Abstract Chronic wound healing is often impaired in conditions such as metabolic syndrome, requiring effective therapeutic interventions to promote tissue regeneration and repair. In this study, we evaluated the wound healing potential of petroleum jelly (P Jelly)-based bioactive glass ointments (PBGCu) with varying copper concentrations (0, 1, and 3 wt%) in both in vitro and in vivo models of wound healing. PBGCu formulations demonstrated high biocompatibility with human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC). Additionally, PBGCu ointments exhibited strong antibacterial activity against Staphylococcus aureus , suggesting their utility for the care of chronic wounds. In both metabolic syndrome mouse and pig models, PBGCu3-treated wounds showed significantly faster wound closure, enhanced epithelial regeneration, and increased dermal thickness compared to saline and P Jelly controls. Histological analysis also revealed 50% increased vascularization (p < 0.0001) and a 90% reduction in scar formation (p < 0.0001) in PBGCu3-treated wounds. These findings show that PBGCu formulations, especially at 3 wt% copper concentration, significantly improve wound healing by promoting epithelial regeneration, dermal tissue formation, and vascularization, while also offering antibacterial protection. The sustained Cu 2+ ions release from PBGCu ointments provides long-term support for tissue regeneration, positioning this ointment composition as a promising therapeutic tool for chronic wound management. Future studies will focus on elucidating the underlying mechanisms and evaluating the therapeutic efficacy of PBGCu formulations in infected wounds. Highlights Developed a Petroleum Jelly–based copper-doped bioactive glass ointment (PBGCu) enabling sustained and controlled Cu²⁺ ion release. PBGCu significantly accelerated wound closure and improved epithelial and dermal tissue regeneration. PBGCu enhanced hair follicle regeneration and tissue remodeling in full-thickness wounds. Validated therapeutic efficacy in both mouse and pig models that support translational relevance. Offers a simple, low-cost, and clinically adaptable topical formulation for metabolic syndrome-related wound complications.

Chronic limb threatening ischemia (CLTI) is a debilitating disease in which chronic ischemia causes skeletal muscle degeneration, fat infiltration, and metabolic dysregulation. This study aims to develop a biomaterials-based strategy for the localized, sustained delivery of sodium butyrate, a gut-derived short-chain fatty acid, using poly(lactic-co-glycolic acid) (PLGA) microspheres. In vitro ischemic culture models demonstrated that butyrate improves cell viability and preserves mitochondrial membrane polarization in both myoblasts and myotubes. Butyrate was successfully encapsulated using a double emulsion method, achieving sustained release over 4 weeks. In a murine hindlimb ischemia model, treatment with butyrate-loaded microspheres improved muscle fiber architecture and reduced fat infiltration, despite no significant changes in limb perfusion. These findings highlight butyrate as a perfusion-independent therapeutic that preserves muscle quality under ischemic conditions and supports its potential as a regenerative strategy for CLTI. This research will serve as the basis for biomaterial-focused therapies for regeneration and provide another therapeutic target to study in CLTI.

Ankle cartilage damage frequently progresses to osteoarthritis (OA), which impairs patient quality of life and creates substantial socioeconomic burdens worldwide. Mesenchymal stem cells (MSCs) offer a promising approach for cartilage regeneration; however, comparative data on the efficacy of MSCs from different tissue sources for ankle cartilage repair remains limited. As an exploratory preclinical study, this investigation aimed to evaluate and compare the effects of bone marrow-derived MSCs (BMMSCs), adipose-derived MSCs (ADMSCs), and synovial fluid-derived MSCs (SFMSCs) in a rat model of full-thickness talar cartilage defects. Full-thickness talar cartilage defects were created by limited-depth drilling in 8-week-old male Sprague-Dawley rats (200-300 g). Animals were randomly assigned to a control group or three treatment groups receiving intra-articular injection of BMMSCs, ADMSCs, or SFMSCs. Outcomes were assessed using behavioral and gait tests, footprint analysis, Micro-CT imaging for subchondral bone parameters (BS/TV, Tb.N, Tb.Sp), ICRS macroscopic scoring, histopathological evaluation, and immunohistochemical staining for chondrogenic markers (Col II, FSTL1, SOX9, Smad3). In this exploratory analysis, the SFMSC group displayed comparatively better functional recovery in behavioral and gait measurements relative to other groups. The footprint length factor was significantly higher in the SFMSC group (6.76 ± 1.15) than in the control group (5.52 ± 1.68, P < 0.05), and the ICRS score was also elevated in the SFMSC group (11.00 ± 1.26 vs. 8.80 ± 1.32 in controls, P < 0.01). No significant differences in subchondral bone Micro-CT parameters were detected among groups (P > 0.05). Histological and immunohistochemical findings suggested that SFMSCs were associated with enhanced hyaline cartilage formation and relatively higher expression of chondrogenic markers at the defect site. Within the constraints of this exploratory preclinical study, SFMSCs showed comparatively favorable effects on talar cartilage repair in rats relative to BMMSCs and ADMSCs. These preliminary findings support SFMSCs as a promising candidate source for stem cell-based ankle cartilage regeneration, providing exploratory evidence that may inform future investigations toward clinical translation.

Critical limb ischemia (CLI), a severe complication of peripheral artery disease, presents a significant clinical challenge due to limited treatment options. Existing preclinical models of CLI frequently neglect biological variables such as age and sex, limiting their capacity to effectively assess novel therapies. This study seeks to address the deficiencies in the widely used murine hindlimb ischemia model for CLI by examining the influence of age and sex on recovery outcomes. Foxn1nu mice with varying age and biological sex were subjected to femoral artery ligation to establish unilateral hindlimb ischemia. Hindlimb perfusion was measured on multiple post-surgery days using Laser Doppler Imaging. Motor function and tissue damage were recorded through gross examination. Gastrocnemius muscles, collected on postsurgical day 36, underwent vascular and muscular histological analysis. All parameters were compared across age and sex groups. Mature adult mice (12–18 weeks old) consistently exhibited significantly heightened motor function impairment and tissue loss, with significantly slower perfusion recovery, in comparison to their younger counterparts (6–12 weeks old). Sexual dimorphism manifested, with females experiencing more pronounced symptoms than males. Histological analysis revealed skeletal muscle pathology in mature adults, similar to the clinical pathology of CLI, including muscle fiber loss, fibrosis, and adipose-like tissue infiltration. Animals of older age and female biological sex exhibited slower vascular regeneration. Age and biological sex significantly influence tissue recovery in the murine hindlimb ischemia model. To better evaluate pro-regenerative therapeutics for CLI, the utilization of older, mixed-sex mice in preclinical studies is recommended. This study underscores the critical impact of age and biological sex on tissue response in the murine hindlimb ischemia model. When evaluating both male and female mice across two distinct age groups, the older female group is significantly associated with worse outcomes in blood perfusion recovery, ambulatory function, tissue loss, and muscular and vascular characteristics. These findings strongly advocate for the use of mature adult mice with mixed biological sex in preclinical investigations. This comprehensive characterization and validation of a widely employed preclinical model significantly enhances the outcome’s credibility while evaluating pro-regenerative therapies for the treatment of critical limb ischemia.

Nuclear morphology plays a critical role in regulating gene expression and cell functions. While most research has focused on the direct effects of nuclear morphology on cell fate, its impact on the cell secretome and surrounding cells remains largely unexplored. In this study, we fabricate implants with a micropillar topography using methacrylated poly(octamethylene citrate)/hydroxyapatite (mPOC/HA) composites to investigate how micropillar-induced nuclear deformation influences cell secretome for osteogenesis and cranial bone regeneration. In vitro, cells with deformed nuclei show enhanced secretion of proteins that support extracellular matrix (ECM) organization, which promotes osteogenic differentiation in neighboring mesenchymal stromal cells (MSCs). In a female mouse model with critical-size cranial defects, nuclear-deformed MSCs on micropillar mPOC/HA implants elevate Col1a2 expression, contributing to bone matrix formation, and drive cell differentiation toward osteogenic progenitor cells. These findings indicate that micropillars modulate the secretome of hMSCs, thereby influencing the fate of surrounding cells through matricrine effects.

Neointimal hyperplasia, a pathological response to arterial interventions or injury, often leads to restenosis and recurrent narrowing or occlusion, particularly in the peripheral vasculature. Its prevalence and negative impact on the long-term success of vascular interventions have driven extensive research aimed at better understanding the condition and developing effective therapies. This review provides a comprehensive overview of emerging bioengineering strategies for treating neointimal hyperplasia in peripheral vessels. These approaches include novel therapeutics and cell-based technologies designed to promote re-endothelialization, modulate vascular smooth muscle cell (VSMC) phenotype, reduce inflammation, scavenge reactive oxygen species (ROS), and enhance biomechanical compatibility between grafts and native vessels. Furthermore, advanced therapeutic delivery modalities are highlighted for their potential to achieve targeted, localized treatment at injury sites. This review also explores underrepresented therapeutic targets beyond traditional approaches, offering new opportunities for intervention. The multifaceted examination underscores the challenge of neointimal hyperplasia and presents a promising roadmap toward more effective treatments, ultimately aiming to improve patient outcomes after vascular interventions.

Mitochondrial dysfunction and oxidative stress are key contributors to impaired vascular healing following stent implantation. Although drug‐eluting stents reduce restenosis, they do not promote tissue regeneration or correct cellular metabolic deficits. A redox‐responsive mitochondrial delivery system is developed for localized therapeutic release from vascular stent surfaces. Intact mitochondria are immobilized onto Nitinol substrates through a stepwise surface modification strategy incorporating a reactive oxygen species (ROS)‐cleavable linker. Upon exposure to elevated ROS levels, mitochondria are released in a dose‐dependent manner and internalized by human endothelial cells and vascular smooth muscle cells. Transmission electron microscopy confirms the integrity of isolated mitochondria, and X‐ray photoelectron spectroscopy and secondary ion mass spectrometry validate successful surface functionalization and grafting. The modified surfaces demonstrate high biocompatibility, with minimal cytotoxicity and reduced platelet adhesion. Released mitochondria restore mitochondrial membrane potential, oxygen consumption rate and enhance cell viability under oxidative stress. Ex vivo assessment in rat aortic tissue provides visual confirmation of mitochondrial uptake in a physiologically relevant context. This platform establishes a stimulus‐responsive, organelle‐based therapeutic strategy that targets mitochondrial dysfunction at the biomaterial–tissue interface and offers potential to enhance vascular regeneration in oxidative microenvironments following endovascular interventions.

Amniotic fluid-derived stem cells (AFSCs) represent a promising yet underutilized resource for research and clinical applications. While AFSCs share phenotypic and functional characteristics with stem cells derived from somatic tissues such as bone marrow, adipose tissue, placenta, and umbilical cord, their unique developmental origin grants them several superior qualities. These include enhanced multipotency, tissue-specific genotypic profiles, and the ability to form single-cell colonies. Such features position AFSCs as highly valuable for translational research and tissue engineering. This review seeks to underscore the distinctive attributes of AFSCs, particularly their relevance in developmental research and engineering. By emphasizing these qualities, we aim to stimulate further exploration into their use in patient-specific induced pluripotent stem cells and organoid development, potentially unlocking their full therapeutic potential. The unique capabilities of AFSCs make them an exceptional candidate for advancing regenerative medicine, offering new avenues for treating a variety of conditions that currently have limited therapeutic options.

Sympathetic innervation plays a critical role in regulating vascular function, yet its influence on vascular regeneration and reinnervation following ischemic injury remains poorly understood. This study develops and validates murine models of localized sympathetic denervation using 6-hydroxydopamine (6-OHDA) to enable study of the sympathetic nervous system's impact on vascular systems during tissue repair. Two methods of 6-OHDA administration were employed: a single topical application during open surgery and minimally invasive weekly subcutaneous injections. The topical application model achieved temporary denervation lasting 1 week without causing vascular damage, while the subcutaneous injection model provided sustained denervation for up to 4 weeks with minimal inflammation and no significant changes to vascular architecture. To investigate the effects of denervation in an ischemic context, these models were combined with a hindlimb ischemia model. Ischemia induced persistent denervation in both 6-OHDA-treated and control limbs, with limited sympathetic nerve regeneration observed over 4 weeks. Despite persistent denervation, microvascular density and perfusion recovery in ischemic muscles were comparable between denervated and control groups. This suggests that ischemia governs vascular regeneration independently of sympathetic input. These results demonstrate that localized 6-OHDA administration provides a versatile tool for achieving controlled sympathetic denervation in peripheral arteries. These models provide a novel platform for studying vascular regeneration and reinnervation under both normal and ischemic conditions, offering novel insights into the interactions between neural regulation and vascular repair processes. This work lays the foundation for future research into neural-vascular crosstalk and new possibilities for developing regenerative therapies targeting the autonomic regulation of vascular health.

Electroactive biomaterials present new opportunities for "smart" vascular grafts capable of supporting tissue integration while enabling electrical stimulation, sensing, or real-time modulation of the vascular environment. In this study, a conductive vascular conduit was engineered by incorporating sulfonated poly(3,4-ethylenedioxythiophene) (S'PEDOT) into extracellular matrix (ECM)-based scaffolds. Initial screening in collagen sponges identified S'PEDOT concentrations that supported biocompatibility with primary endothelial and smooth muscle cells while minimizing platelet adhesion. This strategy was then applied to decellularized rat aortas, which were functionalized with S'PEDOT and evaluated for electrical conductivity, tensile mechanics, and structural integrity. The modified grafts retained native architecture and mechanical compliance while exhibiting significantly enhanced conductivity compared to unmodified controls. In vivo biocompatibility was assessed by subcutaneous implantation in rats, followed by histological and immunohistochemical analyses. The S'PEDOT-modified grafts elicited minimal inflammatory response and preserved tissue architecture. These findings demonstrate a promising approach for integrating conductive polymers into natural scaffolds to develop electroactive vascular grafts, supporting future applications in multifunctional and responsive vascular devices.

Abdominal aortic aneurysm (AAA) is a life-threatening condition with no effective pharmacologic therapies, and increasing evidence suggests that the sympathetic nervous system (SNS) contributes to disease progression. However, the temporal effects of SNS modulation on AAA stability remain unclear. To investigate the impact of SNS modulation on aneurysm outcomes, we induced AAAs in male ApoE-/- mice via continuous subcutaneous infusion of angiotensin II. Using three-dimensional light sheet microscopy, we found that ruptured AAAs exhibited the highest sympathetic innervation in experimental aneurysms. Sympathetic denervation was performed using intraperitoneal 6-hydroxydopamine, administered either serially (before and during AAA induction) or at a delayed time point (after aneurysm formation). Mice were monitored for mortality, and surviving animals were evaluated at day 28 for aortic morphology, nerve density, vascularization, and inflammation. Both serial and delayed denervation significantly reduced sympathetic nerve density, approximating normal levels. Delayed denervation trended toward improved survival (87%) by reducing rupture risk after treatment, whereas serial denervation prevented early ruptures but showed diminishing benefits after midpoint treatment. Both serial denervation and delayed denervation increased intima-media thickness and reduced aortic wall collagen content. Despite these structural changes, no significant differences in vascularization or macrophage infiltration were observed across groups. These findings demonstrate that sympathetic denervation influences aneurysm stability through vascular remodeling, independent of angiogenesis or macrophage infiltration, underscoring the importance of SNS modulation at specific stages of AAA progression.NEW & NOTEWORTHY This study reveals the temporal effects of sympathetic denervation on aneurysm stability in an experimental model of AAA. Using 3-D light sheet microscopy, we demonstrate that ruptured AAAs exhibit the highest sympathetic innervation. Although delayed denervation reduced rupture risk and improved survival, serial denervation altered vascular remodeling. These findings establish SNS modulation as a key regulator of aneurysm progression and highlight the importance of timing in potential therapeutic interventions.

Mitochondrial dysfunction is a key contributor to vascular inflammation in many cardiovascular diseases. This review explores mitochondrial transplantation as a promising strategy for addressing mitochondrial dysfunction and vascular inflammation. We discuss mitochondrial dysfunction across different vascular cell types and current clinical management strategies, highlighting the need for novel approaches that directly target mitochondrial health. We also present recent progress in mitochondrial transplantation across cardiac, neurovascular, and peripheral vascular applications in preclinical settings, as well as ongoing clinical trials. Important technical considerations, such as mitochondria sourcing, delivery routes, and storage, are discussed to facilitate future translation. By reinstating mitochondrial health and hence mitigating vascular inflammation, mitochondrial transplantation holds the potential to provide novel, targeted therapies for cardiovascular diseases, ultimately improving patient outcomes, reducing disease progression, and addressing unmet medical needs in vascular health. The translation of this technology into clinical practice could offer significant advances in the treatment of a wide range of cardiovascular conditions.

Mitochondrial dysfunction contributes to endothelial injury in vascular diseases and interventions. While mitochondrial transplantation offers a promising therapeutic strategy, current approaches lack target specificity, efficient uptake, and long-term retention. This study presents a surface-engineering approach to enhance mitochondria delivery to the vascular endothelium as a step toward novel endothelial repair strategies. Mitochondria were isolated from healthy induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) and surface functionalized with a phospholipid-based coating platform (DSPE-PEG) to enable peptide functionalization. DSPE-PEG was conjugated to either VCAM-1-binding peptide and collagen-binding peptide to enable targeting to dysfunctional and injured endothelium. Mitochondria particle characteristics were measured using flow cytometry, dynamic light scattering and Seahorse. Mitochondrial uptake, retention, and function were assessed in human diabetic aortic endothelial cells (DAECs) using confocal microscopy, flow cytometry, JC-1 staining, and Seahorse metabolic analysis. iPSC-MSCs provided bioenergetically competent mitochondria suitable for therapeutic delivery. DSPE-PEG surface functionalization significantly enhanced mitochondrial uptake in DAECs, compared to uncoated mitochondria. Confocal imaging and quantitative analysis revealed increased cytoplasmic retention and greater colocalization with the endogenous mitochondrial network after 24 h. Functional assays demonstrated improved mitochondrial membrane potential and sustained oxygen consumption in recipient cells, indicating enhanced host mitochondrial function following treatment with surface-engineered mitochondria. This study establishes a proof-of-concept for mitochondria surface engineering to enhance mitochondria transplantation to damaged endothelium, demonstrating improved cellular uptake and bioenergetic restoration. These findings provide a foundation for developing adaptable, cell-free therapeutics for vascular disease.

Introduction: The autonomic nervous system (ANS) drives blood flow regulation in peripheral arteries. This study aims to develop novel models of sympathetic denervation in peripheral arteries and to characterize subsequent vascular remodeling. We hypothesize that sympathetic denervation of murine femoral arteries can be achieved through local administration of neurotoxin 6-hydroxydopamine (6-OHDA). Methods: BALB/c mice underwent local administration of 6-OHDA to the left femoral artery, while the contralateral artery received vehicle solution. Two approaches were tested: (1) Acute: a one-time topical application following surgical exposure of femoral arteries (N=8, male, Fig. A ), and (2) Chronic: weekly subcutaneous injections near the femoral arteries (N=11, male and female, Fig. B ). Hindlimb perfusion was assessed with laser Doppler imaging, and arterial structure and remodeling were evaluated with histology and immunohistochemistry at one, two, or four weeks. Results: One-time 6-OHDA application (Acute) led to temporary femoral artery denervation (reduction in tyrosine hydroxylase) for one week ( Fig. C – D ), with inflammation observed in both 6-OHDA and vehicle-treated arteries. No vascular wall remodeling was observed after temporary denervation. Weekly subcutaneous 6-OHDA injections (Chronic) resulted in persistent denervation throughout the four-week period in male mice, while female mice displayed variable outcomes ( Fig. E – F ). Sustained sympathetic denervation in the chronic model led to reduced hindlimb blood perfusion at low core temperatures ( Fig. G – H ), though no significant differences were detected in vascular smooth muscle cell phenotype or extracellular matrix composition. Conclusions: Local administration of 6-OHDA is an effective means of peripheral artery sympathetic denervation with controllable duration. This innovative approach of targeting the ANS unveils new prospects for advancing vascular therapies.

Objective: Sympathetic innervation plays a pivotal role in regulating cardiovascular health, and its dysregulation is implicated in a wide spectrum of cardiovascular diseases. This study seeks to evaluate the impact of denervation of the abdominal aorta on its morphology and wall homeostasis. Methods: Male and female Sprague-Dawley rats (N = 12), aged 3 months, underwent midline laparotomy for infrarenal aorta exposure. Chemical denervation was induced via a one-time topical application of 10% phenol (n = 6), whereas sham controls received phosphate-buffered saline (n = 6). Animals were allowed to recover and subsequently were sacrificed after 6 months for analysis encompassing morphology, histology, and immunohistochemistry. Results: At 6 months post-treatment, abdominal aortas subjected to phenol denervation still exhibited a significant reduction in nerve fi ber density compared with sham controls. Denervated aortas demonstrated reduced intima-media thickness, increased elastin fragmentation, decreased expression of vascular smooth muscle proteins (a-SMA and MYH11), and elevated adventitial vascular density. Sex-stratified analyses revealed additional dimorphic responses, particularly in aortic collagen and medial cellular density in female animals. Conclusions: Single-timepoint phenol-based chemical denervation induces alterations in abdominal aortic morphology and vascular remodeling over a 6-month period. These fi ndings underscore the potential of the sympathetic nervous system as a therapeutic target for aortic pathologies. Clinical Relevance: Aortic remodeling remains an important consideration in the pathogenesis of aortic disease, including occlusive, aneurysmal, and dissection disease states. The paucity of medical therapies for the treatment of aortic disease has driven considerable interest in elucidating the pathogenesis of these conditions; new therapeutic targets are critically needed. Here, we show significant remodeling after phenol-induced denervation with morphologic, histologic, and immunohistochemical features. Future investigations should integrate sympathetic dysfunction as a potential driver of pathologic aortic wall changes with additional consideration of the sympathetic nervous system as a therapeutic target. (JVSeVascular Science 2024;5:100202.)

Introduction: Aortic remodeling remains an important contributor to the pathogenesis of aortic disease, including occlusive, aneurysmal, and dissection disease states. The paucity of medical therapies has driven considerable interest in elucidating the pathogenesis of these conditions; new therapeutic targets are critically needed. Prior studies have identified sympathetic nervous system signaling (SNS) as a critical regulator of arterial wall homeostasis with potent effects on inflammation and vascular remodeling. Hypothesis: This study seeks to evaluate the impact of sustained denervation on morphology and wall homeostasis of the abdominal aorta. We hypothesized that topical phenol denervation of the rat abdominal aorta would induce sustained reduction in nerve fiber density with concomitant pathologic changes to tissue morphology. Methods: Male and female Sprague-Dawley rats (n=12), aged 3 months, underwent midline laparotomy for infrarenal aorta exposure. Chemical denervation was induced via a one-time topical application of 10% phenol (n=6), while sham controls received phosphate-buffered saline (n=6). Animals were allowed to recover and subsequently sacrificed after 6 months for analysis encompassing morphology, histology, and immunohistochemistry. Results: At 6 months post-treatment, abdominal aortas subjected to phenol denervation exhibited a significant reduction in nerve fiber density compared to sham controls (92.8 ± 20.3 fibers/mm 2 vs 170.4 ± 79.0 fibers/mm 2 , p=0.042). Denervated aortas demonstrated increased elastin fragmentation breakage scores (2.8 ± 1.1 vs 1.1 ± 0.4, p=0.0056). Phenol denervated rats also had significantly decreased expression of vascular smooth muscle proteins α-SMA (32.6 ± 3.9% vs 24.6 ± 3.3%, p=0.0034) and MYH11(14.2 ± 5.7% vs 21.6 ± 3.5%, p=0.023), as well as elevated adventitial microvascular density (58.9 ± 25.2 vessels/mm 2 vs sham control 18.5 ± 8.7 vessels/mm 2 , p=0.0040). Conclusion: Single-timepoint phenol-based chemical denervation induces sustained alterations in abdominal aortic morphology and vascular remodeling over a 6-month period. These findings underscore the potential of the SNS as a therapeutic target for aortic pathologies.

Abdominal aortic aneurysm (AAA) is a significant source of mortality worldwide and carries a mortality of greater than 80% after rupture. Despite extensive efforts to develop pharmacological treatments, there is currently no effective agent to prevent aneurysm growth and rupture. Current treatment paradigms only rely on the identification and surveillance of small aneurysms, prior to ultimate open surgical or endovascular repair. Recently, regenerative therapies have emerged as promising avenues to address the degenerative changes observed in AAA. This review briefly outlines current clinical management principles, characteristics, and pharmaceutical targets of AAA. Subsequently, a thorough discussion of regenerative approaches is provided. These include cellular approaches (vascular smooth muscle cells, endothelial cells, and mesenchymal stem cells) as well as the delivery of therapeutic molecules, gene therapies, and regenerative biomaterials. Lastly, additional barriers and considerations for clinical translation are provided. In conclusion, regenerative approaches hold significant promise for in situ reversal of tissue damages in AAA, necessitating sustained research and innovation to achieve successful and translatable therapies in a new era in AAA management.

Critical limb ischemia (CLI) presents a significant clinical challenge, leading to tissue ischemia and potentially resulting in limb necrosis or amputation. Cell-based regenerative therapies offer promise for improving outcomes in CLI, but their effectiveness is often limited by poor cell survival and engraftment. This study hypothesized that a thermo-responsive polymer, poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN), combined with pro-survival bioactive peptides, can create a protective microenvironment to improve endothelial cell survival and function after their delivery. Through in vitro and in vivo experiments, laminin-derived peptide A5G81 and vascular endothelial growth factor (VEGF)-derived peptide QK are identified as effective in promoting endothelial cell spreading, proliferation, and prolonged survival. PPCN's viscoelastic properties protected against shear stress during injection, while the peptides supported endothelial cell behavior through distinct molecular pathways. Importantly, delivery of endothelial cells with PPCN-A5G81 and PPCN-QK in a murine hindlimb ischemia model resulted in significant improvements in limb perfusion, tissue preservation, and functional outcomes compared to controls. Additionally, this approach enhanced skeletal muscle remodeling following ischemic injury. This innovative biomaterial platform represents a versatile solution for addressing cell survival challenges and advancing regenerative therapies in CLI and other ischemic conditions.

BACKGROUND:Peripheral artery disease (PAD), caused by atherosclerosis, leads to limb ischemia, muscle damage, and impaired mobility in the lower extremities. Recent studies suggest that circadian rhythm disruptions can hinder vascular repair during ischemia, but the specific tissues involved and the impact on muscle health remain unclear. This study investigates the role of the skeletal muscle circadian clock in muscle adaptation to ischemic stress using a surgical mouse model of hindlimb ischemia.METHODS:We performed secondary analysis of publicly available RNA-sequencing data sets derived from patients with PAD to identify the differential expression of circadian-related genes in endothelial cells and ischemic limb skeletal muscles. We used mice with specific genetic loss of the circadian clock activator, BMAL1 (brain and muscle ARNT-like 1), in adult skeletal muscle tissues (Bmal1muscle). Bmal1muscle mice and controls underwent femoral artery ligation surgery to induce hindlimb ischemia. Laser Doppler imaging was used to assess limb perfusion at various time points after the surgery. Muscle tissues were analyzed with RNA sequencing and histological examination to investigate PAD-related muscle pathologies. Additionally, we studied the role of BMAL1 in muscle fiber adaptation to hypoxia using RNA and assay for transposase-accessible chromatin with sequencing analyses in primary myotube culture model.RESULTS:Disrupted expression of circadian rhythm-related genes was observed in existing RNA-sequencing data sets from endothelial cells and ischemic limb skeletal muscles derived from patients with PAD. Genetic loss of Bmal1 specifically in adult mouse skeletal muscle tissues delayed reperfusion recovery following induction of hindlimb ischemia. Histological examination of muscle tissues showed reduced regenerated myofiber number and a decreased proportion of type IIB fast-twitch myofibers in Bmal1muscle mouse muscles in the ischemic limbs but not in their contralateral nonischemic limbs. Transcriptomic analysis revealed abrogated metabolic, angiogenic, and myogenic pathways relevant to hypoxia adaptation in Bmal1muscle mouse muscles. These changes were corroborated in Bmal1-deficient cultured primary myotubes cultured under hypoxic conditions.CONCLUSIONS:Circadian clock in skeletal muscle is crucial for the muscle's response to hypoxia during hindlimb ischemia. Targeting the muscle circadian clock may have therapeutic potential for enhancing muscle response to reduced blood flow and promoting recovery in conditions such as PAD.