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Geometric ring annuloplasty has shown promise during bicuspid aortic valve repair for aortic insufficiency. This study examined early outcomes of bicuspid aortic valve repair associated with proximal aortic aneurysm replacement.From September 2017 to November, 2021, 127 patients underwent bicuspid aortic valve repair with concomitant proximal aneurysm reconstruction. Patient age was 50.6 ± 12.7 years (mean ± standard deviation), male gender was 83%, New York Heart Association Class was 2 (1-2) (median [interquartile range]), and preoperative aortic insufficiency grade was 3 (2-4). Ascending aortic diameter was 50 (46-54) mm, and all patients had ascending aortic replacement. Forty patients had sinus diameters greater than 45 mm, prompting remodeling root procedures. A total of 105 patients had Sievers type 1 valves, 3 patients had type 0, and 7 patients had type 2. A total of 118 patients had primarily right/left fusion, 8 patients had right/nonfusion, and 1 patient had left/nonfusion. Leaflet reconstruction used central leaflet plication and cleft closure, with limited ultrasonic decalcification in 31 patients.Ring size was 23 (21-23) mm, and 26 of 40 root procedures were selective nonfused sinus replacements. Aortic clamp time was 139 (112-170) minutes, and bypass time was 178 (138-217) minutes. Postrepair aortic insufficiency grade was 0 (0-0) (Geometric ring annuloplasty for bicuspid aortic valve repair with proximal aortic aneurysm reconstruction is safe and associated with good early outcomes. Further experience and follow-up will help inform long-term durability.

BACKGROUNDpostoperative atrial fibrillation (POAF) is a common cardiac surgery complication that is associated with increased complications and negative outcomes, but the association between presurgical atrial conduction abnormalities and POAF has not been investigated clinically during premature atrial S1S2 stimulation. This clinical study sought to examine whether intraoperative premature atrial stimulation reveals increased areas of slowed and/or blocked conduction in patients that develop POAF.METHODSHigh-density intraoperative epicardial left atrial mapping was conducted in 20 cardiac surgery patients with no prior history of atrial fibrillation (AF). In 20 patients, 6 (30%) developed POAF. A flexible-array of 240-electrodes was placed on the posterior left atrial wall in between the pulmonary veins. Activation maps were generated for sinus and premature atrial S1S2 stimulated beats. The area of conduction block (CB), conduction delay (CD) and the combination of both (CDCB) for conduction velocity RESULTSFor a premature atrial S2 beat with shortest cycle length captured, conduction velocity maps revealed a significantly higher area for CD (13.19±6.59 versus 6.06±4.22 mm2, p=0.028) and CDCB (17.36±8.75 versus 7.41±6.39 mm2, p=0.034), and a trend toward a larger area for CB (4.17±3.66 versus 1.34±2.86 mm2, p=0.063) in patients who developed POAF in comparison to those that remained in the sinus. Sinus and S1 paced beats did not show substantial differences in abnormal conduction areas between patients with and without POAF.CONCLUSIONIn comparison to sinus and S1 beats, premature atrial S2 beats accentuate conduction abnormalities in cardiac surgery patients that developed POAF.

National and institutional quality initiatives provide benchmarks for evaluating the effectiveness of medical care. However, the dramatic growth in the number and type of medical and organizational quality-improvement standards creates a challenge to identify and understand those that most accurately determine quality in cardiac surgery. It is important that surgeons have knowledge and insight into valid, useful indicators for comparison and improvement. We therefore reviewed the medical literature and have identified improvement initiatives focused on cardiac surgery. We discuss the benefits and drawbacks of existing methodologies, such as comprehensive regional and national databases that aid self-evaluation and feedback, volume-based standards as structural indicators, process measurements arising from evidence-based research, and risk-adjusted outcomes. In addition, we discuss the potential of newer methods, such as patient-reported outcomes and composite measurements that combine data from multiple sources.

Transcatheter aortic valve implantation (TAVI) is now routinely performed in patients with aortic stenosis with low mortality and complication rates. Although periprocedural risks have been substantially minimized, procedure- and contrast-induced acute kidney injury (AKI) remains a major concern. AKI remains a frequent complication of contrast-guided interventional procedures and is associated with a significantly adverse prognosis. We review the currently available clinical data related to AKI, with emphasis on contrast-induced nephropathy (CIN), and discuss a novel, integrated approach aiming to minimize AKI risk in high-risk patients. A stepwise algorithm is also proposed for the management of these complex patients.

Introduction: Postoperative atrial fibrillation (POAF) is a common cardiac surgery complication that is highly associated with increased morbidity and mortality. The relation of presurgical left atrial (LA) conduction abnormalities and POAF during intraoperative premature atrial stimulation (S1S2) pacing is investigated and reported. Hypothesis: Intraoperative premature atrial stimulation reveals increased areas of slowed or blocked conduction in patients that develop POAF. Methods: High-density intraoperative epicardial mapping was conducted in 20 cardiac surgery patients with no history of preoperative atrial fibrillation (AF) both in sinus rhythm (SR) and during S1S2 pacing. A flexible array comprised of 240 electrodes was placed on the posterior LA wall in between the pulmonary veins. For each patient, the area of conduction block (CB), conduction delay (CD) and combined conduction delay and block (CDCB) for conduction velocity Results: In 20 patients, 6 (30%) developed POAF. As shown in the Figure, conduction maps revealed the presence of significantly higher areas of CD (13.2±6.6% vs. 6.1±4.2%, p=0.03) and CDCB (17.5±8.7% vs. 7.4±6.4%, p=0.03), and a trend toward larger CB (4.2±3.8% vs. 1.3±2.9%, p=0.09) in patients that developed POAF for premature atrial beat S2 compared to patients that remained in SR after cardiac surgery. S1 paced beats and SR did not show significant differences in abnormal conduction percentages between patients with and without POAF. Conclusions: Premature atrial stimulation accentuates conduction abnormalities in cardiac surgery patients that develop POAF, revealing a pre-surgical substrate that may indicate greater risk for post-surgical atrial arrhythmias.

Acute changes to left ventricular (LV) hemodynamics after transcatheter aortic valve replacement (TAVR) in humans have been sparsely investigated and informed largely by computer simulation of hypothetical pressure-volume (PV) loops. PV loops are the gold standard for evaluating mechanical

Acute ambient air pollution exposure increases risk of cardiac events. We evaluated sex-and-age-specific effects of PM2.5 on hospital readmission and death among 19,602 Medicare beneficiaries (Nevents = 30,510) who survived cardiovascular events including myocardial infarction (MI), heart failure (HF), ischemic heart disease (IHD), and cardiac arrhythmias in Utah from 1999&ndash, 2009. Fine and Gray regression jointly modeled the effect of PM2.5 on readmission hazard rates while allowing for the competing risk of death. Models were stratified by age and sex and adjusted for Charlson Comorbidity Index, dual enrollment in Medicare Parts A and B, temperature, and household income. There were 2032 cardiac readmissions and 1420 deaths after discharge. Among males age 65&ndash, 74 years admitted for HF, a 10 &mu, m/m3 increase in PM2.5 was associated with a 30% increase in risk of readmission (p = 0.01). Among females age 75&ndash, 84 admitted for HF, PM2.5 was associated with a 22% increase in risk of readmission (p = 0.01). Among females age 75&ndash, 84 years admitted for IHD, PM2.5 was associated with a 25% decrease in readmission (p = 0.01), however this was explained by a 36% increase in risk of death (p = 0.01). Exposure to PM2.5 was associated with increased risk of readmission and death. Associations were dependent upon age, sex, and index condition.

Acute ambient air pollution exposure increases risk of cardiac events. We evaluated sex-and-age-specific effects of PM2.5 on hospital readmission and death among 19,602 Medicare beneficiaries (Nevents = 30,510) who survived cardiovascular events including myocardial infarction (MI), heart failure (HF), ischemic heart disease (IHD), and cardiac arrhythmias in Utah from 1999–2009. Fine and Gray regression jointly modeled the effect of PM2.5 on readmission hazard rates while allowing for the competing risk of death. Models were stratified by age and sex and adjusted for Charlson Comorbidity Index, dual enrollment in Medicare Parts A and B, temperature, and household income. There were 2032 cardiac readmissions and 1420 deaths after discharge. Among males age 65–74 years admitted for HF, a 10 μm/m3 increase in PM2.5 was associated with a 30% increase in risk of readmission (p = 0.01). Among females age 75–84 admitted for HF, PM2.5 was associated with a 22% increase in risk of readmission (p = 0.01). Among females age 75–84 years admitted for IHD, PM2.5 was associated with a 25% decrease in readmission (p = 0.01), however this was explained by a 36% increase in risk of death (p = 0.01). Exposure to PM2.5 was associated with increased risk of readmission and death. Associations were dependent upon age, sex, and index condition.

BACKGROUND Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. METHODS Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. RESULTS PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05). CONCLUSIONS Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

Author(s): Nguyen, Heajung L; Calfon Press, Marcella A; Aboulhosn, Jamil A; Lin, Jeannette P; Benharash, Peyman; Yang, Eric H

Objectives Systemic allergic reactions to nickel alloys in percutaneous atrial septal defect occlusion devices have a poorly defined natural history. We describe our experience of surgical removal of the offending device in a series of patients with nickel allergy and refractory symptoms. Methods Patients with atrial septal defect device explants for nickel allergy were reviewed. Administered questionnaires focused on symptoms, quality of life, and satisfaction along with the 36-Item Short Form Health Survey to measure physical and mental health postsurgery. Results Atrial septal defect devices were removed for nickel allergy in 58 patients during the past 10 years. The median age was 42 years (range, 24-71 years) and 95% were women. Explantation occurred at a median of 8 years (range, 6 months-18 years) after insertion. Symptoms included fatigue (82%), chest pain (78%), headache (73%), and palpitation (58%). Surveys were available for 45 patients: 58% rated their quality of life as poor and 69% were not at all satisfied with their device. Postexplant, all patients reported improvement in their symptoms, with 18 patients (42%) noting complete resolution. In 12 patients prospectively studied, the preoperative scores in physical and mental health domains were lower than the validation group, indicating significant disability. Similarly, there was marked improvement in each domain postremoval. Conclusions Patients with nickel allergy and severe refractory symptoms after atrial septal defect device implantation experience profound resolution of symptoms and improved quality of life after removal. Nickel allergy should be considered before device insertion, and a low threshold should exist for surgical removal for refractory symptoms.

Objective The objective of this study was to determine if the implementation of a value-driven outcomes tool comprising modifiable quality and utilization metrics lowers cost and improves value of coronary artery bypass grafting (CABG) postoperative care. Methods Ten metrics were defined for CABG patients in 2 temporally separated phases. Clinical care protocols were designed and implemented to increase compliance with these metrics. Clinical outcomes and cost data were harvested from the electronic medical record using a proprietary value-driven outcomes tool and verified by a data management team. "Perfect care" was defined as achieving all 10 metrics per patient episode. Results Over a 45-month period, data of 467 consecutive patients who underwent isolated CABG were analyzed. "Perfect care" was successfully achieved in 304 patients (65.1%). There were no observed differences in mortality between patient groups. Linear regression analysis showed a negative correlation between percent compliance with "perfect care" and mean cost. When multivariate analysis was used to adjust for preoperative risk score, mean cost for patients with "perfect care" was 37.0% less than for those without "perfect care." Conclusions In the context of focused institution-specific interventions to target quality and utilization metrics for CABG care, clinical care pathways and protocols informed by innovative tools that link automated tracking of these metrics to cost data might simultaneously promote quality and decrease costs, thereby enhancing value. This descriptive study provides preliminary support for a systematic approach to define, measure, and modulate the drivers of value for cardiothoracic surgery patients.

Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear.We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs.Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs.Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

Reduced expression of bone morphogenetic protein receptor 2 subverts a stress granule response, heightens GM-CSF mRNA translation, and increases inflammatory cell recruitment to exacerbate pulmonary arterial hypertension., Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34–PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)–positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

Although common, the prevalence and impact of treatment on main pulmonary artery (MPA) dilation in Marfan syndrome (MFS) are unclear. We sought to determine the prevalence and progression of MPA dilation (Z score ≥2) and impact of treatment over 3 years using the public dataset from the Pediatric

Background Frostbite injury occurs when exposure to cold results in frozen tissue. To screen drugs and other field therapies that might improve the outcome for a frostbite victim, it would be helpful to have a reliable and cost-effective preclinical in vivo model. Objective We sought to create a novel mouse skin model of induced frostbite injury. This model would allow quantification of the surface area of involved skin, histology of the wound, rate of wound healing, and skin loss in a standardized fashion after the frostbite injury. Methods Thirty-six mice were studied. Standardized 2.9-cm diameter circles were tattooed on the mouse dorsum. Magnets frozen in dry ice (−78.5°C) were used to create a frostbite injury on skin within the circle, either as a continuous 5-minute freeze or as 3 repeated freeze (1-minute) and thaw (3-minute) cycles. Appearance, healing rate, skin surface area loss, and histology were recorded until the wounds were healed. Results The amount of skin surface area loss was approximately 50% for both freeze methods. Although the time to surface skin healing was similar for both freeze methods, the initial healing rate was significantly ( P = .001) slower in mice exposed to the freeze-thaw cycles compared with the continuous freeze model. Histopathology reflected inflammatory changes, cell death, and necrosis. Conclusions This novel in vivo mouse model for frostbite allows quantification of affected skin surface area, histology, healing rate, and skin loss and has the potential of being utilized to screen future treatment modalities.

With the rapidly expanding field of tissue engineering, surgeons have been eager to apply these principles to craniofacial surgery. Tissue engineering strategies combine the use of a cell type placed on a scaffold and subsequently implanted in vivo to address a tissue defect or tissue dysfunction. In this review we will discuss the current clinical need for skeletal and soft tissue engineering faced by craniofacial surgeons and subsequently we will explore cell types and scaffold designs being employed for tissue engineering treatment options. We will conclude by discussing ways to enhance the vascularity of tissue engineered constructs as this will ultimately allow for a definitive repair. Current “stem cell” options include pluripotent stem cells as well as multipotent mesenchymal cells. Biomimetic scaffolds can function to protect and enhance differentiation of stem cells by providing inductive cues. Larger scale studies including prospective, randomized control trials must be performed to determine the optimum cell delivery method and cytokine stimuli for tissue engineering driven strategies to address the clinical needs in skeletal and soft tissue reconstruction.

Before stem cell-based therapies can become a clinical reality, technologies for cell delivery must be developed that can control differentiation and pluripotency, maintain a hospitable environment for cell survival and function, and provide a structural framework for regenerative healing of the target tissue. Insights gained from developmental and stem cell biology should guide the design of devices and techniques to facilitate stem cell-based therapies. Several strategies have been developed for surgical delivery of stem cells, including synthetic and biologic matrices for cell seeding, complex biochemical delivery devices for maintenance and modulation of stem cell properties, and smart constructs with the ability to adapt to the dynamic in vivo environment after implantation. In aggregate, surgical delivery of complex stem cell-seeded constructs has the potential to revolutionize surgical therapies for a wide range of diseases in order to provide a more regenerative platform for tissue and organ healing.

Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood1. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK)1,2. Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders3–5. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK–ERK–MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

Wound repair is a complex biologic process which becomes abnormal in numerous disease states. Althoughin vitromodels have been important in identifying critical repair pathways in specific cell populations,in vivomodels are necessary to obtain a more comprehensive and pertinent understanding of human wound healing. The laboratory mouse has long been the most common animal research tool and numerous transgenic strains and models have been developed to help researchers study the molecular pathways involved in wound repair and regeneration. This paper aims to highlight common surgical mouse models of cutaneous disease and to provide investigators with a better understanding of the benefits and limitations of these models for translational applications.

Diabetic wounds are a significant public health burden, with slow or nonhealing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1alpha (HIF-1alpha), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1alpha activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1alpha transactivation persist even in the setting of constitutive HIF-1alpha protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1alpha (the primary pathway regulating HIF-1alpha/p300 binding) does not alter HIF-1alpha activity. We extend our study of DFO's therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.

Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery, and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow–derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have provasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.

Breast cancer remains one of the most prevalent cancers in women, with an estimated 200,000 new cases of invasive breast cancer and over 50,000 cases of in situ breast cancer expected annually.1 Despite improved early detection and evolving strategies to minimize surgical intervention for diagnosis and treatment of axillary disease associated with breast cancer, many women are still plagued by the disabling complication of upper limb lymphedema. Conservative treatment using massage and compression therapy remains the mainstay for lymphedema; however, these treatments do not offer lasting relief of the condition because they fail to address the underlying pathologic accumulation of excess adipose tissue.2 Several investigators have reported the use of liposuction to treat upper extremity lymphedema, providing an opportunity to examine whether the stromal fraction of lymphedema-associated adipose tissue differs from nonaffected subcutaneous adipose tissue.3 We hypothesize that the stromal vascular fraction of lymphedema tissue has important differences compared with that of healthy subcutaneous fat with regard to gene expression and differentiation capacity. The complication of lymphedema develops gradually as the lymphatic vessels are unable to drain the appropriate amount of lymph and proteins. The remaining lymphatic channels become dilated and overloaded, rendering the valves incompetent.2 Eventually, the entire extremity is affected, and even the most distal vessels become dilated. Concurrently, mononuclear phagocytotic cells and mesenchymal tissue lose their ability to transport proteins, causing these to accumulate. Excess protein creates an osmotic gradient, drawing in additional fluid. Over time, the extremity enlarges and becomes painful and weak. Traditional therapeutic approaches assumed that the enlarged extremity in lymphedema was mainly the cause of excess lymph fluid, and thus noninvasive treatments were focused on compression and enhancing lymph flow. After the first operation on an affected arm, however, surgeons realized that the majority of this excess tissue was adipose tissue and not just edematous tissue.4,5 Therefore, it appears clear that lipoaspiration is needed to address the excess adipose component associated with the chronic lymphedematous arm. Within the stromal vascular fraction of adipose tissue, scientists have identified a group of cells known as adipose-derived stromal cells. As in other mesenchymal populations, adipose-derived stem cells have the capacity to differentiate into skeletal muscle, smooth muscle, fat, cartilage, connective tissues, tendon, and bone.6–9 The adipogenic potential of adipose-derived stem cells has been the focus of many studies, and several articles have been published on the in vitro adipogenic differentiation of adipose-derived stem cells, although we are not aware of studies assessing whether differences in adipose-derived stem cells account for the increased adiposity seen in lymphedema patients.10–12 Similarly, several studies have demonstrated the vasculogenic capacity of adipose-derived stem cells; however, it is unknown whether lymphedema-derived adipose-derived stem cells differ in their vasculogenic potential.13–17 In this study, we set out to characterize functional differences in the adipose-derived stem cells from lymphedematous and healthy adipose tissue, as this might enhance our understanding of the involved molecular mechanisms and provide further insight into the underlying abnormality.

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and non-hematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein (GFP) and non-hematopoietic cells with red fluorescent protein (RFP). Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45− and Cd11b−, representing a previously unidentified population. Both cell types were found to initially peak, then reduce post-healing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time-course of fibrocytes in cutaneous healing.

In this study, we examined the capacity of a biomimetic pullulan-collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.

Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

BACKGROUND Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone. METHODS Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry. RESULTS Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site. CONCLUSIONS Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2–Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.

An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly ‘purified’ cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

Cell-based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from the acute inflammatory environment. Here, a biomimetic hydrogel system is described that is based on the polymer pullulan, a carbohydrate glucan known to exhibit potent antioxidant capabilities. It is shown that pullulan hydrogels are an effective cell delivery system and improve mesenchymal stem cell survival and engraftment in high-oxidative-stress environments. The results suggest that glucan hydrogel systems may prove beneficial for progenitor-cell-based approaches to skin regeneration.

New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan–collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan–collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.

Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings.Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (10 to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate.Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p0.05). This was accompanied by an increase in bone morphogenetic protein expression (p0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p0.05).Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect.

BACKGROUND Human adipose-derived stromal cells possess a great potential for tissue engineering purposes. The authors' laboratory is interested in harnessing human adipose-derived stromal cells for skeletal tissue regeneration and identifying those factors that enhance human adipose-derived stromal cell osteogenic differentiation. The authors hypothesized that insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) would stimulate human adipose-derived stromal cell osteogenesis and that IGF would stimulate adipogenesis. METHODS Adipose-derived stromal cells were harvested from human lipoaspirate. Previously, a microarray analysis examined gene expression throughout osteogenic differentiation. In a candidate fashion, the authors added recombinant IGF-1 and PDGF-alpha individually and in combination. Osteogenesis and adipogenesis were assessed by alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction (RUNX2, ALP, OCN, IGF1, PPARG, LPL, AP2, and GCP1). Finally, intersection between IGF and PDGF signaling pathways was evaluated. RESULTS IGF-1 was observed to increase osteogenic differentiation by all markers (p < 0.01). However, PDGF-alpha when added alone primarily did not affect osteogenic markers. PDGF-alpha positively regulated transcription of IGF1. Addition of PDGF-alpha in combination with or before IGF-1 enhanced osteogenesis more than either alone. IGF-1 increased whereas PDGF-alpha diminished human adipose-derived stromal cell adipogenesis. CONCLUSIONS IGF signaling significantly increased osteogenesis in human adipose-derived stromal cells and may be used for tissue-engineering purposes. The combination of PDGF and IGF may be more beneficial than either alone in driving adipose-derived stromal cell osteogenesis. Future in vivo applications will focus on the combination of adipose-derived stromal cells, biomimetic scaffolds, and recombinant IGF.

Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (

Diabetic wounds are a significant public health burden, with slow or nonhealing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1alpha (HIF-1alpha), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1alpha activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1alpha transactivation persist even in the setting of constitutive HIF-1alpha protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1alpha (the primary pathway regulating HIF-1alpha/p300 binding) does not alter HIF-1alpha activity. We extend our study of DFO's therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.