Post-death Vesicles of Senescent Bone Marrow Mesenchymal Stromal Polyploids Promote Macrophage Aging and Breast Cancer.

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form "spikings" which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCE: Mechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form "spikings" which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk.

Development of pro-angiogenic skin substitutes for wound healing.

Wounds pose significant challenges to public health, primarily due to the loss of the mechanical integrity and barrier function of the skin and impaired angiogenesis, causing physical morbidities and psychological trauma to affect patients. Reconstructing the vasculature of the wound bed is crucial for promoting wound healing, reducing scar formation and enhancing the quality of life for patients. The development of pro-angiogenic skin substitutes has emerged as a promising strategy to facilitate vascularization and expedite the healing process of burn wounds. This review provides an overview of the various types of skin substitutes employed in wound healing, explicitly emphasising those designed to enhance angiogenesis. Synthetic scaffolds, biological matrices and tissue-engineered constructs incorporating stem cells and primary cells, cell-derived extracellular vesicles (EVs), pro-angiogenic growth factors and peptides, as well as gene therapy-based skin substitutes are thoroughly examined. The review summarises the existing challenges, future directions and potential innovations in pro-angiogenic dressing for skin substitutes. It highlights the need for continued research to develop new technologies and combine multiple strategies and factors, and to overcome obstacles and advance the field, ultimately leading to improved outcomes for wound patients.

A bio-instructive parylene-based conformal coating suppresses thrombosis and intimal hyperplasia of implantable vascular devices.

Implantable vascular devices are widely used in clinical treatments for various vascular diseases. However, current approved clinical implantable vascular devices generally have high failure rates primarily due to their surface lacking inherent functional endothelium. Here, inspired by the pathological mechanisms of vascular device failure and physiological functions of native endothelium, we developed a new generation of bioactive parylene (poly(p-xylylene))-based conformal coating to address these challenges of the vascular devices. This coating used a polyethylene glycol (PEG) linker to introduce an endothelial progenitor cell (EPC) specific binding ligand LXW7 (cGRGDdvc) onto the vascular devices for preventing platelet adhesion and selectively capturing endogenous EPCs. Also, we confirmed the long-term stability and function of this coating in human serum. Using two vascular disease-related large animal models, a porcine carotid artery interposition model and a porcine carotid artery-jugular vein arteriovenous graft model, we demonstrated that this coating enabled rapid generation of self-renewable "living" endothelium on the blood contacting surface of the expanded polytetrafluoroethylene (ePTFE) grafts after implantation. We expect this easy-to-apply conformal coating will present a promising avenue to engineer surface properties of "off-the-shelf" implantable vascular devices for long-lasting performance in the clinical settings.

Engineered multi-functional, pro-angiogenic collagen-based scaffolds loaded with endothelial cells promote large deep burn wound healing.

The lack of vascularization associated with deep burns delays the construction of wound beds, increases the risks of infection, and leads to the formation of hypertrophic scars or disfigurement. To address this challenge, we have fabricated a multi-functional pro-angiogenic molecule by grafting integrin αvß3 ligand LXW7 and collagen-binding peptide (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY (LDS), and further employed this to functionalize collagen-based Integra scaffolds. Using a large deep burn wound model in C57/BLK6 mice (8-10 weeks old, 26-32g, n = 39), we demonstrated that LDS-modified collagen-based Integra scaffolds loaded with endothelial cells (ECs) accelerate wound healing rate, re-epithelialization, vascularization, and collagen deposition. Specifically, a 2 cm × 3 cm full-thickness skin burn wound was created 48 h after the burn, and then wounds were treated with four groups of different dressing scaffolds, including Integra + ECs, Integra + LDS, and Integra + LDS + ECs with Integra-only as the control. Digital photos were taken for wound healing measurement on post-treatment days 1, 7, 14, 21, 28, and 35. Post-treatment photos revealed that treatment with the Intgera + LDS + ECs scaffold exhibited a higher wound healing rate in the proliferation phase. Histology results showed significantly increased re-epithelialization, increased collagen deposition, increased thin and mixed collagen fiber content, increased angiogenesis, and shorter wound length within the Integra + LDS + ECs group at Day 35. On Day 14, the Integra + LDS + ECs group showed the same trend. The relative proportions of collagen changed from Day 14 to Day 35 in the Integra + LDS + ECs and Integra + ECs groups demonstrated decreased thick collagen fiber deposition and greater thin and mixed collagen fiber deposition. LDS-modified Integra scaffolds represent a promising novel treatment to accelerate deep burn wound healing, thereby potentially reducing the morbidity associated with open burn wounds. These scaffolds can also potentially reduce the need for autografting and morbidity in patients with already limited areas of harvestable skin.

Evaluation of a biodegradable polyurethane patch for repair of diaphragmatic hernia in a rat model: A pilot study.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) repair is an area of active research. Large defects requiring patches have a hernia recurrence rate of up to 50%. We designed a biodegradable polyurethane (PU)-based elastic patch that matches the mechanical properties of native diaphragm muscle. We compared the PU patch to a non-biodegradable Gore-Tex™ (polytetrafluoroethylene) patch. METHODS: The biodegradable polyurethane was synthesized from polycaprolactone, hexadiisocyanate and putrescine, and then processed into fibrous PU patches by electrospinning. Rats underwent 4 mm diaphragmatic hernia (DH) creation via laparotomy followed by immediate repair with Gore-Tex™ (n = 6) or PU (n = 6) patches. Six rats underwent sham laparotomy without DH creation/repair. Diaphragm function was evaluated by fluoroscopy at 1 and 4 weeks. At 4 weeks, animals underwent gross inspection for recurrence and histologic evaluation for inflammatory reaction to the patch materials. RESULTS: There were no hernia recurrences in either cohort. Gore-Tex™ had limited diaphragm rise compared to sham at 4 weeks (1.3 mm vs 2.9 mm, p = 0.003), but no difference was found between PU and sham (1.7 mm vs 2.9 mm, p = 0.09). There were no differences between PU and Gore-Tex™ at any time point. Both patches formed an inflammatory capsule, with similar thicknesses between cohorts on the abdominal (Gore-Tex™ 0.07 mm vs. PU 0.13 mm, p = 0.39) and thoracic (Gore-Tex™ 0.3 mm vs. PU 0.6 mm, p = 0.09) sides. CONCLUSION: The biodegradable PU patch allowed for similar diaphragmatic excursion compared to control animals. There were similar inflammatory responses to both patches. Further work is needed to evaluate long-term functional outcomes and further optimize the properties of the novel PU patch in vitro and in vivo. LEVEL OF EVIDENCE: Level II, Prospective Comparative Study.

A bioactive material with dual integrin-targeting ligands regulates specific endogenous cell adhesion and promotes vascularized bone regeneration in adult and fetal bone defects.

Significant progress has been made in designing bone materials capable of directing endogenous cells to promote vascularized bone regeneration. However, current strategies lack regulation of the specific endogenous cell populations for vascularized bone regeneration, thus leading to adverse tissue formation and decreased regenerative efficiency. Here, we engineered a biomaterial to regulate endogenous cell adhesion and promote vascularized bone regeneration. The biomaterial works by presenting two synthetic ligands, LLP2A and LXW7, explicitly targeting integrins α4ß1 and αvß3, respectively, expressed on the surfaces of the cells related to bone formation and vascularization, such as mesenchymal stem cells (MSCs), osteoblasts, endothelial progenitor cells (EPCs), and endothelial cells (ECs). In vitro, the LLP2A/LXW7 modified biomaterial improved the adhesion of MSCs, osteoblasts, EPCs, and ECs via integrin α4ß1 and αvß3, respectively. In an adult rat calvarial bone defect model, the LLP2A/LXW7 modified biomaterial enhanced bone formation and vascularization by synergistically regulating endogenous cells with osteogenic and angiogenic potentials, such as DLX5+ cells, osteocalcin+ cells, CD34+/CD45- cells and CD31+ cells. In a fetal sheep spinal bone defect model, the LLP2A/LXW7 modified biomaterial augmented bone formation and vascularization without any adverse effects. This innovative biomaterial offers an off-the-shelf, easy-to-use, and biologically safe product suitable for vascularized bone regeneration in both fetal and adult disease environments.

A bioactive compliant vascular graft modulates macrophage polarization and maintains patency with robust vascular remodeling.

Conventional synthetic vascular grafts are associated with significant failure rates due to their mismatched mechanical properties with the native vessel and poor regenerative potential. Though different tissue engineering approaches have been used to improve the biocompatibility of synthetic vascular grafts, it is still crucial to develop a new generation of synthetic grafts that can match the dynamics of native vessel and direct the host response to achieve robust vascular regeneration. The size of pores within implanted biomaterials has shown significant effects on macrophage polarization, which has been further confirmed as necessary for efficient vascular formation and remodeling. Here, we developed biodegradable, autoclavable synthetic vascular grafts from a new polyurethane elastomer and tailored the grafts' interconnected pore sizes to promote macrophage populations with a pro-regenerative phenotype and improve vascular regeneration and patency rate. The synthetic vascular grafts showed similar mechanical properties to native blood vessels, encouraged macrophage populations with varying M2 to M1 phenotypic expression, and maintained patency and vascular regeneration in a one-month rat carotid interposition model and in a four-month rat aortic interposition model. This innovative bioactive synthetic vascular graft holds promise to treat clinical vascular diseases.

Engineered extracellular vesicles with high collagen-binding affinity present superior in situ retention and therapeutic efficacy in tissue repair.

Although stem cell-derived extracellular vesicles (EVs) have remarkable therapeutic potential for various diseases, the therapeutic efficacy of EVs is limited due to their degradation and rapid diffusion after administration, hindering their translational applications. Here, we developed a new generation of collagen-binding EVs, by chemically conjugating a collagen-binding peptide SILY to EVs (SILY-EVs), which were designed to bind to collagen in the extracellular matrix (ECM) and form an EV-ECM complex to improve EVs' in situ retention and therapeutic efficacy after transplantation. Methods: SILY was conjugated to the surface of mesenchymal stem/stromal cell (MSC)-derived EVs by using click chemistry to construct SILY-EVs. Nanoparticle tracking analysis (NTA), ExoView analysis, cryogenic electron microscopy (cryo-EM) and western-blot analysis were used to characterize the SILY-EVs. Fluorescence imaging (FLI), MTS assay, ELISA and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to evaluate the collagen binding and biological functions of SILY-EVs in vitro. In a mouse hind limb ischemia model, the in vivo imaging system (IVIS), laser doppler perfusion imaging (LDPI), micro-CT, FLI and RT-qPCR were used to determine the SILY-EV retention, inflammatory response, blood perfusion, gene expression, and tissue regeneration. Results:In vitro, the SILY conjugation significantly enhanced EV adhesion to the collagen surface and did not alter the EVs' biological functions. In the mouse hind limb ischemia model, SILY-EVs presented longer in situ retention, suppressed inflammatory responses, and significantly augmented muscle regeneration and vascularization, compared to the unmodified EVs. Conclusion: With the broad distribution of collagen in various tissues and organs, SILY-EVs hold promise to improve the therapeutic efficacy of EV-mediated treatment in a wide range of diseases and disorders. Moreover, SILY-EVs possess the potential to functionalize collagen-based biomaterials and deliver therapeutic agents for regenerative medicine applications.

Bioengineering Extracellular Vesicles for the Treatment of Cardiovascular Diseases.

Cardiovascular diseases (CVD) remain one of the leading causes of mortality worldwide. Despite recent advances in diagnosis and interventions, there is still a crucial need for new multifaceted therapeutics that can address the complicated pathophysiological mechanisms driving CVD. Extracellular vesicles (EVs) are nanovesicles that are secreted by all types of cells to transport molecular cargo and regulate intracellular communication. EVs represent a growing field of nanotheranostics that can be leveraged as diagnostic biomarkers for the early detection of CVD and as targeted drug delivery vesicles to promote cardiovascular repair and recovery. Though a promising tool for CVD therapy, the clinical application of EVs is limited by the inherent challenges in EV isolation, standardization, and delivery. Hence, this review will present the therapeutic potential of EVs and introduce bioengineering strategies that augment their natural functions in CVD.

Engineering Extracellular Microenvironment for Tissue Regeneration.

The extracellular microenvironment is a highly dynamic network of biophysical and biochemical elements, which surrounds cells and transmits molecular signals. Extracellular microenvironment controls are of crucial importance for the ability to direct cell behavior and tissue regeneration. In this review, we focus on the different components of the extracellular microenvironment, such as extracellular matrix (ECM), extracellular vesicles (EVs) and growth factors (GFs), and introduce engineering approaches for these components, which can be used to achieve a higher degree of control over cellular activities and behaviors for tissue regeneration. Furthermore, we review the technologies established to engineer native-mimicking artificial components of the extracellular microenvironment for improved regenerative applications. This review presents a thorough analysis of the current research in extracellular microenvironment engineering and monitoring, which will facilitate the development of innovative tissue engineering strategies by utilizing different components of the extracellular microenvironment for regenerative medicine in the future.

Engineering extracellular vesicles for Alzheimer's disease: An emerging cell-free approach for earlier diagnosis and treatment.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder affecting over five million people globally and has no established cure. Current AD-related treatments only alleviate cognitive and behavioral symptoms and do not address disease onset or progression, underlining the unmet need to create an effective, innovative AD therapeutic. Extracellular vesicles (EVs) have emerged as a new class of nanotherapeutics. These secreted, lipid-bound cellular signaling carriers show promise for potential clinical applications for neurodegenerative diseases like AD. Additionally, analyzing contents and characteristics of patient-derived EVs may address the unmet need for earlier AD diagnostic techniques, informing physicians of altered genetic expression or cellular communications specific to healthy and diseased physiological states. There are numerous recent advances in regenerative medicine using EVs and include bioengineering perspectives to modify EVs, target glial cells in neurodegenerative diseases like AD, and potentially use EVs to diagnose and treat AD earlier. This article is categorized under: Neurological Diseases > Biomedical Engineering Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Stem Cells and Development.

Bioactive extracellular matrix scaffolds engineered with proangiogenic proteoglycan mimetics and loaded with endothelial progenitor cells promote neovascularization and diabetic wound healing.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvß3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

The Unique Properties of Placental Mesenchymal Stromal Cells: A Novel Source of Therapy for Congenital and Acquired Spinal Cord Injury.

Spinal cord injury (SCI) is a devasting condition with no reliable treatment. Spina bifida is the most common cause of congenital SCI. Cell-based therapies using mesenchymal stem/stromal cells (MSCS) have been largely utilized in SCI. Several clinical trials for acquired SCI use adult tissue-derived MSC sources, including bone-marrow, adipose, and umbilical cord tissues. The first stem/stromal cell clinical trial for spina bifida is currently underway (NCT04652908). The trial uses early gestational placental-derived mesenchymal stem/stromal cells (PMSCs) during the fetal repair of myelomeningocele. PMSCs have been shown to exhibit unique neuroprotective, angiogenic, and antioxidant properties, all which are promising applications for SCI. This review will summarize the unique properties and current applications of PMSCs and discuss their therapeutic role for acquired SCI.

Bioengineered extracellular vesicle-loaded bioscaffolds for therapeutic applications in regenerative medicine.

Extracellular vesicle (EV)-based technologies represent a new advancement for disease treatment. EVs can be administered systemically, injected into the injury site directly, or applied locally in conjunction with bioengineered implantable scaffolds. Matrix-bound vesicles (MBVs), a special class of vesicles localized in association with the extracellular matrix (ECM), have been identified as critical bioactive factors and shown to mediate significant regenerative functions of ECM scaffolds. Loading EVs onto bioscaffolds to mimic the MBV-ECM complex has been shown superior to EV bolus injection in recent in vivo studies, such as in providing enhanced tissue regeneration, EV retention rates, and healing efficacy. Different types of natural biomaterials, synthetic polymers, and ceramics have been developed for EV loading, and these EV functionalized biomaterials have been applied in different areas for disease treatment. The EV functionalized scaffolds can be designed to be biodegradable, off-the-shelf biomaterials as a delivery vehicle for EVs. Overall, the bioengineered EV-loaded bioscaffolds represent a promising approach for cell-free treatment in clinical applications.

Nrf2 Regulates CHI3L1 to Suppress Inflammation and Improve Post-Traumatic Osteoarthritis.

INTRODUCTION: Post-traumatic osteoarthritis (PTOA) is an inflammatory condition that occurs following mechanical joint trauma and that results in joint degeneration. This study sought to evaluate the regulatory function of nuclear factor erythroid 2-related factor 2 (Nrf2) in a murine model of anterior cruciate ligament transection (ACLT)-induced PTOA and in an in vitro model of synoviocyte inflammation induced by LPS treatment with the goal of exploring the role of chitinase 3-like-1 (CHI3L1) in this pathogenic context. METHODS: PTOA model mice were intra-articularly injected with Nrf2 overexpression lentiviral vector, and safranin O-fast green staining as well as the Osteoarthritis Research Society International (OARSI) Scoring System were used to evaluate the severity of cartilage damage. Protein expression in the synovial tissue was evaluated by Western blotting, immunohistochemical staining, and ELISA. Additionally, murine synoviocytes were infected with Nrf2 overexpression lentivirus and stimulated with LPS. The levels of inflammatory cytokines were detected by ELISA. ROS levels were measured using dihydroethidium (DHE) dye. RESULTS: We determined that the overexpression of Nrf2 was sufficient to reduce cartilage degradation in the context of PTOA in vivo, and we observed a significant decrease in the expression of matrix metalloproteinase 13 (MMP13) in the articular cartilage of samples from mice overexpressing Nrf2 relative to control mice. Synovial CHI3L1 expression and serum TNF-α, IL-1ß, and IL-6 levels were reduced in animals overexpressing this transcription factor relative to PTOA model controls. Consistent with these findings, murine synoviocytes treated with LPS exhibited dose-dependent increases in ROS, TNF-α, IL-1ß, IL-6, Nrf2, and CHI3L1 levels, whereas Nrf2 overexpression was sufficient to suppress these increases. CONCLUSION: Our data indicated that Nrf2 negatively regulates CHI3L1, suggesting that this signaling axis may regulate PTOA progression and may thus be a viable therapeutic target in individuals affected by this condition.

Exosomal microRNAs from mesenchymal stem/stromal cells: Biology and applications in neuroprotection.

Mesenchymal stem/stromal cells (MSCs) are extensively studied as cell-therapy agents for neurological diseases. Recent studies consider exosomes secreted by MSCs as important mediators for MSCs' neuroprotective functions. Exosomes transfer functional molecules including proteins, lipids, metabolites, DNAs, and coding and non-coding RNAs from MSCs to their target cells. Emerging evidence shows that exosomal microRNAs (miRNAs) play a key role in the neuroprotective properties of these exosomes by targeting several genes and regulating various biological processes. Multiple exosomal miRNAs have been identified to have neuroprotective effects by promoting neurogenesis, neurite remodeling and survival, and neuroplasticity. Thus, exosomal miRNAs have significant therapeutic potential for neurological disorders such as stroke, traumatic brain injury, and neuroinflammatory or neurodegenerative diseases and disorders. This review discusses the neuroprotective effects of selected miRNAs (miR-21, miR-17-92, miR-133, miR-138, miR-124, miR-30, miR146a, and miR-29b) and explores their mechanisms of action and applications for the treatment of various neurological disease and disorders. It also provides an overview of state-of-the-art bioengineering approaches for isolating exosomes, optimizing their yield and manipulating the miRNA content of their cargo to improve their therapeutic potential.

Long non-coding RNA signatures as predictors of prognosis in thyroid cancer: a narrative review.

Thyroid cancer (TC) is the most common endocrine malignancy, with high incidence rates in recent decades. Most TC cases have good prognoses, but a high risk of recurrence and metastases poses challenges, especially for patients with high-risk factors. Currently used prognostic markers for TC involve a combination of genetic factors and overexpressed proteins. Long non-coding RNAs (lncRNAs) regulate several integral biologic processes by playing key roles in the transcription of several downstream targets maintaining cellular behavior. Prior studies have revealed that lncRNAs promote tumor cell proliferation, invasion, metastasis, and angiogenesis, making them important targets for therapeutic intervention in cancer. While the exact molecular mechanisms underlying the role of lncRNAs in modulating TC progression and recurrence is still unclear, it is important to note that some lncRNAs are upregulated in certain cancers, while others are downregulated. In the present study, we review several key lncRNAs, their association with cancer progression, and the important roles they may play as tumor suppressors or tumor promoters in tumorigenesis. We discuss the potential mechanisms of lncRNA-mediated pathogenesis that can be targeted for the treatment of TC, the existing and potential benefits of using lncRNAs as diagnostic and prognostic measures for cancer detection, and tumor burden in patients.

Developing an Injectable Nanofibrous Extracellular Matrix Hydrogel With an Integrin αvß3 Ligand to Improve Endothelial Cell Survival, Engraftment and Vascularization.

Endothelial cell (EC) transplantation via injectable collagen hydrogel has received much attention as a potential treatment for various vascular diseases. However, the therapeutic effect of transplanted ECs is limited by their poor viability, which partially occurs as a result of cellular apoptosis triggered by the insufficient cell-extracellular matrix (ECM) engagement. Integrin binding to the ECM is crucial for cell anchorage to the surrounding matrix, cell spreading and migration, and further activation of intracellular signaling pathways. Although collagen contains several different types of integrin binding sites, it still lacks sufficient specific binding sites for ECs. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LXW7, an integrin αvß3 ligand, which possessed a strong binding affinity to and enhanced functionality of ECs. In this study, to improve the EC-matrix interaction, we developed an approach to molecularly conjugate LXW7 to the collagen backbone, via a collagen binding peptide SILY, in order to increase EC specific integrin binding sites on the collagen hydrogel. Results showed that in the in vitro 2-dimensional (2D) culture model, the LXW7-treated collagen surface significantly improved EC attachment and survival and decreased caspase 3 activity in an ischemic-mimicking environment. In the in vitro 3-dimensional (3D) culture model, LXW7-modified collagen hydrogel significantly improved EC spreading, proliferation, and survival. In a mouse subcutaneous implantation model, LXW7-modified collagen hydrogel improved the engraftment of transplanted ECs and supported ECs to form vascular network structures. Therefore, LXW7-functionalized collagen hydrogel has shown promising potential to improve vascularization in tissue regeneration and may be used as a novel tool for EC delivery and the treatment of vascular diseases.

Extracellular Matrix Mimicking Nanofibrous Scaffolds Modified With Mesenchymal Stem Cell-Derived Extracellular Vesicles for Improved Vascularization.

The network structure and biological components of natural extracellular matrix (ECM) are indispensable for promoting tissue regeneration. Electrospun nanofibrous scaffolds have been widely used in regenerative medicine to provide structural support for cell growth and tissue regeneration due to their natural ECM mimicking architecture, however, they lack biological functions. Extracellular vesicles (EVs) are potent vehicles of intercellular communication due to their ability to transfer RNAs, proteins, and lipids, thereby mediating significant biological functions in different biological systems. Matrix-bound nanovesicles (MBVs) are identified as an integral and functional component of ECM bioscaffolds mediating significant regenerative functions. Therefore, to engineer EVs modified electrospun scaffolds, mimicking the structure of the natural EV-ECM complex and the physiological interactions between the ECM and EVs, will be attractive and promising in tissue regeneration. Previously, using one-bead one-compound (OBOC) combinatorial technology, we identified LLP2A, an integrin α4ß1 ligand, which had a strong binding to human placenta-derived mesenchymal stem cells (PMSCs). In this study, we isolated PMSCs derived EVs (PMSC-EVs) and demonstrated they expressed integrin α4ß1 and could improve endothelial cell (EC) migration and vascular sprouting in an ex vivo rat aortic ring assay. LLP2A treated culture surface significantly improved PMSC-EV attachment, and the PMSC-EV treated culture surface significantly enhanced the expression of angiogenic genes and suppressed apoptotic activity. We then developed an approach to enable "Click chemistry" to immobilize LLP2A onto the surface of electrospun scaffolds as a linker to immobilize PMSC-EVs onto the scaffold. The PMSC-EV modified electrospun scaffolds significantly promoted EC survival and angiogenic gene expression, such as KDR and TIE2, and suppressed the expression of apoptotic markers, such as caspase 9 and caspase 3. Thus, PMSC-EVs hold promising potential to functionalize biomaterial constructs and improve the vascularization and regenerative potential. The EVs modified biomaterial scaffolds can be widely used for different tissue engineering applications.

Rapid endothelialization of small diameter vascular grafts by a bioactive integrin-binding ligand specifically targeting endothelial progenitor cells and endothelial cells.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.