Nanomaterial-Based Scaffolds for Tissue Engineering Applications: A Review on Graphene, Carbon Nanotubes and Nanocellulose.

Nanoscale biomaterials have garnered immense interest in the scientific community in the recent decade. This review specifically focuses on the application of three nanomaterials, i.e., graphene and its derivatives (graphene oxide, reduced graphene oxide), carbon nanotubes (CNTs) and nanocellulose (cellulose nanocrystals or CNCs and cellulose nanofibers or CNFs), in regenerating different types of tissues, including skin, cartilage, nerve, muscle and bone. Their excellent inherent (and tunable) physical, chemical, mechanical, electrical, thermal and optical properties make them suitable for a wide range of biomedical applications, including but not limited to diagnostics, therapeutics, biosensing, bioimaging, drug and gene delivery, tissue engineering and regenerative medicine. A state-of-the-art literature review of composite tissue scaffolds fabricated using these nanomaterials is provided, including the unique physicochemical properties and mechanisms that induce cell adhesion, growth, and differentiation into specific tissues. In addition, in vitro and in vivo cytotoxic effects and biodegradation behavior of these nanomaterials are presented. We also discuss challenges and gaps that still exist and need to be addressed in future research before clinical translation of these promising nanomaterials can be realized in a safe, efficacious, and economical manner.

Versatility of mesenchymal stem cell-derived extracellular vesicles in tissue repair and regenerative applications.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic cells that have been widely explored in the field of regenerative medicine. MSCs possess the ability to secrete soluble factors as well as lipid bound extracellular vesicles (EVs). MSCs have gained increased interest and attention as a result of their therapeutic properties, which are thought to be attributed to their secretome. However, while the use of MSCs as whole cells pose heterogeneity concerns and survival issues post-transplantation, such limitations are absent in cell-free EV-based treatments. EVs derived from MSCs are promising therapeutic agents for a range of clinical conditions and disorders owing to their immunomodulatory, pro-regenerative, anti-inflammatory, and antifibrotic activity. Recent successes with preclinical studies using EVs for repair and regeneration of damaged tissues such as cardiac tissue, lung, liver, pancreas, bone, skin, cornea, and blood diseases are discussed in this review. We also discuss delivery strategies of EVs using biomaterials as delivery vehicles through systemic or local administration. Despite its effectiveness in preclinical investigations, the application of MSC-EV in clinical settings will necessitate careful consideration surrounding issues such as: i) scalability and isolation, ii) biodistribution, iii) targeting specific tissues, iv) quantification and characterization, and v) safety and efficacy of dosage. The future of EVs in regenerative medicine is promising yet still needs further investigation on enhancing the efficacy, scalability, and potency for clinical applications.

Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Peripheral Nerve Decellularization for In Vitro Extracellular Matrix Hydrogel Use: A Comparative Study.

The rise of tissue-engineered biomaterials has introduced more clinically translatable models of disease, including three-dimensional (3D) decellularized extracellular matrix (dECM) hydrogels. Specifically, decellularized nerve hydrogels have been utilized to model peripheral nerve injuries and disorders in vitro; however, there lacks standardization in decellularization methods. Here, rat sciatic nerves of varying preparations were decellularized using previously established methods: sodium deoxycholate (SD)-based, 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS)-based, and apoptosis-mediated. These nerves were characterized for cellular debris removal, ECM retention, and low cytotoxicity with cultured Schwann cells. The best preparations of each decellularization method were digested into dECM hydrogels, and rheological characterization, gelation kinetics, and confocal reflectance imaging of collagen fibril assembly were performed. It was determined that the SD-based method with nerve epineurial removal best maintained the overall ECM composition and mechanical properties of physiological peripheral nerves while efficiently stripping the scaffolds of tissue-specific cells and debris. This method was then utilized as a culture platform for quiescent Schwann cells and cancer-nerve crosstalk. Hydrogel-embedded Schwann cells were found to have high viability and act in a more physiologically relevant manner than those cultured in monolayers, and the hydrogel platform allowed for the activation of Schwann cells following treatment with cancer secreted factors. These findings establish a standard for peripheral nerve decellularization for usage as a dECM hydrogel testbed for in vitro peripheral nerve disease modeling and may facilitate the development of treatments for peripheral nerve disease and injury.

Exploring the Role of Metabolites in Cancer and the Associated Nerve Crosstalk.

Since Otto Warburg's first report on the increased uptake of glucose and lactate release by cancer cells, dysregulated metabolism has been acknowledged as a hallmark of cancer that promotes proliferation and metastasis. Over the last century, studies have shown that cancer metabolism is complex, and by-products of glucose and glutamine catabolism induce a cascade of both pro- and antitumorigenic processes. Some vitamins, which have traditionally been praised for preventing and inhibiting the proliferation of cancer cells, have also been proven to cause cancer progression in a dose-dependent manner. Importantly, recent findings have shown that the nervous system is a key player in tumor growth and metastasis via perineural invasion and tumor innervation. However, the link between cancer-nerve crosstalk and tumor metabolism remains unclear. Here, we discuss the roles of relatively underappreciated metabolites in cancer-nerve crosstalk, including lactate, vitamins, and amino acids, and propose the investigation of nutrients in cancer-nerve crosstalk based on their tumorigenicity and neuroregulatory capabilities. Continued research into the metabolic regulation of cancer-nerve crosstalk will provide a more comprehensive understanding of tumor mechanisms and may lead to the identification of potential targets for future cancer therapies.

Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth-permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug-loaded microparticles for a combinatorial approach to neural injury therapies.

Questionnaire Survey on Current Red Blood Cell Transport and Storage in Korea for Reducing Wastage.

BACKGROUND: To ensure safe red blood cell (RBC) transfusion practice, it is important to comply with storage and transport requirements of RBC units. We conducted a comprehensive survey on the practice of RBC transport and storage to explore the awareness of and compliance with the 30-minute rule, the current status of RBC unit transport, and possible utility of temperature indicators (TIs) to reduce RBC wastage. METHODS: From June to August of 2019, 64 blood bank physicians (14 questions) in 64 secondary- and tertiary-care hospitals and 673 nurses (13 questions) in 42 tertiary-care hospitals replied to a questionnaire survey. The results of the survey were analyzed with descriptive statistics. RESULTS: Among the physicians surveyed, 97.0% (N=62) of hospitals had transfusion guidelines in place. The RBC wastage in 2018 ranged from less than five units to more than 200 units. Among the nurses surveyed, 99.4% (N=669) were aware of and complied with the 30-minute rule; 13.5% (N=91) of the nurses had experience of RBC wastage due to violation of the 30-minute rule. Both physicians (67%, N=43) and nurses (83.1%, N=559) responded that TIs would help reduce RBC wastage. CONCLUSIONS: This is the first survey on the practices related to RBC transport and storage in Korea. This study provides fundamental data on current practice for the blood cold chain, insights into RBC wastage, and highlights the utility of TIs.

Development of novel apoptosis-assisted lung tissue decellularization methods.

Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications. The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with patient-derived cells prior to transplantation, or digested to create thermally-gelling ECM hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergent-based decellularization protocol. Furthermore, thermally-gelling lung ECM hydrogels supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling purposes.

The biology and engineered modeling strategies of cancer-nerve crosstalk.

A recent finding critical to cancer aggravation is the interaction between cancer cells and nerves. There exist two main modes of cancer-nerve interaction: perineural invasion (PNI) and tumor innervation. PNI occurs when cancer cells infiltrate the adjacent nerves, and its relative opposite, tumor innervation, occurs when axons extend into tumor bodies. Like most cancer studies, these crosstalk interactions have mostly been observed in patient samples and animal models at this point, making it difficult to understand the mechanisms in a controlled manner. As such, in recent years in vitro studies have emerged that have helped identify various microenvironmental factors responsible for cancer-nerve crosstalk, including but not limited to neurotrophic factors, neurotransmitters, chemokines, cancer-derived exosomes, and Schwann cells. The versatility of in vitro systems warrants continuous development to increase physiological relevance to study PNI and tumor innervation, for example by utilizing biomimetic three-dimensional (3D) culture systems. Despite the wealth of 3D in vitro cancer models, comparatively there exists a lack of 3D in vitro models of nerve, PNI, and tumor innervation. Native-like 3D in vitro models of cancer-nerve interactions may further help develop therapeutic strategies to curb nerve-mediated cancer aggravation. As such, we provide an overview of the key players of cancer-nerve crosstalk and current in vitro models of the crosstalk, as well as cancer and nerve models. We also discuss a few future directions in cancer-nerve crosstalk research.

Decellularized tissues as platforms for in vitro modeling of healthy and diseased tissues.

Biomedical engineers are at the forefront of developing novel treatments to improve human health, however, many products fail to translate to clinical implementation. In vivo pre-clinical animal models, although the current best approximation of complex disease conditions, are limited by reproducibility, ethical concerns, and poor accurate prediction of human response. Hence, there is a need to develop physiologically relevant, low cost, scalable, and reproducible in vitro platforms to provide reliable means for testing drugs, biomaterials, and tissue engineered products for successful clinical translation. One emerging approach of developing physiologically relevant in vitro models utilizes decellularized tissues/organs as biomaterial platforms for 2D and 3D models of healthy and diseased tissue. Decellularization is a process that removes cellular content and produces tissue-specific extracellular matrix scaffolds that can more accurately recapitulate an organ/tissue's native microenvironment compared to other natural or synthetic materials. Decellularized tissues hold enormous potential for in vitro modeling of various disease phenotypes and tissue responses to drugs or external conditions such as aging, toxin exposure, or even implantation. In this review, we highlight the need for in vitro models, the advantages and limitations of implementing decellularized tissues, and considerations of the decellularization process. We discuss current research efforts towards applying decellularized tissues as platforms to generate in vitro models of healthy and diseased tissues, and where we foresee the field progressing. A variety of organs/tissues are discussed, including brain, heart, kidney, large intestine, liver, lung, skeletal muscle, skin, and tongue. STATEMENT OF SIGNIFICANCE: Many biomedical products fail to reach clinical translation due to animal model limitations. Development of physiologically relevant in vitro models can provide a more economic, scalable, and reproducible means of testing drugs/therapeutics for successful clinical translation. The use of decellularized tissues as platforms for in vitro models holds promise, as these scaffolds can effectively replicate native tissue complexity, but is not widely explored. This review discusses the need for in vitro models, the promise of decellularized tissues as biomaterial substrates, and the current research applying decellularized tissues towards the creation of in vitro models. Further, this review provides insights into the current limitations and future of such in vitro models.

Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Oligonucleotide-functionalized hydrogels for sustained release of small molecule (aptamer) therapeutics.

Natural and synthetic hydrogels have been widely investigated as biomaterial scaffolds to promote tissue repair and regeneration. Nevertheless, the scaffold alone is often insufficient to drive new tissue growth, instead requiring continuous delivery of therapeutics, such as proteins or other biomolecules that work in concert with structural support provided by the scaffold. However, because of the high-water content, hydrogels tend to be permeable and cause rapid release of the encapsulated drug, which could lead to serious complications from local overdose and may result in the significant waste of encapsulated therapeutic(s). To this end, we designed an oligonucleotide-functionalized hydrogel that can provide sustained and controlled delivery of therapeutics for up to 4 weeks. To prove this concept, we successfully achieved sustained release (for over 28 days) of model anti-Nogo receptor (anti-NgR) RNA aptamer from oligonucleotide-functionalized hyaluronic acid-based hydrogel by changing the complementarity between the short antisense sequences and the aptamer. Furthermore, the released aptamer successfully blocked neuro-inhibitory effects of myelin-derived inhibitors and promoted neurite outgrowth from rat dorsal root ganglia in vitro. Because antisense sequences can be designed to bind to proteins, peptides, and aptamer, our oligonucleotide-functionalized hydrogel offers a promising therapeutic delivery system to obtain controlled release (both bolus and sustained) of various therapeutics for the treatment of complex diseases and injury models, such as spinal cord injury. STATEMENT OF SIGNIFICANCE: Producing a therapeutic effect often requires the administration of multiple injections with high dosages. This regimen causes discomfort to the patient and raises cost of treatment. Additionally, systemic delivery of therapeutics often results in adverse effects; therefore, local delivery at the site of injury is desirable. Therefore, in this study, we designed an oligonucleotide-functionalized biomaterial platform using ssDNA oligonucleotides (immobile species) as antisense sequences to increase residence time and fine-tune the release of anti-nogo receptor aptamer (mobile species) for spinal cord injury application. Because antisense sequences can be designed to bind proteins, peptides, and aptamer, our hydrogel offers a promising delivery system to obtain controlled release of various therapeutics for the treatment of complex diseases and injury models.

Novel Sodium Deoxycholate-Based Chemical Decellularization Method for Peripheral Nerve.

Decellularized peripheral nerve has been proven to be an effective clinical intervention for peripheral nerve repair and a preclinical cell carrier after spinal cord injury. However, there are currently a lack of decellularization methods for peripheral nerve that remove cells and maintain matrix similar to the previously established, clinically translated technique (the Hudson method) that relies on the discontinued Triton X-200 detergent. Therefore, the aim of this study was to optimize a novel chemical decellularization method for peripheral nerves based on the currently available anionic detergent sodium deoxycholate. Sprague Dawley rat sciatic nerves were isolated, frozen in buffered solution, and then subject to sequential washes in water, salt buffer, zwitterionic detergents sulfobetaines -10 and -16, and varying concentrations of sodium deoxycholate (SDC). To optimize DNA removal after SDC decellularization, nerves were subjected to deoxyribonuclease (DNase) incubation and salt buffer washes. Immunohistochemical results demonstrated that utilization of 3% SDC in the decellularization process preserved extracellular matrix (ECM) components and structure while facilitating significantly better removal of Schwann cells, axons, and myelin compared with the Hudson method. The addition of a 3-h DNase incubation to the 3% SDC decellularization process significantly removed cellular debris compared with the Hudson method. Proteomic analysis demonstrated that our novel decellularization method based on 3% SDC +3-h DNase used in conjunction with zwitterionic detergents, and salt buffers (new decellularization method using 3% SDC + 3-h DNase, zwitterionic detergents, and salt buffers [SDD method]) produced a similar proteomic profile compared with the Hudson method and had significantly fewer counts of cellular proteins. Finally, cytotoxicity analysis demonstrated that the SDD decellularized scaffolds do not contain significant cytotoxic residuals as eluted media supported metabolically active Schwann cells in vitro. Overall, this study demonstrates that SDD decellularization represents a novel alternative utilizing currently commercially available chemical reagents. Impact Statement Decellularized nerves are clinically relevant materials that can be used for a variety of regenerative applications such as peripheral nerve and spinal cord injury repair. However, discontinuation of key detergents used in a proven chemical decellularization process necessitates the optimization of an equivalent or better method. This research presents the field with a novel chemical decellularization method to replace the previous validated standard. Scaffolds generated from this method provide an extracellular matrix-rich material that can be used in a variety of in vitro applications to understand cellular behavior and in vivo applications to facilitate regeneration after neural injury.

Phosphate supplementation for hypophosphatemia during continuous renal replacement therapy in adults.

BACKGROUND: Hypophosphatemia is common during continuous renal replacement therapy (CRRT) in critically ill patients and can cause generalized muscle weakness, prolonged respiratory failure, and myocardial dysfunction. This study aimed to investigate the efficacy and safety of adding phosphate to the dialysate and replacement solutions to treat hypophosphatemia occurring in intensive CRRT in critically ill patients. METHODS: We retrospectively analyzed 73 patients treated with intensive CRRT (effluent flow ≥35 ml/kg/hr) in the intensive care unit. The control group (group 1, n = 22) received no phosphate supplementation. The treatment groups received dialysate and replacement solution phosphate supplementation at 2.0 mmol/L (group 2, n = 26) or 3.0 mmol/L (group 3, n = 25). RESULTS: The CRRT-induced hypophosphatemia incidence was 59.0%. Correction of hypophosphatemia with phosphate supplementation changed the mean serum phosphorus levels to 1.24 ± 0.37 and 1.44 ± 0.31 mmol/L in groups 2 and 3, respectively (p = .02). The time required for correction was 1.65 ± 0.80 and 1.39 ± 1.43 days for groups 2 and 3, respectively and was significantly longer in group 2 (p = .02). After supplementation, hypophosphatemia, and hyperphosphatemia both occurred in 7% of group 2. Group 3 developed no hypophosphatemia, but 20% developed hyperphosphatemia. The serum phosphate levels in hyperphosphatemia cases returned to normal within 2.0 days (group 2) and 1.0 day (group 3) after stopping phosphate supplementation. CONCLUSION: Phosphate supplementation effectively corrected CRRT-induced hypophosphatemia in critically ill patients with an acute kidney injury. The use of 2 mmol/L phosphate is appropriate in patients with CRRT-induced hypophosphatemia, but a different concentration could be required to prevent hypophosphatemia at the start of CRRT.

Advances in ex vivo models and lab-on-a-chip devices for neural tissue engineering.

The technologies related to ex vivo models and lab-on-a-chip devices for studying the regeneration of brain, spinal cord, and peripheral nerve tissues are essential tools for neural tissue engineering and regenerative medicine research. The need for ex vivo systems, lab-on-a-chip technologies and disease models for neural tissue engineering applications are emerging to overcome the shortages and drawbacks of traditional in vitro systems and animal models. Ex vivo models have evolved from traditional 2D cell culture models to 3D tissue-engineered scaffold systems, bioreactors, and recently organoid test beds. In addition to ex vivo model systems, we discuss lab-on-a-chip devices and technologies specifically for neural tissue engineering applications. Finally, we review current commercial products that mimic diseased and normal neural tissues, and discuss the future directions in this field.

Intrafibrillar, bone-mimetic collagen mineralization regulates breast cancer cell adhesion and migration.

Bone metastasis is a leading cause of death in patients with breast cancer, but the underlying mechanisms are poorly understood. While much work focuses on the molecular and cellular events that drive breast cancer bone metastasis, it is mostly unclear what role bone extracellular matrix (ECM) properties play in this process. Bone ECM primarily consists of mineralized collagen fibrils, which are composed of non-stoichiometric carbonated apatite (HA) and collagen type I. Reduced bone mineral content is epidemiologically linked with increased risk of bone metastasis. Yet elucidating the potential functional impact of collagen mineralization on breast cancer cells has remained challenging because of a lack of model systems that allow studying tumor cell behavior as a function of physiological, intrafibrillar collagen mineralization. Here, we have developed cell culture substrates composed of mineralized collagen type I fibrils using a polymer-induced liquid-precursor (PILP) process. Intrafibrillar HA decreased breast cancer cell adhesion forces and accordingly reduced collagen fiber alignment relative to cells cultured on control collagen. The resulting mineral-mediated changes in collagen network characteristics and mechanosignaling correlated with increased cell motility, but inhibited directed migration of breast cancer cells. These results suggest that physiological mineralization of collagen fibrils reduces tumor cell adhesion with potential functional consequences on skeletal homing of disseminated tumor cells in early stages of breast cancer metastasis.

Recent advances in nanotherapeutic strategies for spinal cord injury repair.

Spinal cord injury (SCI) is a devastating and complicated condition with no cure available. The initial mechanical trauma is followed by a secondary injury characterized by inflammatory cell infiltration and inhibitory glial scar formation. Due to the limitations posed by the blood-spinal cord barrier, systemic delivery of therapeutics is challenging. Recent development of various nanoscale strategies provides exciting and promising new means of treating SCI by crossing the blood-spinal cord barrier and delivering therapeutics. As such, we discuss different nanomaterial fabrication methods and provide an overview of recent studies where nanomaterials were developed to modulate inflammatory signals, target inhibitory factors in the lesion, and promote axonal regeneration after SCI. We also review emerging areas of research such as optogenetics, immunotherapy and CRISPR-mediated genome editing where nanomaterials can provide synergistic effects in developing novel SCI therapy regimens, as well as current efforts and barriers to clinical translation of nanomaterials.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis.

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

Breast cancer-derived extracellular vesicles stimulate myofibroblast differentiation and pro-angiogenic behavior of adipose stem cells.

Adipose-derived stem cells (ASCs) are abundantly present in the mammary microenvironment and can promote breast cancer malignancy by differentiating into myofibroblasts. However, it remains largely unclear which role tumor-derived extracellular vesicles (TEVs) play in this process. Here, we used microfabricated, type I collagen-based 3-D tissue culture platforms to investigate the effect of breast cancer cell-derived TEVs on ASCs myofibroblast differentiation and consequential changes in extracellular matrix remodeling and vascular sprouting. TEVs collected from MDA MB-231 human metastatic breast cancer cells (MDAs) promoted ASC myofibroblast differentiation in both 2-D and 3-D cultures as indicated by increased alpha smooth muscle actin (α-SMA) and fibronectin (Fn) levels. Correspondingly, TEV-treated ASCs were more contractile, secreted more vascular endothelial growth factor (VEGF), and promoted angiogenic sprouting of human umbilical vein endothelial cells (HUVECs). These changes were dependent on transforming growth factor beta (TGF-ß)-related signaling and tumor cell glutaminase activity as their inhibition decreased TEV-related myofibroblastic differentiation of ASCs and related functional consequences. In summary, our data suggest that TEVs are important signaling factors that contribute to ASC desmoplastic reprogramming in the tumor microenvironment, and suggest that tumor cell glutamine metabolism may be used as a therapeutic target to interfere with this process.

Adipose-derived stem cells increase angiogenesis through matrix metalloproteinase-dependent collagen remodeling.

Adipose-derived stem cells (ASCs) are key regulators of new blood vessel formation and widely investigated for their role in tissue regeneration and tumorigenesis. However, the cellular and molecular mechanisms through which ASCs regulate angiogenesis are not well understood. Here, it was our goal to test the functional contribution of ASC-mediated extracellular matrix (ECM) remodeling on endothelial cell invasion. To isolate the effect of ECM-remodeling, ASCs were embedded within 3-D collagen type I hydrogels and pre-cultured for 7 days; controls were not pre-cultured. A confluent monolayer of human umbilical vein endothelial cells (HUVECs) was seeded on top and its invasion into the underlying hydrogel was analyzed. Without pre-culture, ASCs inhibited vascular sprouting by stabilizing the endothelium. In contrast, 7 day pre-culture of ASCs drastically increased invasion by HUVECs. This effect was largely mediated by proteolytic ECM degradation by ASC-derived matrix metalloproteinases (MMPs) rather than vascular endothelial growth factor (VEGF), as our results indicated that blockade of MMPs, but not VEGF, inhibited endothelial sprouting. Collectively, these data suggest that the angiogenic capability of ASCs is modulated by their proteolytic remodeling of the ECM, opening new avenues for pro- and anti-angiogenic therapies.