Tissue-engineered Constructs Research Articles

Spatial Transcriptomics, Proteomics, and Epigenomics as Tools in Tissue Engineering and Regenerative Medicine

Spatial transcriptomics, proteomics, and epigenomics are innovative technologies which offer an unparalleled resolution and wealth of data in understanding and the interpretation of cellular functions and interactions. These techniques allow researchers to investigate gene and protein expressions at an individual cell level, revealing cellular heterogeneity within, for instance, bioengineered tissues and classifying novel and rare cell populations that could be essential for the function of the tissues and in disease processes. It is possible to analyze thousands of cells simultaneously, which gives thorough insights into the transcriptomic view of complex tissues. Spatial transcriptomics combines gene expressions with spatial information, conserving tissue architecture and making the mapping of gene activity across different tissue regions possible. Despite recent advancements in these technologies, they face certain limitations. Single-cell transcriptomics can suffer from technical noise and dropout events, leading to incomplete data. Its applicability has been limited by the complexity of data integration and interpretation, although better resolution and tools for the interpretation of data are developing fast. Spatial proteomics and spatial epigenomics provide data on the distribution of proteins and the gene regulatory aspects in tissues, respectively. The disadvantages of these approaches include rather costly and time-consuming analyses. Nevertheless, combining these techniques promises a more comprehensive understanding of cell function and tissue organization, which can be predicted to be useful in achieving better knowledge of cell guidance in tissue-engineered constructs and a higher quality of tissue technology products.

Biomimetic Versatile Anisotropic, Electroactive Cellulose Hydrogel Scaffolds Tailored from Fern Stem Serving as Nerve Conduit and Cardiac Patch.

Peripheral nerve injury (PNI) and myocardial infarction (MI) are the two most clinically common soft excitable tissue injuries. Both nerve and cardiac tissues exhibit structural anisotropy and electrophysiological activity, providing a wide range of biophysical cues for cell and tissue repair. However, balancing microstructural anisotropy, electroactivity, and biocompatibility is challenging. To address this issue, Dicranopteris linearis (D. linearis) is proposed as a low-perceived value fern plant. Moreover, to enhance its usefulness, it can be designed into a tubular structure and a lamellar structure to bridge the damaged tissue. Therefore, a robust yet simple top-down approach is proposed to designing and fabricating the desired biomimetic versatile hydrogels orienting from the D. linearis to customize for different soft excitable tissue repair applications. These anisotropic electroactive hydrogels performed well as nerve guidance conduits (NGC) and engineered cardiac patches (ECP) in the repair of PNI and MI, respectively. Two birds, one stone. Accordingly, the biomimetic strategy of D. linearis to NGC and D. linearis to ECP is first proposed, opening a new horizon for constructing tissue engineering using natural sources.

Insitu magnetic-field-assisted bioprinting process using magnetorheological bioink to obtain engineered muscle constructs

Tissue-engineered anisotropic cell constructs are promising candidates for treating volumetric muscle loss (VML). However, achieving successful cell alignment within macroscale 3D cell constructs for skeletal muscle tissue regeneration remains challenging, owing to difficulties in controlling cell arrangement within a low-viscosity hydrogel. Herein, we propose the concept of a magnetorheological bioink to manipulate the cellular arrangement within a low-viscosity hydrogel. This bioink consisted of gelatin methacrylate (GelMA), iron oxide nanoparticles, and human adipose stem cells (hASCs). The cell arrangement is regulated by the responsiveness of iron oxide nanoparticles to external magnetic fields. A bioprinting process using ring magnets was developed for in situ bioprinting, resulting in well-aligned 3D cell structures and enhanced mechanotransduction effects on hASCs. In vitro analyses revealed upregulation of cellular activities, including myogenic-related gene expression, in hASCs. When implanted into a VML mouse model, the bioconstructs improved muscle functionality and regeneration, validating the effectiveness of the proposed approach.

Restoration of physiologic loading after engineered disc implantation mitigates immobilization-induced facet joint and paraspinal muscle degeneration.

Intervertebral disc degeneration is commonly associated with back and neck pain, and standard surgical treatments do not restore spine function. Replacement of the degenerative disc with a living, tissue-engineered construct has the potential to restore normal structure and function to the spine. Toward this goal, our group developed endplate-modified disc-like angle-ply structures (eDAPS) that recapitulate the native structure and function of the disc. While our initial large animal studies utilized rigid internal fixation of the eDAPS implanted level to ensure retention of the eDAPS, chronic immobilization does not restore full function and is detrimental to the spinal motion segment. The purpose of this study was to utilize a goat cervical disc replacement model coupled with finite element modeling of goat cervical motion segments to investigate the effects of remobilization (removal of fixation) on the eDAPS, the facet joints and the adjacent paraspinal muscle. Our results demonstrated that chronic immobilization caused notable degeneration of the facet joints and paraspinal muscles adjacent to eDAPS implants. Remobilization improved eDAPS composition and integration and mitigated, but did not fully reverse, facet joint osteoarthritis and paraspinal muscle atrophy and fibrosis. Finite element modeling revealed that these changes were likely due to reduced range of motion and reduced facet loading, highlighting the importance of maintaining normal spine biomechanical function with any tissue engineered disc replacement. STATEMENT OF SIGNIFICANCE: : Back and neck pain are ubiquitous in modern society, and the gold standard surgical treatment of spinal fusion limits patient function. This study advances our understanding of the response of the spinal motion segment to tissue engineered disc replacement with provisional fixation in a large animal model, further advancing the clinical translation of this technology.

Culturing 3D chitosan/gelatin/nano-hydroxyapatite and bone-derived scaffolds in a dynamic environment enhances osteochondral reconstruction.

Bioreactor can provide a dynamic culture environment for the in vitro construction of osteochondral tissue engineering. They facilitate more efficient exchange of nutrients and provide mechanical and other beneficial stimulation. Previous findings demonstrated that rotary flask (RF) bioreactor, rotary cell culture system (RCCS), or electromagnetic field (EMF) mediated scaffold culture could create a favorable dynamic environment for osteochondral tissue engineering. However, it is still unclear whether there is an optimal bioreactor or if bioreactors under multi-parameter coupling conditions are conducive to osteochondral tissue engineering. Based on this, the application of static T-flask (TF), RF, RCCS, and coupling environment of RCCS and EMF for osteochondral tissue engineering were systematically compared. The results showed that the RCCS/EMF culture system achieved the highest level of cellular proliferation and directed differentiation. Compared with the static culture group, the expression levels of chondrogenic factors of Sox9, Col II, and ACAN and osteogenic factors of Runx2, OCN, and Col I in RCCS/EMF culture system were 2.90±0.10, 3.53±0.05, 3.15±0.08, 7.16±0.15, 5.01±0.21 and 3.99±0.17 folds, respectively. The 'Active osteochondral' constructs (The construct is composed of chitosan/gelatin/nano-hydroxyapatite and bone-derived scaffolds) were prepared under different culture modes in vitro and implanted into the femoral condylar defect of New Zealand rabbits. After 12weeks, all culture modes could effectively promote the repair of osteochondral defects, in which the RCCS/EMF intervention had the best effect on the in vivo in-situ repair of osteochondral tissues. Furthermore, the fabricated cartilage and subchondral bone in the RCCS/EMF treatment group were most similar to the surrounding natural tissues, providing a new therapeutic idea for osteochondral tissue engineering.

The impact of mechanical tuning on the printability of decellularized amniotic membrane bioinks for cell-laden bioprinting of soft tissue constructs

Decellularized extracellular matrix (dECM) bioinks hold significant potential in the 3D bioprinting of tissue-engineered constructs (TECs). While 3D bioprinting allows for the creation of custom-designed TECs, the development of bioinks based solely on dAM, without the inclusion of supporting agents or chemical modifications, remains underexplored. In this study, we present the concentration-dependent printability and rheological properties of dAM bioinks, along with an analysis of their in vitro cellular responses. Our findings demonstrate that increasing dAM concentrations, within the range of 1 to 3% w/v, enhances the mechanical moduli of the bioinks, enabling the 3D printing of flat structures with superior shape fidelity. In vitro assays reveal high cell viability across all dAM bioink formulations; however, at 3% w/v, the bioink tends to impede fibroblast proliferation, resulting in round cell morphology. We propose that bioinks containing 2% w/v dAM strike an optimal balance, providing fine-resolved features and a supportive microenvironment for fibroblasts, promoting elongated spindle-like morphology and enhanced proliferation. These results underscore the importance of dAM concentration in regulating the properties and performance of bioinks, particularly regarding cell viability and morphology, for the successful 3D bioprinting of soft tissues.

Abstract A029: Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy response

Abstract Bone metastatic clear cell renal cell carcinoma (BM ccRCC) is a persistent clinical challenge and a source of significant morbidity and lethality for up to 40% of metastatic patients. Despite the recent approval of life-prolonging agents (antiangiogenic agents, immunotherapy) patients with BM ccRCC will eventually progress, due to emerging resistance. Bidirectional communication between tumor cells and bone stroma has emerged as a key determinant of disease progression and recurrence. However, a major challenge in addressing the black box of therapy response in bone is the lack of efficient and informative systems that allow us to analyze the heterotypic stromal-epithelial interaction, further complicated by the anatomical inaccessibility of bone. For these reasons, the evolution of the metastatic niche and its implications in therapy response remain elusive. In order to improve clinical results, it is critical to establish biologically informed pre-clinical models to provide a mechanistic understanding of tumor progression in bone and treatment outcomes. To this purpose, we have established clinically relevant models of ccRCC bone metastasis that span in vivo and ex vivo multiphoton microscopy (iMPM and eMPM), tissue engineered bone window systems, and computational oncology. iMPM displays both sensitivity and time-resolution to identify dynamic interactions between ccRCC cells and bone 3D adaptive niches, which support therapy response and resistance. However, iMPM in bone is limited due to its cortical thickness, increased light scattering, and complex topology. As a novel alternative, we created a tissue-engineered bone construct (TEBC) that, after direct implantation of cancer cells, is combined with an adjacent skin window, allowing for non- destructive intravital examination of tumor growth. To flank these in vivo dynamic analyses, we established a pipeline for ex vivo extraction of topological information related to the molecular and cellular niches involved in tumor progression and response to treatment (eMPM). This method allows reconstruction of whole lesions, including zones more distant from the bone interface, paired with an extensive panel of molecular markers. To further investigate the effects of a broad multi-parameter space on tumor regression or persistence upon treatment, in silico, we developed an in vivo-inspired agent-based model (ABM) of ccRCC in bone. A computational approach combined to ad hoc biological experimentation can refine the experimental design, test clinically relevant hypotheses (including impact of treatments on disease progression) and predict scenarios that guide biological testing towards more successful outcomes. Consequently, by integrating these approaches, we are currently studying the progression of the bone metastatic niche and its response to therapy, with the ultimate goal of overcoming therapy failure. Citation Format: Sergio Barrios, Luca Marsilio, Stefan Maksimovic, Matthew Campbell, Stefano Casarin, Eleonora Dondossola. Integrated modeling of renal cancer bone metastasis to illuminate tumor progression and therapy response [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A029.

Clinical translation of tissue-engineered oesophageal grafts: are patients ready for us?

PurposeWe sought to engage with expert patient/carers to understand attitudes towards use of tissue engineering (TE) for long-gap oesophageal atresia (OA).MethodsAn in-person engagement event for 70 patients/parents was held by the OA patient group, TOFS. Attitudes towards TE were assessed before and after a talk on use of TE oesophagi in a pre-clinical OA model. Perceptions were assessed using a 5-point Likert scale (median [range]) and compared using Mann–Whitney test.Results43 attendees responded; 56% parents/caregivers, 21% patients, 7% healthcare workers, 16% unreported. Most (85%) had some awareness of TE but for 15%, it was a new concept. Attendees were receptive to TE; 89% reported no concerns about growth of their/child(s) cells in a lab and 61% reported no concerns about using animal products. Perceptions of TE significantly improved after the presentation from 4 (2–5, n = 32) to 5 (3–5, n = 28) p < 0.0001, and 96% would like to be involved in focus groups on development of a TE product for use in OA.ConclusionInput from key stakeholders is essential to introduction of TE constructs clinically. The overall response to TE constructs was positive, and informs development of an OA-specific focus group to guide translation.

Improved visualisation of ACP-engineered osteoblastic spheroids: a comparative study of contrast-enhanced micro-CT and traditional imaging techniques

This study investigates osteoblastic cell spheroid cultivation methods, exploring flat-bottom, U-bottom, and rotary flask techniques with and without amorphous calcium phosphate (ACP) supplementation to replicate the 3D bone tissue microenvironment. ACP particles derived from eggshell waste exhibit enhanced osteogenic activity in 3D models. However, representative imaging of intricate 3D tissue-engineered constructs poses challenges in conventional imaging techniques due to notable scattering and absorption effects in light microscopy, and hence limited penetration depth. We investigated contrast-enhanced micro-CT as a methodological approach for comprehensive morphological 3D-analysis of thein-vitromodel and compared the technique with confocal laser scanning microscopy, scanning electron microscopy and classical histology. Phosphotungstic acid and iodine-based contrast agents were employed for micro-CT imaging in laboratory and synchrotron micro-CT imaging. Results revealed spheroid shape variations and structural integrity influenced by cultivation methods and ACP particles. The study underscores the advantage of 3D spheroid models over traditional 2D cultures in mimicking bone tissue architecture and cellular interactions, emphasising the growing demand for novel imaging techniques to visualise 3D tissue-engineered models. Contrast-enhanced micro-CT emerges as a promising non-invasive imaging method for tissue-engineered constructs containing ACP particles, offering insights into sample morphology, enabling virtual histology before further analysis.

Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by increased levels of inflammation that primarily manifests in the joints. Macrophages act as key drivers for the progression of RA, contributing to the perpetuation of chronic inflammation and dysregulation of pro-inflammatory cytokines such as interleukin 1 (IL-1). The goal of this study was to develop a macrophage-based cell therapy for biologic drug delivery in an autoregulated manner.MethodsFor proof-of-concept, we developed “smart” macrophages to mitigate the effects of IL-1 by delivering its inhibitor, IL-1 receptor antagonist (IL-1Ra). Bone marrow-derived macrophages were lentivirally transduced with a synthetic gene circuit that uses an NF-κB inducible promoter upstream of either the Il1rn or firefly luciferase transgenes. Two types of joint like cells were utilized to examine therapeutic protection in vitro, miPSCs derived cartilage and isolated primary mouse synovial fibroblasts while the K/BxN mouse model of RA was utilized to examine in vivo therapeutic protection.ResultsThese engineered macrophages were able to repeatably produce therapeutic levels of IL-1Ra that could successfully mitigate inflammatory activation in co-culture with both tissue-engineered cartilage constructs and synovial fibroblasts. Following injection in vivo, macrophages homed to sites of inflammation and mitigated disease severity in the K/BxN mouse model of RA.ConclusionThese findings demonstrate the successful development of engineered macrophages that possess the ability for controlled, autoregulated production of IL-1 based on inflammatory signaling such as via the NF-κB pathway to mitigate the effects of this cytokine for applications in RA or other inflammatory diseases. This system provides proof of concept for applications in other immune cell types as self-regulating delivery systems for therapeutic applications in a range of diseases.

High-throughput Generation of Collagen Microbeads for 3D Cell Culture and Extracellular Vesicle Production.

Collagen type I, a fundamental component of natural extracellular matrices across species, is an attractive material for the development of tissue engineering constructs. Nevertheless, the poor mechanical properties and thermal instability hinder its use for specific construction of 3D cultures and 3D printing. In this study, we present a microfluidic high-throughput approach for producing high-quality and uniform collagen microbeads without introducing any chemical modification. We achieved rapid and uniform collagen droplet fabrication with sizes spanning from 50 μm to 1200 μm in a production rate of up to 10000 droplets per minute. The resulting collagen microbeads can serve as numerous microbioreactors which are suspended in the culture medium without precipitation and are ideal for 3D cell growth. We demonstrated excellent cell compatibility, facilitating cell attachment and proliferation, as well as promoting extracellular vesicle secretion from collagen microbeads. This technology is facile and versatile for high throughput 3D cell culture, heterogeneous tissue modeling, and extracellular vesicle production, which is essential in drug delivery and drug screening.

Targeted CRISPR regulation of ZNF865 enhances stem cell cartilage deposition, tissue maturation rates, and mechanical properties in engineered intervertebral discs

Cell and tissue engineering based approaches have garnered significant interest for treating intervertebral disc degeneration and associated low back pain due to the substantial limitations of currently available clinical treatments. Herein we present a clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation strategy to improve the therapeutic potential of cell and tissue engineering therapies for treating intervertebral disc disease. Recently, we discovered a zinc finger (ZNF) protein, ZNF865 (BLST), which is associated with no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates cell cycle progression and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity. We also demonstrate that targeted ZNF865 regulation can enhance protein production and fibrocartilage-like matrix deposition in human adipose-derived stem cells (hASCs). Furthermore, we demonstrate CRISPR-engineered hASCs ability to increase GAG and collagen II matrix deposition in human-size tissue-engineered discs by 8.5-fold and 88.6-fold, respectively, while not increasing collagen X expression compared to naive hASCs dosed with growth factors. With this increased tissue deposition, we observe significant improvements in compressive mechanical properties, generating a stiffer and more robust tissue. Overall, we present novel biology on ZNF865 and display the power of CRISPR-cell engineering to enhance strategies treating musculoskeletal disease. Statement of significanceThis work reports on a novel gene, ZNF865 (also known as BLST), that when regulated with CRISPRa, improves cartilagenous tissue deposition in human sized tissue engineering constructs. Producing tissue engineering constructs at human scale has proven difficult, and this strategy presents a broadly applicable tool to enhance a cells ability to produce tissue at these scales, as we saw an ∼8–88 fold increase in tissue deposition and significantly improved biomechanics in large tissue engineered intervertebral disc compared to traditional growth factor differentiation methods. Additionally, this work begins to elucidate the biology of this novel zinc finger protein, which appears to be critical in regulating cell function and activity.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

Functional outcome of cell seeded tracheal scaffold after mechanical stress in vitro

Tracheal tissue engineering is still facing major challenges: realization of efficient vascularization and mechanical properties comparable to native trachea need to be achieved. In this study, we present a strategy for the manufacturing of a construct for tracheal tissue engineering by conditioning through cell seeding followed by mechanical stimulation in vitro. Scaffolds derived from porcine trachea decellularized with supercritical carbon dioxide were seeded with stem cells of different tissue sources and cultured in a bioreactor for 21 days under mechanical stimulation. Enhanced chondrogenic development was demonstrated, with improved sulphated glycosaminoglycan secretion and cellular alignment which resulted in mechanical properties resembling native trachea. This method may provide a useful addition to tracheal tissue engineering strategies aimed at optimizing cartilage formation.

Mueller matrix analysis of a biologically sourced engineered tissue construct as polarimetric phantom.

The polarimetric properties of biological tissues are often difficult to ascertain independent of their complex structural and organizational features. Conventional polarimetric tissue phantoms have well-characterized optical properties but are overly simplified. We demonstrate that an innovative, biologically sourced, engineered tissue construct better recapitulates the desired structural and polarimetric properties of native collagenous tissues, with the added benefit of potential tunability of the polarimetric response. We bridge the gap between non-biological polarimetric phantoms and native tissues. We aim to evaluate a synthesized tissue construct for its effectiveness as a phantom that mimics the polarimetric properties in typical collagenous tissues. We use a fibroblast-derived, ring-shaped engineered tissue construct as an innovative tissue phantom for polarimetric imaging. We perform polarimetry measurements and subsequent analysis using the Mueller matrix decomposition and Mueller matrix transformation methods. Scalar polarimetric parameters of the engineered tissue are analyzed at different time points for both a control group and for those treated with the transforming growth factor . Second-harmonic generation (SHG) imaging and three-dimensional collagen fiber organization analysis are also applied. We identify linear retardance and circular depolarization as the parameters that are most sensitive to the tissue culture time and the addition of . Aside from a statistically significant increase over time, the behavior of linear retardance and circular depolarization indicates that the addition of accelerates the growth of the engineered tissue, which is consistent with expectations. We also find through SHG images that collagen fiber organization becomes more aligned over time but is not susceptible to the addition of . The engineered tissue construct exhibits changes in polarimetric properties, especially linear retardance and circular depolarization, over culture time and under treatments. This tissue construct has the potential to act as a controlled modular optical phantom for polarimetric-based methods.

Microgels With Electrostatically Controlled Molecular Affinity to Direct Morphogenesis.

Concentration gradients of soluble signaling molecules-morphogens-determine the cellular organization in tissue development. Morphogen-releasing microgels have shown potential to recapitulate this principle in engineered tissue constructs, however, with limited control over the molecular cues in space and time. Inspired by the functionality of sulfated glycosaminoglycans (sGAGs) in morphogen signaling in vivo, a library of sGAG-based microgels is developed and designated as µGel Units to Instruct Development (µGUIDEs). Adjustment of the microgel's sGAG sulfation patterns and concentration enabled the programming of electrostatic affinities that control the release of morphogens. Based on computational analyses of molecular transport processes, µGUIDEs provided unprecedented precision in the spatiotemporal modulation of vascular endothelial growth factor (VEGF) gradients in a microgel-in-gel vasculogenesis model and kidney organoid cultures. The versatile approach offers new options for creating morphogen signaling centers to advance the understanding of tissue and organ development.

3D Bioprinting for Engineered Tissue Constructs and Patient-Specific Models: Current Progress and Prospects in Clinical Applications.

Advancements in bioprinting technology are driving the creation of complex, functional tissue constructs for use in tissue engineering and regenerative medicine. Various methods, including extrusion, jetting, and light-based bioprinting, have their unique advantages and drawbacks. Over the years, researchers and industry leaders have made significant progress in enhancing bioprinting techniques and materials, resulting in the production of increasingly sophisticated tissue constructs. Despite this progress, challenges still need to be addressed in achieving clinically relevant, human-scale tissue constructs, presenting a hurdle to widespread clinical translation. However, with ongoing interdisciplinary research and collaboration, the field is rapidly evolving and holds promise for personalized medical interventions. Continued development and refinement of bioprinting technologies have the potential to address complex medical needs, enabling the development of functional, transplantable tissues and organs, as well as advanced in vitro tissue models.

Glycosaminoglycan Concentration and Sulfation Patterns of Biohybrid Polymer Matrices Direct Microvascular Network Formation and Stability

AbstractComplexation with sulfated glycosaminoglycans (GAGs) regulates the signaling of pro‐angiogenic growth factors in vivo. To use this principle in modulating microvascular network formation and stability in vitro, a library of biohybrid hydrogels containing heparin, a highly sulfated GAG, or its selectively desulfated derivatives, is prepared. The hydrogels are applied to systematically investigate the effects of GAG concentration and sulfation patterns on embedded cultures of human umbilical vein endothelial cells (HUVECs) and cocultures of HUVECs with mesenchymal stromal cells (MSCs). Formation and morphology of microvascular networks, stromal cell expansion, and extracellular matrix (ECM) protein deposition are found to be significantly influenced by the hydrogel's GAG concentrations and sulfation patterns, presumably through differences in the resulting availability and bioactivity of GAG‐binding growth factors. In particular, cocultures in hydrogels with either low heparin concentration (500 µm) or higher concentrations of heparin desulfated at the 6O or N position resulted in the formation of dense, stable microvascular networks. The respective conditions also displayed enhanced stromal cell proliferation and ECM protein deposition, leading to increased matrix stiffness. Therefore, precisely tuning the affinity of biohybrid materials for pro‐angiogenic factors effectively enhances the vascularization of engineered tissue constructs in vitro.

Autoinduction-Based Quantification of In Situ TGF-β Activity in Native and Engineered Cartilage.

Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify in situ activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.

Tissue engineering of Cornea via Type 1 Collagen Biomaterials – A perspective

Engineering corneal tissue has advanced significantly in recent years. Engineering a biocompatible, mechanically stable, and optically clear tissue presents significant engineering hurdles. Two fundamental strategies have been explored by scholars to address these issues: cell-based methods for controlling cells their own extracellular matrix and scaffold-based methods for supplying dense, transparent matrices for cell growth. Both approaches have had some level of success. Additionally, new developments in innervating a tissue-engineered construct have been developed. Future research must concentrate on enhancing the mechanical stability of engineered constructions and the host reaction to implantation. Type 1 Collagen Biomaterial has been used for the construction of scaffolds or implantation for the cornea repair. Various methods for fabrication of the scaffolds have been described and mentioned tissue engineering application for cornea repair and regeneration. Given this correspondence, the type 1 collagen was a potential biomaterial for tissue engineering scaffold fabrication for effective regeneration of cornea.