High-Axial-Aspect Tannic Acid Microparticles Facilitate Gelation and Injectability of Collagen-Based Hydrogels.

Injectable collagen-based hydrogels offer great promise for tissue engineering and regeneration, but their use is limited by poor mechanical strength. Herein, we incorporate tannic acid (TA) to tailor the rheology of the corresponding hydrogels while simultaneously adding the therapeutic benefits inherent to this polyphenolic component. TA in the solution form and needle-shaped TA microparticles are combined with collagen and the respective systems studied for their time-dependent sol-gel transitions (from storage to body temperatures, 4-37 °C) as a function of TA concentration. Compared to systems incorporating TA microparticles, those with dissolved TA, applied at a similar concentration, generate a less significant enhancement of the elastic modulus. Premature gelation at a low temperature and associated colloidal arrest of the system are proposed as a main factor explaining this limited performance. A higher yield stress (elastic stress method) is determined for systems loaded with TA microparticles compared to the system with dissolved TA. These results are interpreted in terms of the underlying interactions of TA with collagen, as probed by spectroscopy and isothermal titration calorimetry. Importantly, hydrogels containing TA microparticles show high cell viability (human dermal fibroblasts) and comparative cellular activity relative to the collagen-only hydrogel. Overall, composite hydrogels incorporating TA microparticles demonstrate a new, simple, and better-performance alternative to cell culturing and difficult implantation scenarios.

Toward beta cell replacement for diabetes.

The discovery of insulin more than 90 years ago introduced a life-saving treatment for patients with type 1 diabetes, and since then, significant progress has been made in clinical care for all forms of diabetes. However, no method of insulin delivery matches the ability of the human pancreas to reliably and automatically maintain glucose levels within a tight range. Transplantation of human islets or of an intact pancreas can in principle cure diabetes, but this approach is generally reserved for cases with simultaneous transplantation of a kidney, where immunosuppression is already a requirement. Recent advances in cell reprogramming and beta cell differentiation now allow the generation of personalized stem cells, providing an unlimited source of beta cells for research and for developing autologous cell therapies. In this review, we will discuss the utility of stem cell-derived beta cells to investigate the mechanisms of beta cell failure in diabetes, and the challenges to develop beta cell replacement therapies. These challenges include appropriate quality controls of the cells being used, the ability to generate beta cell grafts of stable cellular composition, and in the case of type 1 diabetes, protecting implanted cells from autoimmune destruction without compromising other aspects of the immune system or the functionality of the graft. Such novel treatments will need to match or exceed the relative safety and efficacy of available care for diabetes.

Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages for in vitro studies. These findings could help select appropriate markers when testing macrophage behavior in vitro and highlight those markers that may confuse interpretation of results from experiments employing macrophages from different sources.

Response of human macrophages to wound matrices in vitro.

Chronic wounds remain a major burden to the global healthcare system. Myriad wound matrices are commercially available but their mechanisms of action are poorly understood. Recent studies have shown that macrophages are highly influenced by their microenvironment, but it is not known how different biomaterials affect this interaction. Here, it was hypothesized that human macrophages respond differently to changes in biomaterial properties in vitro with respect to phenotype, including pro-inflammatory M1, anti-inflammatory M2a, known for facilitating extracellular matrix deposition and proliferation, and M2c, which has recently been associated with tissue remodeling. Using multiple donors, it was found that collagen scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) promoted the least inflammatory phenotype in primary human macrophages compared with scaffolds cross-linked with formaldehyde or glutaraldehyde. Importantly, gene expression analysis trends were largely conserved between donors, especially TNFa (M1), CCL22 (M2a), and MRC1 (M2a). Then the response of primary and THP1 monocyte-derived macrophages to four commercially available wound matrices were compared-Integra Dermal Regeneration Template (Integra), PriMatrix Dermal Repair Scaffold (PriMatrix), AlloMend Acellular Dermal Matrix (AlloMend), and Oasis Wound Matrix (Oasis). Gene expression trends were different between primary and THP1 monocyte-derived macrophages for all six genes analyzed in this study. Finally, the behavior of primary macrophages cultured onto the wound matrices over time was analyzed. Integra and Oasis caused down-regulation of M2a markers CCL22 and TIMP3. PriMatrix caused up-regulation of TNFa (M1) and CD163 (M2c) and down-regulation of CCL22 and TIMP3 (both M2a). AlloMend caused up-regulation in CD163 (M2c). Lastly, Oasis promoted the largest increase in the combinatorial M1/M2 score, defined as the sum of M1 genes divided by the sum of M2 genes. This preliminary study suggested that biomaterials influenced the wound microenvironment to affect macrophage phenotype.

Characterization and biological evaluation of a novel flavonoid-collagen antioxidant hydrogel with cytoprotective properties.

Reactive oxygen species (ROS) play a critical and important role during wound healing but excess ROS at the wound site can lead to cellular damage and sub-optimal healing. Minimizing oxidative damage to the wound site and any supplemental therapeutic cells can be achieved by delivering exogenous antioxidants. Collagen hydrogels are ideal wound care materials due to their biocompatibility, high water content, and porous, three-dimensional architecture. Yet, they lack the inherent antioxidant activity that could help mitigate excess ROS at a wound site. This work formulates and evaluates the in vitro biocompatibility and antioxidant capabilities of collagen-fibroblast hydrogels combined with the polyphenolic antioxidant luteolin. Collagen solutions mixed with luteolin readily assembled into robust hydrogels with increasing gel strength due to increasing concentrations of luteolin. SEM images confirmed a mean pore size of 2.2 µm and a drastically different macromolecular ultrastructure with extensive fine crosslinking relative to collagen. Adequate cell viability and metabolic activity of dermal fibroblasts cultured within the gels were measured across all formulations, resulting in higher antioxidant activity and more than double the protection to cells from oxidative damage than traditional collagen hydrogels. Given these results, luteolin-collagen hydrogels demonstrate the potential for superior wound-healing properties when compared to collagen alone.

Human Cell-Derived Matrix Composite Hydrogels with Diverse Composition for Use in Vasculature-on-chip Models.

Microphysiological and organ-on-chip platforms seek to address critical gaps in human disease models and drug development that underlie poor rates of clinical success for novel interventions. While the fabrication technology and model cells used to synthesize organs-on-chip have advanced considerably, most platforms rely on animal-derived or synthetic extracellular matrix as a cell substrate, limiting mimicry of human physiology and precluding use in modeling diseases in which matrix dynamics play a role in pathogenesis. Here, the development of human cell-derived matrix (hCDM) composite hydrogels for use in 3D microphysiologic models of the vasculature is reported. hCDM composite hydrogels are derived from human donor fibroblasts and maintain a complex milieu of basement membrane, proteoglycans, and nonfibrillar matrix components. The use of hCDM composite hydrogels as 2D and 3D cell culture substrates is demonstrated, and hCDM composite hydrogels are patterned to form engineered human microvessels. Interestingly, hCDM composite hydrogels are enriched in proteins associated with vascular morphogenesis as determined by mass spectrometry, and functional analysis demonstrates proangiogenic signatures in human endothelial cells cultured in these hydrogels. In conclusion, this study suggests that human donor-derived hCDM composite hydrogels could address technical gaps in human organs-on-chip development and serve as substrates to promote vascularization.

Macrophages modulate the viability and growth of human mesenchymal stem cells.

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro-inflammatory (M1) and anti-inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro-inflammatory (M1) and anti-inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL-1ß, IL-6, TNF-α, and IFN-γ (for M1), and IL-10, TGF-ß1, TGF-ß3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN-γ; M2: IL-4 and IL-13) and common chemokines (SDF-1 and MCP-1) found during inflammation were also studied. Indirect and direct co-cultures were conducted using M1 and M2 polarized human THP-1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti-inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro-inflammatory (M1) environment at specific concentrations.

Towards a standardized multi-tissue decellularization protocol for the derivation of extracellular matrix materials.

The goal of tissue decellularization is to efficiently remove unwanted cellular components, such as DNA and cellular debris, while retaining the complex structural and molecular milieu within the extracellular matrix (ECM). Decellularization protocols to date are centered on customized tissue-specific and lab-specific protocols that involve consecutive manual steps which results in variable and protocol-specific ECM material. The differences that result from the inconsistent protocols between decellularized ECMs affect consistency across batches, limit comparisons between results obtained from different laboratories, and could limit the transferability of the material for consistent laboratory or clinical use. The present study is the first proof-of-concept towards the development of a standardized protocol that can be used to derive multiple ECM biomaterials (powders and hydrogels) via a previously established automated system. The automated decellularization method developed by our group was used due to its short decellularization time (4 hours) and its ability to reduce batch-to-batch variability. The ECM obtained using this first iteration of a unified protocol was able to produce ECM hydrogels from skin, lung, muscle, tendons, cartilage, and laryngeal tissues. All hydrogels formed in this study were cytocompatible and showed gelation and rheological properties consistent with previous ECM hydrogels. The ECMs also showed unique proteomic composition. The present study represents the first step towards developing standardized protocols that can be used on multiple tissues in a fast, scalable, and reproducible manner.

Scalable and High-Throughput In Vitro Vibratory Platform for Vocal Fold Tissue Engineering Applications.

The vocal folds (VFs) are constantly exposed to mechanical stimulation leading to changes in biomechanical properties, structure, and composition. The development of long-term strategies for VF treatment depends on the characterization of related cells, biomaterials, or engineered tissues in a controlled mechanical environment. Our aim was to design, develop, and characterize a scalable and high-throughput platform that mimics the mechanical microenvironment of the VFs in vitro. The platform consists of a 24-well plate fitted with a flexible membrane atop a waveguide equipped with piezoelectric speakers which allows for cells to be exposed to various phonatory stimuli. The displacements of the flexible membrane were characterized via Laser Doppler Vibrometry (LDV). Human VF fibroblasts and mesenchymal stem cells were seeded, exposed to various vibratory regimes, and the expression of pro-fibrotic and pro-inflammatory genes was analyzed. Compared to current bioreactor designs, the platform developed in this study can incorporate commercial assay formats ranging from 6- to 96-well plates which represents a significant improvement in scalability. This platform is modular and allows for tunable frequency regimes.

Low-dose intrapulmonary drug delivery device for studies on next-generation therapeutics in mice.

Although nebulizers have been developed for delivery of small molecules in human patients, no tunable device has been purpose-built for targeted delivery of modern large molecule and temperature-sensitive therapeutics to mice. Mice are used most of all species in biomedical research and have the highest number of induced models for human-relevant diseases and transgene models. Regulatory approval of large molecule therapeutics, including antibody therapies and modified RNA highlight the need for quantifiable dose delivery in mice to model human delivery, proof-of-concept studies, efficacy, and dose-response. To this end, we developed and characterized a tunable nebulization system composed of an ultrasonic transducer equipped with a mesh nebulizer fitted with a silicone restrictor plate modification to control the nebulization rate. We have identified the elements of design that influence the most critical factors to targeted delivery to the deep lungs of BALB/c mice. By comparing an in silico model of the mouse lung with experimental data, we were able to optimize and confirm the targeted delivery of over 99% of the initial volume to the deep portions of the mouse lung. The resulting nebulizer system provides targeted lung delivery efficiency far exceeding conventional nebulizers preventing waste of expensive biologics and large molecules during proof-of-concept and pre-clinical experiments involving mice. (Word Count =207).

Optimizing dynamic interactions between a cardiac patch and inflammatory host cells.

Damaged heart muscle has only a minimal ability for regeneration following myocardial infarction in which cardiomyocytes are lost to ischemia. The most clinically promising approach to regeneration of cardiac muscle currently under investigation is that of injecting cardiogenic repair cells or implanting a preformed tissue-engineered patch. While major advances are being made in the derivation of functional human cardiomyocytes and the development of tissue-engineering modalities for cardiac repair, the host environment into which the repair cells are placed is largely overlooked. Within seconds of myocardial ischemia, hypoxia sets in in the myocardium and the inflammatory response starts, characterized by rapid deployment of circulating cells and the release of paracrine and autocrine signals. Therefore, the inflammatory conditions under which these interactions take place, the design of the scaffold material used, and the maturity of the implanted cells will determine the outcomes of any stem cell-based therapy. We discuss here the interactions between implanted and inflammatory cells of the host, which are critical for the design of effective heart repair therapies.

Bioreactors for Vocal Fold Tissue Engineering.

It is estimated that almost one-third of the United States population will be affected by a vocal fold (VF) disorder during their lifespan. Promising therapies to treat VF injury and scarring are mostly centered on VF tissue engineering strategies such as the injection of engineered biomaterials and cell therapy. VF tissue engineering, however, is a challenging field as the biomechanical properties, structure, and composition of the VF tissue change upon exposure to mechanical stimulation. As a result, the development of long-term VF treatment strategies relies on the characterization of engineered tissues under a controlled mechanical environment. In this review, we highlight the importance of bioreactors as a powerful tool for VF tissue engineering with a focus on the current state of the art of bioreactors designed to mimic phonation in vitro. We discuss the influence of the phonatory environment on the development, function, injury, and healing of the VF tissue and its importance for the development of efficient therapeutic strategies. A concise and comprehensive overview of bioreactor designs, principles, operating parameters, and scalability are presented. An in-depth analysis of VF bioreactor data to date reveals that mechanical stimulation significantly influences cell viability and the expression of proinflammatory and profibrotic genes in vitro. Although the precision and accuracy of bioreactors contribute to generating reliable results, diverse gene expression profiles across the literature suggest that future efforts should focus on the standardization of bioreactor parameters to enable direct comparisons between studies. Impact statement We present a comprehensive review of bioreactors for vocal fold (VF) tissue engineering with a focus on the influence of the phonatory environment on the development, function, injury, and healing of the VFs and the importance of mimicking phonation on engineered VF tissues in vitro. Furthermore, we put forward a strong argument for the continued development of bioreactors in this area with an emphasis on the standardization of bioreactor designs, principles, operating parameters, and oscillatory regimes to enable comparisons between studies.

Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins.

Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue, resulting in junction-specific features. The ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue-specific pre-hydrogel digests. The ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities that were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissue-specific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. The ECM conditioned media had only a minor effect on the upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with a varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, another junction marker, type XXII collagen, had similar expression patterns as paxillin, with smaller effect sizes. Using tissue-specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscle-tendon junctions to study the expression of myotendinous junction-specific proteins. Impact statement The muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, the junction is often overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction-specific features and markers that should be included in tissue models of the muscle-tendon unit, while gaining a deeper understanding of the natural junction. This research aims at informing future methods to engineer a more relevant multi-tissue platform to study the muscle-tendon unit.

Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an In Vitro Hydrogel Model.

Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded in vitro and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1 in vitro. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture in vitro, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft in vivo models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future in vivo studies using human dermal fibroblasts in an in vivo model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible in vitro testing constructs.

Mast Cell-Biomaterial Interactions and Tissue Repair.

Tissue engineers often use biomaterials to provide structural support along with mechanical and chemical signals to modulate the wound healing process. Biomaterials that are implanted into the body interact with a heterogeneous and dynamic inflammatory environment that is present at the site of injury. Whether synthetically derived, naturally derived, or a combination of both, it is important to assess biomaterials for their ability to modulate inflammation to understand their potential clinical use. One important, but underexplored cell in the context of biomaterials is the mast cell (MC). MCs are granulocytic leukocytes that engage in a variety of events in both the innate and adaptive immune systems. Although highly recognized for their roles in allergic reactions, MCs play an important role in wound healing by recognizing antigens through pattern recognition receptors and the high-affinity immunoglobulin E receptor (FceRI) and releasing granules that affect cell recruitment, fibrosis, extracellular matrix deposition, angiogenesis, and vasculogenesis. MCs also mediate the foreign body response, contributing to the incorporation or rejection of implants. Studies of MC-biomaterial interactions can aid in the elucidation of MC roles during the host tissue response and tissue repair. This review is designed for those in the tissue engineering and biomaterial fields who are interested in exploring the role MCs may play in wound-biomaterial interactions and wound healing. With this review, we hope to inspire more research in the MC-biomaterial space to accelerate the design and construction of optimized implants. Impact statement Mast cells (MCs) are highly specialized inflammatory cells that have crucial, but not fully understood, roles in wound healing and tissue repair. Upon stimulation, they recognize foreign antigens and release granules that help orchestrate the inflammatory response after tissue damage or biomaterial implantation. This review summarizes the current use of MCs in biomaterial research along with literature from the past decade focusing on MC interactions with materials used for tissue repair and regeneration. Studying MC-biomaterial interactions will help (i) further understand the process of inflammation and (ii) design biomaterials and tissue-engineered constructs for optimal repair and regeneration.

Transcriptome-targeted analysis of human peripheral blood-derived macrophages when cultured on biomaterial meshes.

Surgical meshes are commonly used to repair defects and support soft tissues. Macrophages (Mφs) are critical cells in the wound healing process and are involved in the host response upon foreign biomaterials. There are various commercially available permanent and absorbable meshes used by surgeons for surgical interventions. Polypropylene (PP) meshes represent a permanent biomaterial that can elicit both inflammatory and anti-inflammatory responses. In contrast, poly-4-hydroxybutyrate (P4HB) based meshes are absorbable and linked to positive clinical outcomes but have a poorly characterized immune response. This study evaluated the in vitro targeted transcriptomic response of human Mφs seeded for 48 h on PP and P4HB surgical meshes. The in vitro measured response from human Mφs cultured on P4HB exhibited inflammatory and anti-inflammatory gene expression profiles typically associated with wound healing, which aligns with in vivo animal studies from literature. The work herein provides in vitro evidence for the early transcriptomic targeted signature of human Mφs upon two commonly used surgical meshes. The findings suggest a transition from an inflammatory to a non-inflammatory phenotype by P4HB as well as an upregulation of genes annotated under the pathogen response pathway.

Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds.

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulationin vitrosuggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.

Microphysiological System for High-Throughput Computer Vision Measurement of Microtissue Contraction.

The ability to measure microtissue contraction in vitro can provide important information when modeling cardiac, cardiovascular, respiratory, digestive, dermal, and skeletal tissues. However, measuring tissue contraction in vitro often requires the use of high number of cells per tissue construct along with time-consuming microscopy and image analysis. Here, we present an inexpensive, versatile, high-throughput platform to measure microtissue contraction in a 96-well plate configuration using one-step batch imaging. More specifically, optical fiber microprobes are embedded in microtissues, and contraction is measured as a function of the deflection of optical signals emitted from the end of the fibers. Signals can be measured from all the filled wells on the plate simultaneously using a digital camera. An algorithm uses pixel-based image analysis and computer vision techniques for the accurate multiwell quantification of positional changes in the optical microprobes caused by the contraction of the microtissues. Microtissue constructs containing 20,000-100,000 human ventricular cardiac fibroblasts (NHCF-V) in 6 mg/mL collagen type I showed contractile displacements ranging from 20-200 µm. This highly sensitive and versatile platform can be used for the high-throughput screening of microtissues in disease modeling, drug screening for therapeutics, physiology research, and safety pharmacology.

Dermal Extracellular Matrix-Derived Hydrogels as an In Vitro Substrate to Study Mast Cell Maturation.

Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.

Platelet-like particles improve fibrin network properties in a hemophilic model of provisional matrix structural defects.

Following injury, a fibrin-rich provisional matrix is formed to stem blood loss and provide a scaffold for infiltrating cells, which rebuild the damaged tissue. Defects in fibrin network formation contribute to impaired healing outcomes, as evidenced in hemophilia. Platelet-fibrin interactions greatly influence fibrin network structure via clot contraction, which increases fibrin density over time. Previously developed hemostatic platelet-like particles (PLPs) are capable of mimicking platelet functions including binding to fibrin fibers, augmenting clotting, and inducing clot retraction. In this study, we aimed to apply PLPs within a plasma-based in vitro hemophilia B model of deficient fibrin network structure to determine the ability of PLPs to improve fibrin structure and wound healing responses within hemophilia-like abnormal fibrin network formation. PLP impact on structurally deficient clot networks was assessed via confocal microscopy, a micropost deflection model, atomic force microscopy and an in vitro wound healing model of early cell migration within a provisional fibrin matrix. PLPs improved clot network density, force generation, and stiffness, and promoted fibroblast migration within an in vitro model of early wound healing under hemophilic conditions, indicating that PLPs could provide a biomimetic platform for improving wound healing events in disease conditions that cause deficient fibrin network formation.