metastasis dynamics

Abstract Prostate cancer (PCa) ranks as the second most frequently diagnosed cancer in men and the fifth leading cause of male cancer-related deaths globally Advanced PCa exhibits a propensity for bone metastasis which significantly contributes to morbidity. It is challenging to understand its mechanisms and develop effective treatments due to complicated tumor microenvironments. The current study addresses this gap by focusing on the construction of in vitro and in vivo models to mimic tissue-specific microenvironments resembling primary prostate tumor sites and metastatic bone tissue, shedding light on the complex processes of cancer cell dissemination, osteomimicry, and bone metastasis. To recapitulate the complex interplay between primary tumor sites and bone microenvironments, the study employs a combination of tunable extracellular matrix mechanics, bone-derived growth factors, and bone marrow-derived stromal cells. These engineered niches emulate the contexts of both primary tumor and bone tissue. LNCAP-C4-2B prostate cancer cells are introduced into these niches to analyze various aspects, including cell proliferation, osteoblastic differentiation, dissemination, and in vivo tumor formation. The study introduces a 3D organoid culture that mimics the pre-metastatic niche, providing a platform for studying PCa cell proliferation and dissemination. A negative relationship is observed between 3D matrix stiffness and cell proliferation, while a positive link is identified with cell dissemination. Bone matrix-derived soluble factors (BMSF) are identified as key modulators of LNCAP-C4-2B cell behavior, suppressing LNCAP-C4-2B proliferation but enhancing osteomimicry in vitro, Notably, bone marrow-derived stromal cells, primed with bone matrix-derived growth factors, amplify tumor cell proliferation and chemotaxis in vitro, contributing to increased tumor formation in vivo. In vivo experiments demonstrate BMSFs eliciting local inflammatory responses and inhibiting in vivo tumor formation. Moreover, osteogenic bone marrow stromal cells are shown to play a promotive role in LNCAP-C4-2B proliferation and migration, emphasizing the influence of the bone microenvironment on the prostate-to-bone progression of PCa cells. In conclusion, this study highlights the potential of the engineered tissue models for dissecting tumor progression and screening therapeutic interventions. By leveraging tissue-engineered soft and hard tissues, our approach offers a versatile toolbox for comprehensively investigating PCa progression and bone metastasis. These insights extend to potential therapeutic screening applications, providing a valuable resource for advancing our understanding of cancer pathogenesis and treatment strategies. Citation Format: Bo Han, Zhi Yang, Shuqing Zhao, Gangning Liang, Ba Xuan Hoang, Josephine Fang. Engineering tissue models to elucidate prostate cancer bone metastasis dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1501.

Abstract 4214: Tissue-engineered 3D model for elucidating the role of the bone niche in osteosarcoma therapy response

Abstract Introduction. Osteosarcoma (OS) is an aggressive pediatric bone cancer whose 5-year survival rate has remained stagnant for nearly 40 years, and often exhibits high therapy resistance. Experimental models that can elucidate how the OS bone niche modulates OS signaling and drug response could accelerate discovery of new targeted therapies. Our group has recently reported a biomaterials-based 3D in vitro model of OS with bone matrix mimicking cues: a gelatin microribbon scaffold coated with hydroxyapatite particles (GelHA µRBs). We demonstrated this 3D OS model can better mimic in vivo signaling and drug responses than standard 2D culture. However, the potential of this 3D model for probing OS-bone niche crosstalk has not yet been investigated. We hypothesize that OS-bone crosstalk will modulate OS drug response and signaling. To test this hypothesis, here we aim to establish a 3D model to assess the effect of tissue engineered (TE) bone on OS drug response and signaling. Methods. Gel µRBs were fabricated by wet spinning as previously described. To generate tissue engineered bone, human mesenchymal stem cells (hMSCs) were encapsulated (15 M/mL) in GelHA µRBs and cultured in osteogenic medium for 28 days. A PDX-derived OS line (OS052) was used. Three groups were studied: OS monoculture, separate coculture (paracrine only), and OS cells seeded directly on TE bone (paracrine plus direct contact). For all groups, 96K OS cells were seeded per sample. OS cells were labeled with firefly luciferase to allow tracking of OS cell viability and proliferation using bioluminescence. Gemcitabine, a standard chemotherapy drug for OS, was used as the model drug (0.001 to 1 uM). Outcome analyses include cell viability, proliferation, gene expression, and immunostaining. Results. TE bone enhances OS resistance to gemcitabine in both separate coculture and direct seeding on bone, as shown by bioluminescence. At 1 uM, OS cell death in monoculture, separate coculture, and seeding on bone was on average 98.19%, 82.05%, and 64.44%. These results indicate that both paracrine signaling from osteoblasts and direct contact with bone ECM contribute to OS drug resistance. TE bone did not significantly impact OS proliferation over 7 days as shown by bioluminescence. Ongoing studies will assess the effect of TE bone on OS expression of key proteins in the bone niche (RANK/RANKL/OPG) and OS response to cabozantinib, a cMET inhibitor whose antiproliferative effect may be osteoblast-mediated. Conclusions. Here we report a tissue engineered 3D model to study the crosstalk between OS cells and bone niche. The results support our hypothesis that bone niche contributes to OS drug resistance, which is mediated through both paracrine signals and ECM. This model can be used to further elucidate the mechanisms through which the bone niche influences OS progression and drug resistance, and for screening potential drug candidates that target such crosstalk. Citation Format: Abena Darkwah Peasah, Callan Monette, Jeehee Lee, Michelle Tai, E. Alejandro Sweet-Cordero, Fan Yang. Tissue-engineered 3D model for elucidating the role of the bone niche in osteosarcoma therapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4214.

Abstract 2830: Intravital multiphoton microscopy of bone metastatic renal cell carcinoma

Abstract Clear cell renal carcinoma (ccRCC) represents 80% of renal neoplasms, with bone as a major site for distant spread (35-40% of patients). These secondary lesions cause a variety of skeletal-related complications, including pain, spinal cord compression, hypercalcemia, mobility issues and fractures, posing a significant negative impact on patient quality of life and survival. Furthermore, bone is well-known as a site of therapy resistance, which confers to patients treated with systemic therapies significantly worse time-to-treatment failure and progression-free and overall survival compared to those without bone metastases. Because of the complexity of the bone microenvironment and the paucity of suitable models, preclinical investigation of bone metastasis and therapy response is challenging. Preclinical models of bone metastasis that integrate tissue engineering and advanced imaging techniques can facilitate the study of the mechanism of tumor progression and therapy response. We recently developed a window model amenable to intravital multiphoton microscopy (MPM) to longitudinally monitor human ccRCC lesions in tissue engineered bone constructs (TEBCs) in immunodeficient mice. TEBCs were generated by functionalizing polymeric polycaprolactone scaffolds with bone morphogenetic protein 7. Human ccRCC lesions, after implantation into the bone cavity, were longitudinally monitored for growth and niche development, using multi-parameter recording of collagen/bone matrix (SHG), bone surface (THG), blood vessels/stromal phagocytes (fluorescent dextran), and tumor cells (GFP). However, this model lacked key components of the immune system (including B and T-cells), which are of major interest in the study of tumor progression and (immune) therapy response. Therefore, we developed a tissue-engineered preclinical model that incorporate species-specific interactions between cancer and bone stromal cells, and account for an intact immune system. To this purpose, we optimized TEBC formation in C57BL/6 fluorescent reporter mice (Sp7-mCherry, osteoblasts; Trap-td-Tomato, osteoclasts; GFP, immune cells, αSMA-RFP fibroblasts; and Flk1-GFP, blood vessels), coupled to detection by intravital or ex vivo multiphoton microscopy analysis, to monitor the evolution and fate of the TEBC. Interestingly, the bone marrow in the core of the TEBC was replaced by a desmoplastic tissue with features like the foreign body response, including foreign body giant cells, which was not present in immunodeficient mice. By applying dual color GFP/αSMA-RFP mice, which expressed red fluorescence in activated fibroblasts, we confirmed the fibrotic nature of this core. To avoid its formation, we generated scaffold-free ossicle, by direct implantation of BMP7 and VEGF in the subcutaneous tissue. To conclude, we established a protocol for generating ectopic ossicles in immunocompetent mice. Citation Format: Stefan Maksimovic, Nina Constanza Boscolo, Sergio Barrios, Antonios Mikos, Matthew T. Campbell, Eleonora Dondossola. Intravital multiphoton microscopy of bone metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2830.

Abstract 6773: Tissue engineered colorectal cancer microspheres for drug screening

Abstract Three-dimensional (3D) disease models have garnered widespread interest for use in later stages of the drug discovery process, such as preclinical efficacy and toxicology studies, due to their pathophysiologically relevant properties. However, there is a need and opportunity for 3D cancer models to be used earlier in the drug screening process. To meet this need, the 3D models must strike a balance between throughput, which includes scalability and uniformity, and physiological relevance, such as the ability to modulate key attribute of the tumor microenvironment. Here we report the creation of 3D colorectal cancer (CRC) tissue models, referred to as VivoSpheres, and demonstrated their relevance to cancer drug screening. The VivoSphere production platform couples tissue engineering toolkits with microfluidics, enabling the scalable production of engineered cancer microspheres. The model supports the long-term maintenance of the cancer cell phenotype. In a preliminary study, we were able to generate more physiologically relevant drug responses. We formed CRC VivoSpheres by encapsulating HT-29 CRC cells within poly(ethylene glycol)-fibrinogen hydrogel microspheres using our previously developed microfluidic platform. CRC VivoSpheres were rapidly produced with high cell densities (20 × 106 cells/ml) and high uniformity on day 0 with a coefficient of variation (COV) < 7%. This high uniformity was maintained for 15 days (COV ≤ 10%), which is critical for long-term dose studies. The cells maintained high viability and showed high proliferative capability with a significant increase in colony size and expression of Ki67 up to day 29. The encapsulated cells maintained the CRC phenotype over time with the expression of CD44 (cancer stem cell marker) and CK20 (CRC marker). After establishing shipping conditions that maintained cell viability for remote use, the HT-29 VivoSpheres were shipped to the oncology team at Southern Research for drug testing. The CRC VivoSpheres were treated with DMSO, GANT61, and SRI-38832, the latter two of which are GLI1 inhibitors. Phase contrast images and western blot were used to assess the response of CRC VivoSpheres to the treatments. Oncogenic GLI1 transcription activity and NBS1 overexpression have been found to contribute to chemotherapeutic resistance, negating the anti-tumor effects of 5-fluorouracil. While 2D cultured HT-29s responded to treatment with GANT61, HT-29 VivoSpheres continued to express NBS1 following GANT1 treatment, but downregulated NBS1 in response to the GLI1 inhibitor SRI-38832, which is the same response Southern Research has seen in in vivo tumor models. In conclusion, we have developed tissue-engineered 3D CRC models that hold promise for use in drug screening. These models have demonstrated an initial capability to reproduce the CRC phenotype and mimic in vivo drug response. Citation Format: Yuan Tian, Mohammadjafar Hashemi, Benjamin Anbiah, Ruowen Zhang, Iman Hassani, Rebecca Boohaker, Elizabeth Lipke. Tissue engineered colorectal cancer microspheres for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6773.

Abstract 6896: Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells

Abstract Melanoma is a uniquely difficult cancer to treat due to complex tumor heterogeneity and resistance, where upon dissemination, survival drops to less than 17%. Drug resistance has arisen for the current standard of care and combinatorial therapies, expediting the need for new efficacious agents. The lack of physiologically relevant preclinical models has impeded the development of these therapeutics. Potential melanoma drugs are typically assayed using cell lines on tissue culture plastic, then tested for in vivo efficacy and side effects in animals. This paradigm is inherently unsuitable for the generation of effective therapies as typical cell culture platforms fail to replicate the complex microenvironments known to play critical roles in tumor pathology and rodent models are prohibitively expensive for optimizing novel and combination therapies. Thus, our goal is to synthesize tissue-engineered melanoma models using biomimetic polymers that recapitulate patient tumor characteristics to assess combination and novel therapies that attenuate resistance and disrupt metastatic processes. In this study, patient melanoma cells, which were unable to recapitulate drug responses on traditional cell culture platforms, were cultured in a controlled spatial and temporal manner on polyacrylamide hydrogels. The hydrogel scaffolds feature stiffnesses spanning that of physiological tissue, with microcontact printed human fibronectin for biomimetic protein presentation to create a microtumor array. Cells cultured on the hydrogel showed a 2.5-fold increase in expression of the cancer stem cell biomarkers, CD271 and ABCB5, which play a key role in melanoma survival and recurrence, compared to cells cultured on plastic or glass substrates. Further study identified BRAF mutant, drug resistant patient cells, regained resistance to standard of care targeted therapies, trametinib and dabrafenib. Through hydrogel microarrays featuring physiological similarities, the technology has enabled the exploration of heterogeneous cell populations with novel and combinatorial therapeutics. Citation Format: Yi L. Liu, Justyna Gleba, Chantal Kopecky, Matthew Pawlush, John A. Copland, Kristopher A. Kilian. Biomimetic hydrogels at physiological stiffness reprogram patient derived melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6896.

Vascular study of decellularized porcine long bones: Characterization of a tissue engineering model

IntroductionMassive bone allografts enable the reconstruction of critical bone defects in numerous conditions (e.g. tumoral, infection or trauma). Unfortunately, their biological integration remains insufficient and the reconstruction may suffer from several postoperative complications. Perfusion-decellularization emerges as a tissue engineering potential solution to enhance osseointegration. Therefore, an intrinsic vascular study of this novel tissue engineering tool becomes essential to understand its efficacy and applicability. Material and methods32 porcine long bones (humeri and femurs) were used to assess the quality of their vascular network prior and after undergoing a perfusion-decellularization protocol. 12 paired bones were used to assess the vascular matrix prior (N = 6) and after our protocol (N = 6) by immunohistochemistry. Collagen IV, Von Willebrand factor and CD31 were targeted then quantified. The medullary macroscopic vascular network was evaluated with 12 bones: 6 were decellularized and the other 6 were, as control, not treated. All 12 underwent a contrast-agent injection through the nutrient artery prior an angio CT-scan acquisition. The images were processed and the length of medullary vessels filled with contrast agent were measured on angiographic cT images obtained in control and decellularized bones by 4 independent observers to evaluate the vascular network preservation. The microscopic cortical vascular network was evaluated on 8 bones: 4 control and 4 decellularized. After injection of gelatinous fluorochrome mixture (calcein green), non-decalcified fluoroscopic microscopy was performed in order to assess the perfusion quality of cortical vascular lacunae. ResultsThe continuity of the microscopic vascular network was assessed with Collagen IV immunohistochemistry (p-value = 0.805) while the decellularization quality was observed through CD31 and Von Willebrand factor immunohistochemistry (p-values <0.001). The macroscopic vascular network was severely impaired after perfusion-decellularization; nutrient arteries were still patent but the amount of medullary vascular channels measured was significantly higher in the control group compared to the decellularized group (p-value <0.001). On average, the observers show good agreement on these results, except in the decellularized group where more inter-observer discrepancies were observed. The microscopic vascular network was observed with green fluoroscopic signal in almost every canals and lacunae of the bone cortices, in three different bone locations (proximal metaphysis, diaphysis and distal metaphysis). ConclusionDespite the aggressiveness of the decellularization protocol on medullary vessels, total porcine long bones decellularized by perfusion retain an acellular cortical microvascular network. By injection through the intact nutrient arteries, this latter vascular network can still be used as a total bone infusion access for bone tissue engineering in order to enhance massive bone allografts prior implantation.

Fabrication of vascularized tissue-engineered bone models using triaxial bioprinting.

Bone tissue is a highly vascularized tissue. When constructing tissue-engineered bone models, both the osteogenic and angiogenic capabilities of the construct should be carefully considered. However, fabricating a vascularized tissue-engineered bone to promote vascular formation and bone generation, while simultaneously establishing nutrition channels to facilitate nutrient exchange within the constructs, remains a significant challenge. Triaxial bioprinting, which not only allows the independent encapsulation of different cell types while simultaneously forming nutrient channels, could potentially emerge as a strategy for fabricating vascularized tissue-engineered bone. Moreover, bioinks should also be applied in combination to promote both osteogenesis and angiogenesis. In this study, employing triaxial bioprinting, we used a blend bioink of gelatin methacryloyl (GelMA), sodium alginate (Alg), and different concentrations of nano beta-tricalcium phosphate (nano β-TCP) encapsulated MC3T3-E1 preosteoblasts as the outer layer, a mixed bioink of GelMA and Alg loaded with human umbilical vein endothelial cells (HUVEC) as the middle layer, and gelatin as a sacrificial material to form nutrient channels in the inner layer to fabricate vascularized bone constructs simulating the microenvironment for bone and vascular tissues. The results showed that the addition of nano β-TCP could adjust the mechanical, swelling, and degradation properties of the constructs. Biological assessments revealed the cell viability of constructs containing different concentrations of nano β-TCP was higher than 90% on day 7, The cell-laden constructs containing 3% (w/v) nano β-TCP exhibited better osteogenic (higher Alkaline phosphatase activity and larger Osteocalcin positive area) and angiogenic (the gradual increased CD31 positive area) potential. Therefore, using triaxial bioprinting technology and employing GelMA, Alg, and nano β-TCP as bioink components could fabricate vascularized bone tissue constructs, offering a novel strategy for vascularized bone tissue engineering.

The Assessment of a Novel Endoscopic Ultrasound-Compatible Cryocatheter to Ablate Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease that may be treated utilizing thermal therapies. Cryoablation is an effective, minimally invasive therapy that has been utilized for the treatment of various cancers, offering patients a quicker recovery and reduced side effects. Cryoablation has been utilized on a limited basis for the treatment of PDAC. With the recent reports on the success of cryoablation, there is a growing interest in the use of cryoablation as a standalone, minimally invasive procedure to treat PDAC. While offering a promising path, the application of cryoablation to PDAC is limited by current technologies. As such, there is a need for the development of new devices to support advanced treatment strategies for PDAC. To this end, this study investigated the performance of a new endoscopic ultrasound-compatible cryoablation catheter technology, FrostBite. We hypothesized that FrostBite would enable the rapid, effective, minimally invasive delivery of ultra-cold temperatures to target tissues, resulting in effective ablation via an endoscopic approach. Thermal properties and ablative efficacy were evaluated using a heat-loaded gel model, tissue-engineered models (TEMs), and an initial in vivo porcine study. Freeze protocols evaluated included single and repeat 3 and 5 min applications. Isotherm assessment revealed the generation of a 2.2 cm diameter frozen mass with the -20 °C isotherm reaching a diameter of 1.5 cm following a single 5 min freeze. TEM studies revealed the achievement of temperatures ≤ -20 °C at a diameter of 1.9 cm after a 5 min freeze. Fluorescent imaging conducted 24 h post-thaw demonstrated a uniformly shaped ellipsoidal ablative zone with a midline diameter of 2.5 cm, resulting in a total ablative volume of 6.9 cm3 after a single 5 min freeze. In vivo findings consistently demonstrated the generation of ablative areas measuring 2.03 cm × 3.2 cm. These studies demonstrate the potential of the FrostBite cryocatheter as an endoscopic ultrasound-based treatment option. The data suggest that FrostBite may provide for the rapid, effective, controllable freezing of cancerous pancreatic and liver tissues. This ablative power also offers the potential of improved safety margins via the minimally invasive nature of an endoscopic ultrasound-based approach or natural orifice transluminal endoscopic surgery (NOTES)-based approach. The results of this pre-clinical feasibility study show promise, affirming the need for further investigation into the potential of the FrostBite cryocatheter as an advanced, minimally invasive cryoablative technology.

Biofabrication of biomimetic undulating microtopography at the dermal-epidermal junction and its effects on the growth and differentiation of epidermal cells.

The undulating microtopography located at the junction of the dermis and epidermis of the native skin is called rete ridges (RRs), which plays an important role in enhancing keratinocyte function, improving skin structure and stability, and providing three-dimensional (3D) microenvironment for skin cells. Despite some progress in recent years, most currently designed and manufactured tissue-engineered skin models still cannot replicate the RRs, resulting in a lack of biological signals in the manufactured skin models. In this study, a composite manufacturing method including electrospinning, 3D printing, and functional coating was developed to produce the epidermal models with RRs. Polycaprolactone (PCL) nanofibers were firstly electrospun to mimic the extracellular matrix environment and be responsible for cell attachment. PCL microfibers were then printed onto top of the PCL nanofibers layer by 3D printing to quickly prepare undulating microtopography and finally the entire structures were dip-coated with gelatin hydrogel to form a functional coating layer. The morphology, chemical composition, and structural properties of the fabricated models were studied. The results proved that the multi-process composite fabricated models were suitable for skin tissue engineering. Live and dead staining, cell counting kit-8 (CCK-8) as well as histology (haematoxylin and eosin (HE) methodology) and immunofluorescence (primary and secondary antibodies combination assay) were used to investigate the viability, metabolic activity, and differentiation of skin cells forin vitroculturing.In vitroresults showed that each model had high cell viability, good proliferation, and the expression of differentiation marker. It was worth noting that the sizes of the RRs affected the cell growth status of the epidermal models. In addition, the unique undulation characteristics of the epidermal-dermal junction can be reproduced in the developed epidermal models. Overall, thesein vitrohuman epidermal models can provide valuable reference for skin transplantation, screening and safety evaluation of drugs and cosmetics.

Vascularized 3D Human Skin Models in the Forefront ofDermatological Research.

In vitro engineered skin models are emerging as an alternative platform to reduce and replace animal testing in dermatological research. Despite the progress made in recent years, considerable challenges still exist for the inclusion of diverse cell types within skin models. Blood vessels, in particular, are essential in maintaining tissue homeostasis and are one of many primary contributors to skin disease inception and progression. Substantial efforts in the past have allowed the successful fabrication of vascularized skin models that are currently utilized for disease modeling and drugs/cosmetics testing. This review first discusses the need for vascularization within tissue-engineered skin models, highlighting their role in skin grafting and disease pathophysiology. Second, the review spotlights the milestones and recent progress in the fabrication and utilization of vascularized skin models. Additionally, advances including the use of bioreactors, organ-on-a-chip devices, and organoid systems are briefly explored. Finally, the challenges and future outlook for vascularized skin models are addressed.

Decellularized extracellular matrix for three-dimensional bioprinted in vitro disease modeling

Precise in vitro models in tissue engineering have attracted the attention of researchers seeking to understand physiological consequences from native tissues as well as the mechanism of diseases in vitro. To construct delicate native tissue-like in vitro models, a proper combination of biomimetic materials and a biofabrication strategy is required. Conventional biomaterials, such as collagens, laminins, and synthetic polymers, have been widely adapted in tissue recapitulation; however, they lack tissue specificity in the context of biophysical properties and native-like extracellular matrix composition. The lack of tissue specificity accounts for the pathophysiological discrepancy between preclinical model and actual human patient. Thus, biomaterials should be improved for attaining physiological similarity between disease models and patients. Additionally, a biofabrication technique is essential for building mature cellular or tissue structures with a sophisticated bioassembly process. Among the biofabrication techniques, bioprinting stands as a promising approach for constructing three-dimensional (3D) cellular structures using specific cell types and biomaterials. Combining multifunctional bioinks and bioprinting is expected to enhance tissue specificity with regard to structural recapitulation. From this viewpoint, decellularized extracellular matrix (dECM) bioink has been increasingly used to achieve tissue specificity and manufacturability in 3D bioprinting. Progress in this domain requires the clarification of tissue-specific decellularization method and the development of a proper 3D bioprinting method, in conjunction with the improvement of the compatibility between dECM and bioprinting. In this review, we introduce the production methods and characteristics of dECM in the context of tissue specificity and examine state-of-the-art dECM-incorporated 3D-bioprinted in vitro models for disease investigation. We also recommend a strategy for improving dECM for use in therapeutic studies based on simulations of the pathophysiological microenvironment.

Soft Tissue Reconstruction.

Autologous fat transplantation has revolutionized soft tissue reconstruction, but conventional methods remain unpredictable as graft resorption rates are high due to lack of vascularization. The advent of adipose-derived stem cells (ASCs) has led to improvement of fat grafting outcomes, in part to their ability to undergo facile differentiation into adipose tissue, their angiogenic properties, and their ability to express and secrete multiple growth factors. This chapter discusses the isolation and characterization of human ASCs, its expansion in vitro, and relevant in vivo models for adipose tissue engineering.

A self-assembly and cellular migration based fabrication of high-density 3D tubular constructs of barrier forming membranes.

Three-dimensional (3D) in vitro models, superior in simulating physiological conditions compared to 2D models, offer intricate cell-cell and cell-ECM interactions with diverse signaling cues like fluid shear stress and growth factor gradients. Yet, developing 3D tissue barrier models, specifically perfusable luminal structures with dense, multicellular constructs maintained for extended durations with oxygen and nutrients, remains a technical challenge. Here, we describe a molding-based approach for the fabrication of free-standing, perfusable, high cellular density tissue constructs using a self-assembly and migration process to form functional barriers. This technique utilizes a polytetrafluoroethylene (PTFE)-coated stainless-steel wire, held by stainless steel needles, as a template for a perfusable channel within an elongated PDMS well. Upon adding a bio-ink mix of cells and collagen, it self-assembles into a high cell density layer conformally around the wire. Removing the wire reveals a hollow construct, connectable to an inlet and outlet for perfusion. This scalable method allows creating varied dimensions and multicellular configurations. Notably, post-assembly, cells such as human umbilical vein endothelial cells (HUVECs) migrate to the surface and form functional barriers with adherens junctions. Permeability tests and fluorescence imaging confirm that these constructs closely mimic in vivo endothelial barrier permeability, exhibiting the lowest permeability among all in vitro models in the literature. Unlike traditional methods involving uneven post-seeding of endothelial cells leading to subpar barriers, our approach is a straightforward alternative for fabricating complex perfusable 3D tissue constructs and effective tissue barriers for use in various applications, including tissue engineering, drug screening, and disease modeling.

Engineering Biomimetic Nanoparticles through Extracellular Vesicle Coating in Cancer Tissue Models.

Using nanoparticles (NPs) in drug delivery has exhibited promising therapeutic potential in various cancer types. Nevertheless, several challenges must be addressed, including the formation of the protein corona, reduced targeting efficiency and specificity, potential immune responses, and issues related to NP penetration and distribution within 3-dimensional tissues. To tackle these challenges, we have successfully integrated iron oxide nanoparticles into neuroblastoma-derived extracellular vesicles (EVs) using the parental labeling method. We first developed a tissue-engineered (TE) neuroblastoma model, confirming the viability and proliferation of neuroblastoma cells for at least 12 days, supporting its utility for EV isolation. Importantly, EVs from long-term cultures exhibited no differences compared to short-term cultures. Concurrently, we designed Rhodamine (Rh) and Polyacrylic acid (PAA)-functionalized magnetite nanoparticles (Fe3O4@PAA-Rh) with high crystallinity, purity, and superparamagnetic properties (average size: 9.2 ± 2.5 nm). We then investigated the internalization of Fe3O4@PAA-Rh nanoparticles within neuroblastoma cells within the TE model. Maximum accumulation was observed overnight while ensuring robust cell viability. However, nanoparticle internalization was low. Taking advantage of the enhanced glucose metabolism exhibited by cancer cells, glucose (Glc)-functionalized nanoparticles (Fe3O4@PAA-Rh-Glc) were synthesized, showing superior cell uptake within the 3D model without inducing toxicity. These glucose-modified nanoparticles were selected for parental labeling of the TE models, showing effective NP encapsulation into EVs. Our research introduces innovative approaches to advance NP delivery, by partially addressing the challenges associated with 3D systems, optimizing internalization, and enhancing NP stability and specificity through EV-based carriers. Also, our findings hold the promise of more precise and effective cancer therapies while minimizing potential side effects.

Cardiovascular Tissue Engineering Models for Atherosclerosis Treatment Development.

In the early years of tissue engineering, scientists focused on the generation of healthy-like tissues and organs to replace diseased tissue areas with the aim of filling the gap between organ demands and actual organ donations. Over time, the realization has set in that there is an additional large unmet need for suitable disease models to study their progression and to test and refine different treatment approaches. Increasingly, researchers have turned to tissue engineering to address this need for controllable translational disease models. We review existing and potential uses of tissue-engineered disease models in cardiovascular research and suggest guidelines for generating adequate disease models, aimed both at studying disease progression mechanisms and supporting the development of dedicated drug-delivery therapies. This involves the discussion of different requirements for disease models to test drugs, nanoparticles, and drug-eluting devices. In addition to realistic cellular composition, the different mechanical and structural properties that are needed to simulate pathological reality are addressed.

Investigating the Impact of Mechanical Properties and Cell-Collagen Interaction on NIH3T3 Function: A Comparative Study on Different Substrates and Culture Environments.

This study investigates the intricate dynamics of matrix stiffness, substrate composition, and cell-cell interactions and elucidates their cumulative effects on fibroblast behavior in different culture contexts. Three primary substrate types were examined: non-coated, collagen-coated, and collagen hydrogel, within both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid cultures. The research provides several key insights. First, 3D spheroid culture, which promotes robust cell-cell interactions, emerges as a critical factor in maintaining fibroblast functionality. Second, substrate stiffness significantly influences results, with the soft collagen hydrogel showing superior support for fibroblast function. Notably, fibroblasts cultured on collagen hydrogel in 2D exhibit comparable functionality to those in 3D, highlighting the importance of substrate mechanical properties. Third, surface composition, as exemplified by collagen coating, showed a limited effect compared to the other factors studied. These findings provide a basis for innovative applications in regenerative medicine, tissue engineering, and drug testing models, and offer valuable insights into harnessing the potential of fibroblasts and advancing biomedical sciences.

In Vitro Models of Head and Neck Cancer: From Primitive to Most Advanced.

For several decades now, researchers have been trying to answer the demand of clinical oncologists to create an ideal preclinical model of head and neck squamous cell carcinoma (HNSCC) that is accessible, reproducible, and relevant. Over the past years, the development of cellular technologies has naturally allowed us to move from primitive short-lived primary 2D cell cultures to complex patient-derived 3D models that reproduce the cellular composition, architecture, mutational, or viral load of native tumor tissue. Depending on the tasks and capabilities, a scientific laboratory can choose from several types of models: primary cell cultures, immortalized cell lines, spheroids or heterospheroids, tissue engineering models, bioprinted models, organoids, tumor explants, and histocultures. HNSCC in vitro models make it possible to screen agents with potential antitumor activity, study the contribution of the tumor microenvironment to its progression and metastasis, determine the prognostic significance of individual biomarkers (including using genetic engineering methods), study the effect of viral infection on the pathogenesis of the disease, and adjust treatment tactics for a specific patient or groups of patients. Promising experimental results have created a scientific basis for the registration of several clinical studies using HNSCC in vitro models.

A tissue-engineered model of the blood-tumor barrier during metastatic breast cancer

Metastatic brain cancer has poor prognosis due to challenges in both detection and treatment. One contributor to poor prognosis is the blood–brain barrier (BBB), which severely limits the transport of therapeutic agents to intracranial tumors. During the development of brain metastases from primary breast cancer, the BBB is modified and is termed the ‘blood-tumor barrier’ (BTB). A better understanding of the differences between the BBB and BTB across cancer types and stages may assist in identifying new therapeutic targets. Here, we utilize a tissue-engineered microvessel model with induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (iBMECs) and surrounded by human breast metastatic cancer spheroids with brain tropism. We directly compare BBB and BTB in vitro microvessels to unravel both physical and chemical interactions occurring during perivascular cancer growth. We determine the dynamics of vascular co-option by cancer cells, modes of vascular degeneration, and quantify the endothelial barrier to antibody transport. Additionally, using bulk RNA sequencing, ELISA of microvessel perfusates, and related functional assays, we probe early brain endothelial changes in the presence of cancer cells. We find that immune cell adhesion and endothelial turnover are elevated within the metastatic BTB, and that macrophages exert a unique influence on BTB identity. Our model provides a novel three-dimensional system to study mechanisms of cancer-vascular-immune interactions and drug delivery occurring within the BTB.