Endogenous Extracellular Matrix Research Articles

Photoresponsive Hydrogels for Tissue Engineering.

Hydrophilic and biocompatible hydrogels are widely applied as ideal scaffolds in tissue engineering. The "smart" gelation material can alter its structural, physiochemical, and functional features in answer to various endo/exogenous stimuli to better biomimic the endogenous extracellular matrix for the engineering of cells and tissues. Light irradiation owns a high spatial-temporal resolution, complete biorthogonal reactivity, and fine-tunability and can thus induce physiochemical reactions within the matrix of photoresponsive hydrogels with good precision, efficiency, and safety. Both gel structure (e.g., geometry, porosity, and dimension) and performance (like conductivity and thermogenic or mechanical properties) can hence be programmed on-demand to yield the biochemical and biophysical signals regulating the morphology, growth, motility, and phenotype of engineered cells and tissues. Here we summarize the strategies and mechanisms for encoding light-reactivity into a hydrogel and demonstrate how fantastically such responsive gels change their structure and properties with light irradiation as desired and thus improve their applications in tissue engineering including cargo delivery, dynamic three-dimensional cell culture, and tissue repair and regeneration, aiming to provide a basis for more and better translation of photoresponsive hydrogels in the clinic.

Modified EBP-bFGF targeting endogenous renal extracellular matrix protects against renal ischemia-reperfusion injury in rats.

Acute kidney injury (AKI) is a life-threatening disease primarily caused by renal ischemia-reperfusion (I/R) injury, which can result in renal failure. Currently, growth factor therapy is considered a promising and effective approach for AKI treatment. Basic fibroblast growth factor (bFGF), an angiogenic factor with potent activity, efficiently stimulates angiogenesis and facilitates regeneration of renal tissue. However, the unrestricted diffusion of bFGF restricts its clinical application in AKI treatment. Therefore, developing a novel sustained released system for bFGF could enhance its potential in treating AKI. In this study, we genetically engineered a multifunctional recombinant protein by fusing bFGF with a specific peptide (EBP). EBP-bFGF effectively binds to the extracellular matrix in the injured kidney, enabling slow release of bFGF in AKI. Furthermore, following orthotopic injection into I/R rats' ischemic kidneys, EBP-bFGF exhibited stable retention within the tissue. Additionally, EBP-bFGF suppressed apoptosis of renal cells, reduced renal fibrosis, and facilitated recovery of renal function. These findings suggest that EBP-bFGF delivery system represents a promising strategy for treating AKI.

Molecular Mechanisms of Reelin in the Enteric Nervous System and the Microbiota-Gut-Brain Axis: Implications for Depression and Antidepressant Therapy.

Current pharmacological treatments for depression fail to produce adequate remission in a significant proportion of patients. Increasingly, other systems, such as the microbiome-gut-brain axis, are being looked at as putative novel avenues for depression treatment. Dysbiosis and dysregulation along this axis are highly comorbid with the severity of depression symptoms. The endogenous extracellular matrix protein reelin is present in all intestinal layers as well as in myenteric and submucosal ganglia, and its receptors are also present in the gut. Reelin secretion from subepithelial myofibroblasts regulates cellular migration along the crypt-villus axis in the small intestine and colon. Reelin brain expression is downregulated in mood and psychotic disorders, and reelin injections have fast antidepressant-like effects in animal models of depression. This review seeks to discuss the roles of reelin in the gastrointestinal system and propose a putative role for reelin actions in the microbiota-gut-brain axis in the pathogenesis and treatment of depression, primarily reflecting on alterations in gut epithelial cell renewal and in the clustering of serotonin transporters.

Double-Network DNA Macroporous Hydrogel Enables Aptamer-Directed Cell Recruitment to Accelerate Bone Healing.

Recruiting endogenous bone marrow mesenchymal stem cells (BMSCs) in vivo to bone defect sites shows great promise in cell therapies for bone tissue engineering, which tackles the shortcomings of delivering exogenous stem cells, including limited sources, low retention, stemness loss, and immunogenicity. However, it remains challenging to efficiently recruit stem cells while simultaneously directing cell differentiation in the dynamic microenvironment and promoting neo-regenerated tissue ingrowth to achieve augmented bone regeneration. Herein, a synthetic macroporous double-network hydrogel presenting nucleic acid aptamer and nano-inducer enhances BMSCs recruitment, and osteogenic differentiation is demonstrated. An air-in-water template enables the rapid construction of highly interconnective macroporous structures, and the physical self-assembly of DNA strands and chemical cross-linking of gelatin chains synergistically generate a resilient double network. The aptamer Apt19S and black phosphorus nanosheets-specific macroporous hydrogel demonstrate highly efficient endogenous BMSCs recruitment, cell differentiation, and extracellular matrix mineralization. Notably, the enhanced calvarial bone healing with promising matrix mineralization and new bone formation is accompanied by adapting this engineered hydrogel to the bone defects. The findings suggest an appealing material approach overcoming the traditional limitations of cell-delivery therapy that can inspire the future design of next-generation hydrogel for enhanced bone tissue regeneration.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Proregenerative extracellular matrix hydrogel mitigates pathological alterations of pelvic skeletal muscles after birth injury.

Pelvic floor disorders, including pelvic organ prolapse and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Childbirth, specifically vaginal delivery, has been recognized as the most important potentially modifiable risk factor for PFM injury; however, the precise mechanisms of PFM dysfunction after parturition remain elusive. In this study, we demonstrated that PFMs exhibit atrophy and fibrosis in parous women with symptomatic pelvic organ prolapse. These pathological alterations were recapitulated in a preclinical rat model of simulated birth injury (SBI). The transcriptional signature of PFMs after injury demonstrated an impairment in muscle anabolism, persistent expression of genes that promote extracellular matrix (ECM) deposition, and a sustained inflammatory response. We also evaluated the administration of acellular injectable skeletal muscle ECM hydrogel for the prevention of these pathological alterations. Treatment of PFMs with the ECM hydrogel either at the time of birth injury or 4 weeks after injury mitigated PFM atrophy and fibrosis. By evaluating gene expression, we demonstrated that these changes are mainly driven by the hydrogel-induced enhancement of endogenous myogenesis, ECM remodeling, and modulation of the immune response. This work furthers our understanding of PFM birth injury and demonstrates proof of concept for future investigations of proregenerative biomaterial approaches for the treatment of injured pelvic soft tissues.

Piezoelectric biomaterials for neural tissue engineering

AbstractNerve injury caused by trauma or iatrogenic trauma can lead to loss of sensory and motor function, resulting in paralysis of patients. Inspired by endogenous bioelectricity and extracellular matrix, various external physical and chemical stimuli have been introduced to treat nerve injury. Benefiting from the self‐power feature and great biocompatibility, piezoelectric biomaterials have attracted widespread attention in biomedical applications, especially in neural tissue engineering. Here, we provide an overview of the development of piezoelectric biomaterials for neural tissue engineering. First, several types of piezoelectric biomaterials are introduced, including inorganic piezoelectric nanomaterials, organic piezoelectric polymers, and their derivates. Then, we focus on the in vitro and in vivo external energy‐driven piezoelectric effects involving ultrasound, mechanical movement, and other external field‐driven piezoelectric effects. Neuroengineering applications of the piezoelectric biomaterials as in vivo grafts for the treatment of central nerve injury and peripheral nerve injury are also discussed and highlighted. Finally, the current challenges and future development of piezoelectric biomaterials for promoting nerve regeneration and treating neurological diseases are presented.

Sono-promoted drug penetration and extracellular matrix modulation potentiate sonodynamic therapy of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) exhibits difficult penetration of most drugs, leading to a very poor therapeutic outcome with a quite low five-year survival rate. The foremost reason is the highly-dense extracellular matrix (ECM) with abundant collagen and fibronectin secreted by the activated pancreatic stellate cells (PSCs). Here, we constructed a sono-responsive polymeric perfluorohexane (PFH) nanodroplet to elicit a deep drug penetration in PDAC via the combination of exogenous ultrasonic (US) exposure and endogenous ECM modulation for potent sonodynamic therapy (SDT) of PDAC. Under US exposure, the rapid drug release and deep penetration in PDAC tissues were realized. The released and well penetrated all-trans retinoic acid (ATRA) as an inhibitor of activated PSCs successfully reduced the secretion of ECM components to form a non-dense matrix conducive to drug diffusion. Meanwhile, the sonosensitizer, manganese porphyrin (MnPpIX), was triggered to produce robust reactive oxygen species (ROS) to exert the SDT effect under US exposure. Furthermore, oxygen (O2) delivered by PFH nanodroplets alleviated tumor hypoxia and enhanced the eradication of cancer cells. Overall, the sono-responsive polymeric PFH nanodroplets were successfully developed as an efficient strategy for PDAC therapy. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma (PDAC) is a representative refractory cancer with a highly dense extracellular matrix (ECM), making it difficult for most drugs to penetrate the nearly impenetrable desmoplastic stroma. Seeking methods for deep drug penetration is an extremely pressing matter for the treatment of PDAC and many other solid tumors. Herein, we designed a fluoroalkane-modified polymer to prepare a sono-responsive polymeric perfluorohexane (PFH) nanodroplet for loading sonosensitizers, and inhibitors of activated PSCs and O2. Under ultrasonic exposure, the nanodroplet elicited deep drug penetration in PDAC via ultrasonic disturbance and stromal remodeling, inducing potent sonodynamic therapy (SDT) of PDAC. By combining exogenous ultrasonic exposure and endogenous ECM modulation, this work successfully alleviated the severe physiological barrier of PDAC and achieved a favourable treatment effect.

Endogenous Extracellular Matrix Regulates the Response of Osteosarcoma 3D Spheroids to Doxorubicin

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

The adaptation of bovine embryonic stem cells to the changes of feeder layers.

Although the feeder-free culture system has been established, the microenvironment provided by the feeder cells still possesses a unique advantage in maintaining the long-term stability and the rapid proliferation of pluripotent stem cells (PSCs). The aim of this study is to discover the adaptive ability of PSCs upon changes of feeder layers. In this study, the morphology, pluripotent marker expression, differentiation ability of bovine embryonic stem cells (bESCs) cultured on low-density, or methanol fixed mouse embryonic fibroblasts were examined by immunofluorescent staining, Western blotting, real-time reverse transcription polymerase chain reaction, and RNA-seq. The results showed that the changes of feeder layers did not induce the rapid differentiation of bESCs, while resulting in the differentiation initiation and alteration of pluripotent state of bESCs. More importantly, the expression of endogenous growth factors and extracellular matrix were increased, and the expression of cell adhesion molecules was altered, which indicated that bESCs may compensate some functions of the feeder layers upon its changes. This study shows the PSCs have the self-adaptive ability responded to the feeder layer alteration.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

Optimal regeneration and repair of critical size articular cartilage driven by endogenous CLECSF1

Due to the lack of blood supply and nerve in articular cartilage, it is a great challenge to repair critical size cartilage defect, to maintain the phenotype of new chondrocytes and to restore joint function. We find that the endogenous cartilage extracellular matrix specific protein C-type lectin (CLECSF1) can be applied for the repair of cartilage defect. The endogenous expression of CLECSF1 can be achieved by integrating its coding gene into the recombinant lentiviral vectors. These vectors are used to transfect into bone marrow mesenchymal stem cells (BMSCs) and then these transfected BMSCs are seeded in a scaffold. We reveal that such scaffold can promote chondrogenic differentiation, enhance cartilage extracellular matrix deposition and maintain chondrocyte phenotype. Furthermore, such scaffold can repair critical size cartilage defect and finally achieve a full recovery of both structure and function. Therefore, we believe that such scaffold can be an ideal alternative strategy for the treatment of critical size cartilage defect in future.

Bioengineered Wound Healing Skin Models: The Role of Immune Response and Endogenous ECM to Fully Replicate the Dynamic of Scar Tissue Formation In Vitro.

The healing of deep skin wounds is a complex phenomenon evolving according with a fine spatiotemporal regulation of different biological events (hemostasis, inflammation, proliferation, remodeling). Due to the spontaneous evolution of damaged human dermis toward a fibrotic scar, the treatment of deep wounds still represents a clinical concern. Bioengineered full-thickness skin models may play a crucial role in this direction by providing a deep understanding of the process that leads to the formation of fibrotic scars. This will allow (i) to identify new drugs and targets/biomarkers, (ii) to test new therapeutic approaches, and (iii) to develop more accurate in silico models, with the final aim to guide the closure process toward a scar-free closure and, in a more general sense, (iv) to understand the mechanisms involved in the intrinsic and extrinsic aging of the skin. In this work, the complex dynamic of events underlaying the closure of deep skin wound is presented and the engineered models that aim at replicating such complex phenomenon are reviewed. Despite the complexity of the cellular and extracellular events occurring during the skin wound healing the gold standard assay used to replicate such a process is still represented by planar in vitro models that have been largely used to identify the key factors regulating the involved cellular processes. However, the lack of the main constituents of the extracellular matrix (ECM) makes these over-simplistic 2D models unable to predict the complexity of the closure process. Three-dimensional bioengineered models, which aim at recreating the closure dynamics of the human dermis by using exogenous biomaterials, have been developed to fill such a gap. Although interesting mechanistic effects have been figured out, the effect of the inflammatory response on the ECM remodelling is not replicated yet. We discuss how more faithful wound healing models can be obtained by creating immunocompetent 3D dermis models featuring an endogenous ECM.

A 3D in vitro model of the device-tissue interface: functional and structural symptoms of innate neuroinflammation are mitigated by antioxidant ceria nanoparticles

Objective. The recording instability of neural implants due to neuroinflammation at the device-tissue interface is a primary roadblock to broad adoption of brain-machine interfaces. While a multiphasic immune response, marked by glial scaring, oxidative stress (OS), and neurodegeneration, is well-characterized, the independent contributions of systemic and local ‘innate’ immune responses are not well-understood. We aimed to understand and mitigate the isolated the innate neuroinflammatory response to devices. Approach. Three-dimensional primary neural cultures provide a unique environment for studying the drivers of neuroinflammation by decoupling the innate and systemic immune systems, while conserving an endogenous extracellular matrix and structural and functional network complexity. We created a three-dimensional in vitro model of the device-tissue interface by seeding primary cortical cells around microwires. Live imaging of both dye and Adeno-Associated Virus (AAV) - mediated functional, structural, and lipid peroxidation fluorescence was employed to characterize the neuroinflammatory response. Main results. Live imaging of microtissues over time revealed independent innate neuroinflammation, marked by increased OS, decreased neuronal density, and increased functional connectivity. We demonstrated the use of this model for therapeutic screening by directly applying drugs to neural tissue, bypassing low bioavailability through the in vivo blood brain barrier. As there is growing interest in long-acting antioxidant therapies, we tested efficacy of ‘perpetual’ antioxidant ceria nanoparticles, which reduced OS, increased neuronal density, and protected functional connectivity. Significance. Our three-dimensional in vitro model of the device-tissue interface exhibited symptoms of OS-mediated innate neuroinflammation, indicating a significant local immune response to devices. The dysregulation of functional connectivity of microcircuits surround implants suggests the presence of an observer effect, in which the process of recording neural activity may fundamentally change the neural signal. Finally, the demonstration of antioxidant ceria nanoparticle treatment exhibited substantial promise as a neuroprotective and anti-inflammatory treatment strategy.

Functional periodontal regeneration using biomineralized extracellular matrix/stem cell microspheroids

Engineering complete periodontal tissue regeneration remains a considerable clinical challenge because the native periodontium is composed of hard mineralized and soft unmineralized tissues. Stem cell therapies that use endogenous extracellular matrix (ECM) have the potential for structural and functional periodontal regeneration. Here, mineralized ECM/dental pulp stem cell microspheroids (MMCMs) mimicking native mesenchymal microspheroid precursors were developed, and showed an enhanced Young’s modulus ranging from 0.4 GPa to 1.9 GPa. Furthermore, biomineralization promoted cell viability, inhibited cell apoptosis, and enhanced ECM secretion and osteogenic differentiation potential in dental pulp stem cells. After implantation in the full periodontal defect, MMCMs regenerated complete periodontium with native-like hierarchically organized periodontal ligament collagen fibers inserted into the newly formed alveolar bone and cementum; they also recruited CD90+CD73+ host mesenchymal stem cells. Mechanistically, MMCMs promoted functional periodontal regeneration through activation of the transforming growth factor beta 1 (TGF-β1)/Smad3 signaling pathway. Inhibition of TGF-β1 signaling pathway significantly impaired osteogenic potential and matrix secretion of MMCMs, while exogenous TGF-β1 treatment enhanced the osteogenesis and periodontal regeneration outcomes of MMCMs. These results indicate the promising application of MMCMs in complex tissue regeneration.

Advances in Electrospun Nerve Guidance Conduits for Engineering Neural Regeneration.

Injuries to the peripheral nervous system result in devastating consequences with loss of motor and sensory function and lifelong impairments. Current treatments have largely relied on surgical procedures, including nerve autografts to repair damaged nerves. Despite improvements to the surgical procedures over the years, the clinical success of nerve autografts is limited by fundamental issues, such as low functionality and mismatching between the damaged and donor nerves. While peripheral nerves can regenerate to some extent, the resultant outcomes are often disappointing, particularly for serious injuries, and the ongoing loss of function due to poor nerve regeneration is a serious public health problem worldwide. Thus, a successful therapeutic modality to bring functional recovery is urgently needed. With advances in three-dimensional cell culturing, nerve guidance conduits (NGCs) have emerged as a promising strategy for improving functional outcomes. Therefore, they offer a potential therapeutic alternative to nerve autografts. NGCs are tubular biostructures to bridge nerve injury sites via orienting axonal growth in an organized fashion as well as supplying a supportively appropriate microenvironment. Comprehensive NGC creation requires fundamental considerations of various aspects, including structure design, extracellular matrix components and cell composition. With these considerations, the production of an NGC that mimics the endogenous extracellular matrix structure can enhance neuron–NGC interactions and thereby promote regeneration and restoration of function in the target area. The use of electrospun fibrous substrates has a high potential to replicate the native extracellular matrix structure. With recent advances in electrospinning, it is now possible to generate numerous different biomimetic features within the NGCs. This review explores the use of electrospinning for the regeneration of the nervous system and discusses the main requirements, challenges and advances in developing and applying the electrospun NGC in the clinical practice of nerve injuries.

Engineered Cell-Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues.

The clinical translation of mesenchymal stromal cell (MSC)-based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue-forming potential by promoting cell-cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell-secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell-secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell-matrix and cell-cell interactions. ECM-loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes-associated protein (YAP) activation via integrin α2β1 binding. ECM-loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage-specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM-loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM-loaded spheroids as building blocks for the repair of musculoskeletal tissues.

Open-source human skin model with an in vivo-like barrier for drug testing.

There is a global trend towards the development of physiologically relevant in vitro skin models to reduce or replace animal testing in the evaluation of therapeutic drug candidates. However, only commercial reconstructed human epidermis models (RHEm) have undergone formal validation. Although these commercial models are suitable for a wide range of applications, they are costly, lack flexibility, and the protocols used to generate them are not transparent. In this study, we present an open-source full-thickness skin model (FTSm) and assess its potential for drug testing. The FTSm was developed using endogenous extracellular matrix to recreate the dermal compartment, avoiding animal-derived hydrogels. An RHEm based on an open-source protocol was evaluated in parallel. The integrity of the skin barrier was analyzed by challenging the surface with detergents and measuring cell viability as well as by trans-epithelial electrical resistance (TEER) measurements. Skin irritation studies were performed based on OECD guidelines and complemented with an evaluation of the impact on the skin barrier by TEER measurement. The permeation of a dye through the developed models and a commercial membrane (Strat-M®) was compared using Franz diffusion cells and an infinite dose approach. The FTSm demonstrated structural and barrier properties comparable to native human skin. Although the RHEm showed a better performance in drug testing, the FTSm presented better barrier properties than commercial models as reported in the literature. These skin models can be a valuable contribution to accelerating the development and dissemination of alternatives to animal testing, avoiding the limitations of commercial models.

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry.

The goal of this study was to develop strategies to localize human collagen-based hydrogels within an infarcted mouse heart, as well as analyze its impact on endogenous extracellular matrix (ECM) remodeling. Collagen is a natural polymer that is abundantly used in bioengineered hydrogels because of its biocompatibility, cell permeability, and biodegradability. However, without the use of tagging techniques, collagen peptides derived from hydrogels can be difficult to differentiate from the endogenous ECM within tissues. Imaging mass spectrometry is a robust tool capable of visualizing synthetic and natural polymeric molecular structures yet is largely underutilized in the field of biomaterials outside of surface characterization. In this study, our group leveraged a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technique to enzymatically target collagen and other ECM peptides within the tissue microenvironment that are both endogenous and hydrogel-derived. Using a multimodal approach of fluorescence microscopy and ECM-IMS techniques, we were able to visualize and relatively quantify significantly abundant collagen peptides in an infarcted mouse heart that were localized to regions of therapeutic hydrogel injection sites. On-tissue MALDI MS/MS was used to putatively identify sites of collagen peptide hydroxyproline site occupancy, a post-translational modification that is critical in collagen triple helical stability. Additionally, the technique could putatively identify over 35 endogenously expressed ECM peptides that were expressed in hydrogel-injected mouse hearts. Our findings show evidence for the use of MALDI-IMS in assessing the therapeutic application of collagen-based biomaterials.