Sex as a Biological Variable in Tissue Engineering and Regenerative Medicine.

Although sex differences have been noted in cellular function and behavior, therapy efficacy, and disease incidence and outcomes, the adoption of sex as a biological variable in tissue engineering and regenerative medicine remains limited. Furthering the development of personalized, precision medicine requires considering biological sex at the bench and in the clinic. This review provides the basis for considering biological sex when designing tissue-engineered constructs and regenerative therapies by contextualizing sex as a biological variable within the tissue engineering triad of cells, matrices, and signals. To achieve equity in biological sex within medicine requires a cultural shift in science and engineering research, with active engagement by researchers, clinicians, companies, policymakers, and funding agencies.

Review: Human stem cell-based 3D in vitro angiogenesis models for preclinical drug screening applications.

Vascular diseases are the underlying pathology in many life-threatening illnesses. Human cellular and molecular mechanisms involved in angiogenesis are complex and difficult to study in current 2D in vitro and in vivo animal models. Engineered 3D in vitro models that incorporate human pluripotent stem cell (hPSC) derived endothelial cells (ECs) and supportive biomaterials within a dynamic microfluidic platform provide a less expensive, more controlled, and reproducible platform to better study angiogenic processes in response to external chemical or physical stimulus. Current studies to develop 3D in vitro angiogenesis models aim to establish single-source systems by incorporating hPSC-ECs into biomimetic extracellular matrices (ECM) and microfluidic devices to create a patient-specific, physiologically relevant platform that facilitates preclinical study of endothelial cell-ECM interactions, vascular disease pathology, and drug treatment pharmacokinetics. This review provides a detailed description of the current methods used for the directed differentiation of human stem cells to endothelial cells and their use in engineered 3D in vitro angiogenesis models that have been developed within the last 10 years.

Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development.

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

What Can an Organ-on-a-Chip Teach Us About Human Lung Pathophysiology?

The intertwined relationship between structure and function has been key to understanding human organ physiology and disease pathogenesis. An organ-on-a-chip (organ chip) is a bioengineered microfluidic cell culture device lined by living cells and tissues that recapitulates organ-level functions in vitro. This is accomplished by recreating organ-specific tissue-tissue interfaces and microenvironmental biochemical and mechanical cues while providing dynamic perfusion through endothelium-lined vascular channels. In this review, we discuss how this emerging technology has contributed to the understanding of human lung structure-function relationships at the cell, tissue, and organ levels.

Fabrication and Characterization Techniques of In Vitro 3D Tissue Models.

The culturing of cells in the laboratory under controlled conditions has always been crucial for the advancement of scientific research. Cell-based assays have played an important role in providing simple, fast, accurate, and cost-effective methods in drug discovery, disease modeling, and tissue engineering while mitigating reliance on cost-intensive and ethically challenging animal studies. The techniques involved in culturing cells are critical as results are based on cellular response to drugs, cellular cues, external stimuli, and human physiology. In order to establish in vitro cultures, cells are either isolated from normal or diseased tissue and allowed to grow in two or three dimensions. Two-dimensional (2D) cell culture methods involve the proliferation of cells on flat rigid surfaces resulting in a monolayer culture, while in three-dimensional (3D) cell cultures, the additional dimension provides a more accurate representation of the tissue milieu. In this review, we discuss the various methods involved in the development of 3D cell culture systems emphasizing the differences between 2D and 3D systems and methods involved in the recapitulation of the organ-specific 3D microenvironment. In addition, we discuss the latest developments in 3D tissue model fabrication techniques, microfluidics-based organ-on-a-chip, and imaging as a characterization technique for 3D tissue models.

A 3D, Compartmental Tumor-Stromal Microenvironment Model of Patient-Derived Bone Metastasis.

Bone is a frequent site of tumor metastasis. The bone-tumor microenvironment is heterogeneous and complex in nature. Such complexity is compounded by relations between metastatic and bone cells influencing their sensitivity/resistance to chemotherapeutics. Standard chemotherapeutics may not show efficacy for every patient, and new therapeutics are slow to emerge, owing to the limitations of existing 2D/3D models. We previously developed a 3D interface model for personalized therapeutic screening, consisting of an electrospun poly lactic acid mesh activated with plasma species and seeded with stromal cells. Tumor cells embedded in an alginate-gelatin hydrogel are overlaid to create a physiologic 3D interface. Here, we applied our 3D model as a migration assay tool to verify the migratory behavior of different patient-derived bone metastasized cells. We assessed the impact of two different chemotherapeutics, Doxorubicin and Cisplatin, on migration of patient cells and their immortalized cell line counterparts. We observed different migratory behaviors and cellular metabolic activities blocked with both Doxorubicin and Cisplatin treatment; however, higher efficiency or lower IC50 was observed with Doxorubicin. Gene expression analysis of MDA-MB231 that migrated through our 3D hybrid model verified epithelial-mesenchymal transition through increased expression of mesenchymal markers involved in the metastasis process. Our findings indicate that we can model tumor migration in vivo, in line with different cell characteristics and it may be a suitable drug screening tool for personalized medicine approaches in metastatic cancer treatment.

Minimally Manipulated Bone Marrow-Derived Cells Can Be Used for Tissue Engineering In Situ and Simultaneous Formation of Personalized Tissue Models.

Red bone marrow and autologous bone tissue (bone fragments and bone chips) of the donor were harvested intraoperatively during autoplasty of talus bone defect. Titanium chips were obtained by grinding a fragment of a microporous titanium-coated hip arthroplasty (Zimmer). Bone marrow mononuclear cells were isolated in the operating room, and bone and titanium fragments were incubated with a suspension of mononuclear cells. The quality of revitalization was assessed by fluorescence microscopy and histological examination after culturing of adherent cells on the bone and titanium fragments. During culturing on bone chips, bone marrow mononuclear fraction cells demonstrated significantly higher metabolic activity than bone marrow cells (p=0.04). Mononuclear fraction cells were also capable of stable colonization of titanium fragments with the formation of composite tissue model.

Population-based Bayesian regularization for microstructural diffusion MRI with NODDIDA.

PURPOSE: Information on the brain microstructure can be probed by Diffusion Magnetic Resonance Imaging (dMRI). Neurite Orientation Dispersion and Density Imaging with Diffusivities Assessment (NODDIDA) is one of the simplest microstructural model proposed. However, the estimation of the NODDIDA parameters from clinically plausible dMRI acquisition is ill-posed, and different parameter sets can describe the same measurements equally well. A few approaches to resolve this problem focused on developing better optimization strategies for this non-convex optimization. However, this fundamentally does not resolve ill-posedness. This article introduces a Bayesian estimation framework, which is regularized through knowledge from an extensive dMRI measurement set on a population of healthy adults (henceforth population-based prior). METHODS: We reformulate the problem as a Bayesian maximum a posteriori estimation, which includes as a special case previous approach using non-informative uniform priors. A population-based prior is estimated from 35 subjects of the MGH Adult Diffusion data (Human Connectome Project), acquired with an extensive acquisition protocol including high b-values. The accuracy and robustness of different approaches with and without the population-based prior is tested on subsets of the MGH dataset, and an independent dataset from a clinically comparable scanner, with only clinically plausible dMRI measurements. RESULTS: The population-based prior produced substantially more accurate and robust parameter estimates, compared to the conventional uniform priors, for clinically feasible protocols, without introducing any evident bias. CONCLUSIONS: The use of the proposed Bayesian population-based prior can lead to clinically feasible and robust estimation of NODDIDA parameters without changing the acquisition protocol.

Resolving degeneracy in diffusion MRI biophysical model parameter estimation using double diffusion encoding.

PURPOSE: Biophysical tissue models are increasingly used in the interpretation of diffusion MRI (dMRI) data, with the potential to provide specific biomarkers of brain microstructural changes. However, it has been shown recently that, in the general Standard Model, parameter estimation from dMRI data is ill-conditioned even when very high b-values are applied. We analyze this issue for the Neurite Orientation Dispersion and Density Imaging with Diffusivity Assessment (NODDIDA) model and demonstrate that its extension from single diffusion encoding (SDE) to double diffusion encoding (DDE) resolves the ill-posedness for intermediate diffusion weightings, producing an increase in accuracy and precision of the parameter estimation. METHODS: We analyze theoretically the cumulant expansion up to fourth order in b of SDE and DDE signals. Additionally, we perform in silico experiments to compare SDE and DDE capabilities under similar noise conditions. RESULTS: We prove analytically that DDE provides invariant information non-accessible from SDE, which makes the NODDIDA parameter estimation injective. The in silico experiments show that DDE reduces the bias and mean square error of the estimation along the whole feasible region of 5D model parameter space. CONCLUSIONS: DDE adds additional information for estimating the model parameters, unexplored by SDE. We show, as an example, that this is sufficient to solve the previously reported degeneracies in the NODDIDA model parameter estimation.

Expression and enzyme activity of cytochrome P450 enzymes CYP3A4 and CYP3A5 in human skin and tissue-engineered skin equivalents.

CYP3A4 and CYP4A5 share specificity for a wide range of xenobiotics with the CYP3 subfamily collectively involved in the biotransformation of approximately 30% of all drugs. CYP3A4/5 mRNA transcripts have been reported in the skin, yet knowledge of their protein expression and function is lacking. In this study, we observed gene and protein expression of CYP3A4/5 in both human skin and tissue-engineered skin equivalents (TESEs), and enzyme activity was detected using the model substrate benzyl-O-methyl-cyanocoumarin. Mass spectrometric analysis of TESE lysates following testosterone application revealed a time-dependent increase in metabolite production, confirming the functional expression of these enzymes in skin.

Bioprinting technologies for disease modeling.

There is a great need for the development of biomimetic human tissue models that allow elucidation of the pathophysiological conditions involved in disease initiation and progression. Conventional two-dimensional (2D) in vitro assays and animal models have been unable to fully recapitulate the critical characteristics of human physiology. Alternatively, three-dimensional (3D) tissue models are often developed in a low-throughput manner and lack crucial native-like architecture. The recent emergence of bioprinting technologies has enabled creating 3D tissue models that address the critical challenges of conventional in vitro assays through the development of custom bioinks and patient derived cells coupled with well-defined arrangements of biomaterials. Here, we provide an overview on the technological aspects of 3D bioprinting technique and discuss how the development of bioprinted tissue models have propelled our understanding of diseases' characteristics (i.e. initiation and progression). The future perspectives on the use of bioprinted 3D tissue models for drug discovery application are also highlighted.

Multi-atlas and label fusion approach for patient-specific MRI based skull estimation.

PURPOSE: MRI-based skull segmentation is a useful procedure for many imaging applications. This study describes a methodology for automatic segmentation of the complete skull from a single T1-weighted volume. METHODS: The skull is estimated using a multi-atlas segmentation approach. Using a whole head computed tomography (CT) scan database, the skull in a new MRI volume is detected by nonrigid image registration of the volume to every CT, and combination of the individual segmentations by label-fusion. We have compared Majority Voting, Simultaneous Truth and Performance Level Estimation (STAPLE), Shape Based Averaging (SBA), and the Selective and Iterative Method for Performance Level Estimation (SIMPLE) algorithms. RESULTS: The pipeline has been evaluated quantitatively using images from the Retrospective Image Registration Evaluation database (reaching an overlap of 72.46 ± 6.99%), a clinical CT-MR dataset (maximum overlap of 78.31 ± 6.97%), and a whole head CT-MRI pair (maximum overlap 78.68%). A qualitative evaluation has also been performed on MRI acquisition of volunteers. CONCLUSION: It is possible to automatically segment the complete skull from MRI data using a multi-atlas and label fusion approach. This will allow the creation of complete MRI-based tissue models that can be used in electromagnetic dosimetry applications and attenuation correction in PET/MR.

In the beginning there were soft collagen-cell gels: towards better 3D connective tissue models?

In the 40 years since Elsdale and Bard's analysis of fibroblast culture in collagen gels we have moved far beyond the concept that such 3D fibril network systems are better models than monolayer cultures. This review analyses key aspects of that progression of models, against a background of what exactly each model system tries to mimic. This story tracks our increasing understanding of fibroblast responses to soft collagen gels, in particularly their cytoskeletal contraction, migration and integrin attachment. The focus on fibroblast mechano-function has generated models designed to directly measure the overall force generated by fibroblast populations, their reaction to external loads and the role of the matrix structure. Key steps along this evolution of 3D collagen models have been designed to mimic normal skin, wound repair, tissue morphogenesis and remodelling, growth and contracture during scarring/fibrosis. As new models are developed to understand cell-mechanical function in connective tissues the collagen material has become progressively more important, now being engineered to mimic more complex aspects of native extracellular matrix structure. These have included collagen fibril density, alignment and hierarchical structure, controlling material stiffness and anisotropy. But of these, tissue-like collagen density is key in that it contributes to control of the others. It is concluded that across this 40 year window major progress has been made towards establishing a family of 3D experimental collagen tissue-models, suitable to investigate normal and pathological fibroblast mechano-functions.

Essential Role of Anisotropy in Bioengineered Cardiac Tissue Models.

As regulatory bodies encourage alternatives to animal testing, there is renewed interest in engineering disease models, particularly for cardiac tissues. The aligned organization of cells in the mammalian heart controls the electrical and ionic currents and its ability to efficiently circulate blood to the body. Although the development of engineered cardiac systems is rising, insights into the topographical aspects, in particular, the necessity to design in vitro cardiac models incorporating cues for unidirectional cell growth, is lacking. This review first summarizes the widely used methods to organize cardiomyocytes (CMs) unidirectionally and the ways to quantify the resulting cellular alignment. The behavior of CMs in response to alignment is described, with emphasis on their functions and underlying mechanisms. Lastly, the limitations of state-of-the-art techniques to modulate CM alignment in vitro and opportunities for further development in the future to improve the cardiac tissue models that more faithfully mimic the pathophysiological hallmarks are outlined. This review serves as a call to action for bioengineers to delve deeper into the in vivo role of cellular organization in cardiac muscle tissue and draw inspiration to effectively mimic in vitro for engineering reliable disease models.

A 3D In Vitro Cortical Tissue Model Based on Dense Collagen to Study the Effects of Gamma Radiation on Neuronal Function.

Studies on gamma radiation-induced injury have long been focused on hematopoietic, gastrointestinal, and cardiovascular systems, yet little is known about the effects of gamma radiation on the function of human cortical tissue. The challenge in studying radiation-induced cortical injury is, in part, due to a lack of human tissue models and physiologically relevant readouts. Here, a physiologically relevant 3D collagen-based cortical tissue model (CTM) is developed for studying the functional response of human iPSC-derived neurons and astrocytes to a sub-lethal radiation exposure (5 Gy). Cytotoxicity, DNA damage, morphology, and extracellular electrophysiology are quantified. It is reported that 5 Gy exposure significantly increases cytotoxicity, DNA damage, and astrocyte reactivity while significantly decreasing neurite length and neuronal network activity. Additionally, it is found that clinically deployed radioprotectant amifostine ameliorates the DNA damage, cytotoxicity, and astrocyte reactivity. The CTM provides a critical experimental platform to understand cell-level mechanisms by which gamma radiation (GR) affects human cortical tissue and to screen prospective radioprotectant compounds.

A comprehensive review on 3D tissue models: Biofabrication technologies and preclinical applications.

The limitations of traditional two-dimensional (2D) cultures and animal testing, when it comes to precisely foreseeing the toxicity and clinical effectiveness of potential drug candidates, have resulted in a notable increase in the rate of failure during the process of drug discovery and development. Three-dimensional (3D) in-vitro models have arisen as substitute platforms with the capacity to accurately depict in-vivo conditions and increasing the predictivity of clinical effects and toxicity of drug candidates. It has been found that 3D models can accurately represent complex tissue structure of human body and can be used for a wide range of disease modeling purposes. Recently, substantial progress in biomedicine, materials and engineering have been made to fabricate various 3D in-vitro models, which have been exhibited better disease progression predictivity and drug effects than convention models, suggesting a promising direction in pharmaceutics. This comprehensive review highlights the recent developments in 3D in-vitro tissue models for preclinical applications including drug screening and disease modeling targeting multiple organs and tissues, like liver, bone, gastrointestinal tract, kidney, heart, brain, and cartilage. We discuss current strategies for fabricating 3D models for specific organs with their strengths and pitfalls. We expand future considerations for establishing a physiologically-relevant microenvironment for growing 3D models and also provide readers with a perspective on intellectual property, industry, and regulatory landscape.

Fabrication of ECM protein coated hollow collagen channels to study peripheral nerve regeneration.

Peripheral nerve injury is a prevalent clinical problem that often leads to lifelong disability and reduced quality of life. Although peripheral nerves can regenerate, recovery after severe injury is slow and incomplete. The current gold standard treatment, autologous nerve transplantation, has limitations including donor site morbidity and poor functional outcomes, highlighting the need for improved repair strategies. We developed a reproducible in vitro hollow channel collagen gel construct to investigate peripheral nerve regeneration (PNR) by exploring the influence of key extracellular matrix (ECM) proteins on axonal growth and regeneration. Channels were coated with ECM proteins: collagen IV, laminin, or fibronectin and seeded with dorsal root ganglia (DRG) collected from E16 rat embryos to compare the ability of the ECM proteins to enhance axonal growth. Robust axonal extension and Schwann cell (SC) infiltration were observed in fibronectin-coated channels, suggesting its superiority over other ECM proteins. Differential effects of ECM proteins on axons and SCs indicated direct growth stimulation beyond SC-mediated guidance. In vitro laceration injury modeling further confirmed fibronectin's superior pro-regenerative effects, showcasing its potential in enhancing axonal regrowth post-injury. Advancing in vitro modeling that closely replicates native microenvironments will accelerate progress in overcoming the limitations of current nerve repair approaches.

Characterization of a Human Respiratory Mucosa Model to Study Odorant Metabolism.

Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p = 0.0022), benzyl alcohol (10.05 ± 0.88%, p = 0.0008), and benzoic acid (8.49 ± 0.57%, p = 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.

Mechanism of Ocular Penetration of Lipophilic Drugs from Lipophilic Vehicles.

Since eyedrops have conventionally been formulated in aqueous vehicles, ocular pharmacokinetic studies are generally performed using aqueous buffers to identify physicochemical properties of the drug and the vehicles that influence drug absorption. In recent years, biocompatible lipophilic vehicles are increasingly finding application in ocular drug delivery; however, the mechanism of drug penetration from these non-aqueous vehicles is poorly understood. This study aims to compare ocular penetration of the model lipophilic drug curcumin when incorporated into lipophilic vehicles. To elucidate whether intrinsic solubility in the lipophilic vehicle influences ocular penetration, a curcumin solution and suspension were prepared in medium chain triglycerides (MCT) and squalane, respectively. Ocular penetration and distribution of curcumin from both vehicles was compared and evaluated qualitatively and quantitatively ex vivo. Significantly greater and faster penetration was observed from the squalane suspension than from the MCT solution in all ocular tissues. Our results suggest that the ability of lipophilic drugs to partition out of lipophilic vehicles and into cell membranes, rather than their intrinsic solubility in the lipophilic vehicle, determines the rate and extent of their ocular penetration.

Functionalized Gelatin/Polysaccharide Hydrogels for Encapsulation of Hepatocytes.

Liver diseases represent a considerable burden to patients and healthcare systems. Hydrogels play an important role in the engineering of soft tissues and may be useful for embedding hepatocytes for different therapeutic interventions or the development of in vitro models to study the pathogenesis of liver diseases or testing of drugs. Here, we developed two types of hydrogels by crosslinking hydrazide-functionalized gelatin with either oxidized dialdehyde hyaluronan or alginate through the formation of hydrazone bonds. Gel formulations were studied through texture analysis and rheometry, showing mechanical properties comparable to those of liver tissue while also demonstrating long-term stability. The biocompatibility of hydrogels and their ability to host hepatocytes was studied in vitro in comparison to pure gelatin hydrogels crosslinked by transglutaminase using the hepatocellular line HepG2. It was found that HepG2 cells could be successfully embedded in the hydrogels, showing no signs of gel toxicity and proliferating in a 3D environment comparable to pure transglutaminase cross-linked gelatin hydrogels used as control. Altogether, hydrazide gelatin in combination with oxidized polysaccharides makes stable in situ gelling systems for the incorporation of hepatocytes, which may pave the way for use in liver tissue engineering and drug testing.