Vasculogenic properties of adventitial Sca-1+CD45+ progenitor cells in mice: a potential source of vasa vasorum in atherosclerosis.

The cellular origins of vasa vasorum are ill-defined and may involve circulating or local progenitor cells. We previously discovered that murine aortic adventitia contains Sca-1+CD45+ progenitors that produce macrophages. Here we investigated whether they are also vasculogenic. In aortas of C57BL/6 mice, Sca-1+CD45+ cells were localised to adventitia and lacked surface expression of endothelial markers (<1% for CD31, CD144, TIE-2). In contrast, they did show expression of CD31, CD144, TIE-2 and VEGFR2 in atherosclerotic ApoE-/- aortas. Although Sca-1+CD45+ cells from C57BL/6 aorta did not express CD31, they formed CD31+ colonies in endothelial differentiation media and produced interconnecting vascular-like cords in Matrigel that contained both endothelial cells and a small population of macrophages, which were located at branch points. Transfer of aortic Sca-1+CD45+ cells generated endothelial cells and neovessels de novo in a hindlimb model of ischaemia and resulted in a 50% increase in perfusion compared to cell-free control. Similarly, their injection into the carotid adventitia of ApoE-/- mice produced donor-derived adventitial and peri-adventitial microvessels after atherogenic diet, suggestive of newly formed vasa vasorum. These findings show that beyond its content of macrophage progenitors, adventitial Sca-1+CD45+ cells are also vasculogenic and may be a source of vasa vasorum during atherogenesis.

Effect of Preprocedural Thrombocytopenia on Prognosis After Percutaneous Coronary Intervention.

OBJECTIVE: To assess early and late outcomes, including bleeding, in patients with thrombocytopenia undergoing percutaneous coronary intervention (PCI). PATIENTS AND METHODS: We performed a retrospective single-center study of patients with preprocedural thrombocytopenia (platelet count ≤100,000/µL; n=204) undergoing PCI between 2003 and 2015. Inhospital and late outcomes were compared with those of a matched control group without thrombocytopenia (n=1281). RESULTS: The most common causes of thrombocytopenia were liver disease, immune-mediated disease, and hematologic malignant neoplasms. Inhospital bleeding events after PCI were similar in patients with thrombocytopenia and matched controls (24 of 146 [16.4%] vs 179 of 1281 [14.0%]; P=.40) and were largely classified as minor using the Bleeding Academic Research Consortium (BARC) classification (89% BARC 1 or 2). There was no significant difference in inhospital death (4 of 146 [2.7%] vs 71 of 1281 [2.0%]; P=.56), but patients with thrombocytopenia had higher rates of platelet and red blood cell transfusion (18 of 146 [12.3%] vs 93 of 1281 [7.2%]; P=.05). During long-term follow-up, Kaplan-Meier estimated rates of bleeding events (BARC ≥2) were higher for thrombocytopenia (at 5 years, 7.9% vs 3.6%; P=.03). Patients with thrombocytopenia had a similar risk of long-term cardiac mortality, but significantly higher rates of noncardiac mortality (at 5 years, 28% vs 21%; P=.02). CONCLUSION: This study suggests that short-term outcomes after PCI in patients with thrombocytopenia were favorable. On long-term follow-up, thrombocytopenia was associated with a higher risk of long-term noncardiac mortality and bleeding.

Low-Dose Gamma Irradiation of Decellularized Heart Valves Results in Tissue Injury In Vitro and In Vivo.

BACKGROUND: Decellularized heart valves are emerging as a potential alternative to current bioprostheses for valve replacement. Whereas techniques of decellularization have been thoroughly examined, terminal sterilization techniques have not received the same scrutiny. METHODS: This study evaluated low-dose gamma irradiation as a sterilization method for decellularized heart valves. Incubation of valves and transmission electron microscopy evaluation after different doses of gamma irradiation were used to determine the optimal dose of gamma irradiation. Quantitative evaluation of mechanical properties was done by tensile mechanical testing of isolated cusps. Sterilized decellularized heart valves were tested in a sheep model (n = 3 [1 at 1,500 Gy and 2 at 3,000 Gy]) of pulmonary valve replacement. RESULTS: Valves sterilized with gamma radiation between 1,000 Gy and 3,000 Gy were found to be optimal with in vitro testing. However, in vivo testing showed deteriorating valve function within 2 months. On explant, the valve with 1,500 Gy gamma irradiation showed signs of endocarditis with neutrophils on hematoxylin and eosin staining, and positive gram stain resembling streptococcus infection. The 3,000 Gy valves had no evidence of infection, but the hematoxylin and eosin staining showed evidence of wound remodeling with macrophages and fibroblasts. Tensile strength testing showed decreased strength (0 Gy: 2.53 ± 0.98 MPa, 1,500 Gy: 2.03 ± 1.23 MPa, and 3,000 Gy: 1.26 ± 0.90 MPa) with increasing levels of irradiation. CONCLUSIONS: Low-dose gamma irradiation does not maintain the mechanical integrity of valves, and the balance between sterilization and damage may not be able to be achieved with gamma irradiation. Other methods of terminal sterilization must be pursued and evaluated.

Prediction of Cardiac and Noncardiac Mortality After Percutaneous Coronary Intervention.

BACKGROUND: Current risk models for predicting long-term mortality after percutaneous coronary intervention are restricted to all-cause mortality. We sought to develop novel risk models for the prediction of cardiac and noncardiac mortality after percutaneous coronary intervention. METHODS AND RESULTS: We retrospectively evaluated patients who underwent index percutaneous coronary intervention at Mayo Clinic from 2003 to 2008. Long-term deaths were ascertained through scheduled prospective surveillance. Cause of death was determined via telephone interviews, medical records, and autopsy reports. Fine and Gray extension of Cox proportional hazards models was used to model cause-specific cumulative incidence. Candidate variables and interactions were chosen a priori, without variable selection methods. Resulting models were mapped to an integer-based risk score. The study comprised 6636 patients followed up over a median of 62 months (25th, 75th percentiles: 45, 77 months). There were 1488 deaths, 518 (35%) cardiac, 938 (63%) noncardiac, and 32 (2%) unknown. The 5-year predicted cardiac mortality ranged from 0.6% to 97%, with a corrected c-statistic of 0.82. Risk factors for cardiac death included age, body mass index, ejection fraction, and history of congestive heart failure. The integer score for noncardiac death included age, medicine index, body mass index, current smoker, noncardiac Charlson index and cardiac Charlson index, and accommodated significant age-based interactions for smoking and the 2 Charlson indices. Predicted noncardiac mortality at 5 years ranged from 0.2% to 81%, with a corrected c-statistic of 0.77. CONCLUSIONS: We report novel risk models to predict cardiac and noncardiac long-term mortality after percutaneous coronary intervention.

Incidence of symptomatic venous thromboembolism in patients with hemophilia undergoing joint replacement surgery: a retrospective study.

INTRODUCTION: Venous thromboembolism (VTE) is a recognized complication after joint replacement surgery, and prophylaxis is routinely used in patients without bleeding disorders. However, for patients with hemophilia, pharmacologic prophylaxis is highly variable and controversial because of the inherent bleeding risk. AIM: To review our institutional experience with outcomes of total knee or hip arthroplasty with regard to symptomatic VTE and use of VTE prophylaxis in patients with hemophilia and without inhibitors. METHODS: We reviewed records of 42 consecutive patients with hemophilia A or B who underwent 71 hip or knee replacements over a 39-year period. We also reviewed the literature to estimate the incidence of VTE after arthroplasty in the hemophilia population. RESULTS: All patients used compression stockings for up to 6weeks after surgery; additionally, 6 cases (10.5%; 57 with available data) used sequential intermittent compression devices and 2 (2.8%) postoperatively received low-molecular-weight heparin. One patient (1.4%) who received low-molecular-weight heparin had a symptomatic, lower-extremity, deep venous thrombosis 10days after hip replacement for traumatic fracture. None of the other 70 surgical cases had symptomatic VTE within 3months after the procedure. Analysis of pooled data from published series of hemophilia patients undergoing arthroplasty showed an estimated incidence of symptomatic VTE of 0.5%. CONCLUSION: Our study suggests that in patients with hemophilia, joint replacement surgery can be performed safely without routine pharmacologic VTE prophylaxis and without increasing risk of thromboembolic events. Pharmacologic VTE prophylaxis may be considered in select patients with known additional risk factors for VTE.

An update on stem cell therapies for acute coronary syndrome.

Well into the second decade since its conception, cell transplantation continues to undergo intensive evaluation for the treatment of myocardial infarction. At a mechanistic level, its objectives remain to replace lost cardiac cell mass with new functioning cardiomyocytes and vascular cells, thereby minimizing infarct size and scar formation, and improving clinical outcomes by preventing adverse left ventricular remodeling and recurrent ischemic events. Many different cell types, including pluripotent stem cells and various adult-derived progenitor cells, have been shown to have therapeutic potential in preclinical studies, while early phase human trial experience has provided divergent outcomes and fundamental lessons, emphasizing that there remain key issues to address and challenges to overcome before cell therapy can be applied to wider clinical practice. The purpose of this review is to provide a balanced update on recent seminal developments in this exciting field and look to the next important steps to ensure its forward progression.

Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature.

RATIONALE: Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. OBJECTIVE: We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. METHODS AND RESULTS: Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1(+)CD45(+) cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67(+)) Lin(-)c-Kit(+)CD135(-)CD115(+)CX3CR1(+)Ly6C(+)CD11b(-) subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1(+)CD45(+) adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE(-/-) and LDL-R(-/-) mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. CONCLUSIONS: The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

Bone marrow mononuclear cell therapy for acute myocardial infarction: a perspective from the cardiovascular cell therapy research network.

To understand the role of bone marrow mononuclear cells in the treatment of acute myocardial infarction, this overview offers a retrospective examination of strengths and limitations of 3 contemporaneous trials with attention to critical design features and provides an analysis of the combined data set and implications for future directions in cell therapy for acute myocardial infarction.

Trends in cause of death after percutaneous coronary intervention.

BACKGROUND: The impact of changing demographics on causes of long-term death after percutaneous coronary intervention (PCI) remains incompletely defined. METHODS AND RESULTS: We evaluated trends in cause-specific long-term mortality after index PCI performed at a single center from 1991 to 2008. Deaths were ascertained by scheduled prospective surveillance. Cause was determined via telephone interviews, medical records, autopsy reports, and death certificates. Competing-risks analysis of cause-specific mortality was performed using 3 time periods of PCI (1991-1996, 1997-2002, and 2003-2008). Final follow-up was December 31, 2012. A total of 19 077 patients survived index PCI hospitalization, of whom 6988 subsequently died (37%, 4.48 per 100 person-years). Cause was determined in 6857 (98.1%). Across 3 time periods, there was a 33% decline in cardiac deaths at 5 years after PCI (incidence: 9.8%, 7.4%, and 6.6%) but a 57% increase in noncardiac deaths (7.1%, 8.5%, and 11.2%). Only 36.8% of deaths in the recent era were cardiac. Similar trends were observed regardless of age, extent of coronary disease, or PCI indication. After adjustment for baseline variables, there was a 50% temporal decline in cardiac mortality but no change in noncardiac mortality. The decline in cardiac mortality was driven by fewer deaths from myocardial infarction/sudden death (P<0.001) but not heart failure (P=0.85). The increase in noncardiac mortality was primarily attributable to cancer and chronic diseases (P<0.001). CONCLUSIONS: This study found a marked temporal switch from predominantly cardiac to predominantly noncardiac causes of death after PCI over 2 decades. The decline in cardiac mortality was independent of changes in baseline clinical characteristics. These findings have implications for patient care and clinical trial design.

Outcome of repair of myocardial bridging at the time of septal myectomy.

BACKGROUND: Myocardial bridging describes systolic compression of the muscular investment of a portion of an epicardial coronary artery. We evaluated the outcome of muscular bridge unroofing of the left anterior descending artery at the time of septal myectomy in patients with hypertrophic cardiomyopathy. METHODS: We conducted a case-controlled study of 36 patients (23 men; median age, 42 years) with hypertrophic cardiomyopathy and myocardial bridging. Group 1 patients had septal myectomy and concomitant unroofing (n = 13), group 2 patients underwent myectomy alone (n = 10), and group 3 patients were treated medically (n = 13). RESULTS: Angina was more prevalent preoperatively in group 1, 46% compared with 20% in group 2. Preoperative left ventricular outflow tract gradients of 67.8 ± 58.2 mm Hg and 74.1 ± 19.7 mm Hg were reduced to 1.9 ± 2.9 mm Hg in group 1 (p < 0.0001) and to 5.6 ± 8.8 mm Hg in group 2 (p < 0.0001). In the surgical groups, there were no early deaths or complications related to unroofing. Survival at 10 years was 83.3% in group 1 (p = 0.297), 100.0% in group 2, and 67.9% in group 3; there were no late sudden deaths. At follow-up, 77% in group 1 were asymptomatic compared with 70% of patients in group 2 (p = 0.19). There was no recurrent angina in group 1. CONCLUSIONS: Myocardial unroofing can be performed safely at the time of septal myectomy for left ventricular outflow tract obstruction. Angina was improved, but we found no difference in late survival compared with patients who had myocardial bridging and myectomy alone. Unroofing should be considered in patients with angina who have significant left anterior descending artery bridging and require myectomy.

Effect of the use and timing of bone marrow mononuclear cell delivery on left ventricular function after acute myocardial infarction: the TIME randomized trial.

CONTEXT: While the delivery of cell therapy after ST-segment elevation myocardial infarction (STEMI) has been evaluated in previous clinical trials, the influence of the timing of cell delivery on the effect on left ventricular function has not been analyzed. OBJECTIVES: To determine the effect of intracoronary autologous bone marrow mononuclear cell (BMC) delivery after STEMI on recovery of global and regional left ventricular function and whether timing of BMC delivery (3 days vs 7 days after reperfusion) influences this effect. DESIGN, SETTING, AND PATIENTS: A randomized, 2 × 2 factorial, double-blind, placebo-controlled trial, Timing In Myocardial infarction Evaluation (TIME) enrolled 120 patients with left ventricular dysfunction (left ventricular ejection fraction [LVEF] ≤ 45%) after successful primary percutaneous coronary intervention (PCI) of anterior STEMI between July 17, 2008, and November 15, 2011, as part of the Cardiovascular Cell Therapy Research Network sponsored by the National Heart, Lung, and Blood Institute. INTERVENTIONS: Intracoronary infusion of 150 × 106 BMCs or placebo (randomized 2:1) within 12 hours of aspiration and cell processing administered at day 3 or day 7 (randomized 1:1) after treatment with PCI. MAIN OUTCOME MEASURES: The primary end points were change in global (LVEF) and regional (wall motion) left ventricular function in infarct and border zones at 6 months measured by cardiac magnetic resonance imaging and change in left ventricular function as affected by timing of treatment on day 3 vs day 7. The secondary end points included major adverse cardiovascular events as well as changes in left ventricular volumes and infarct size. RESULTS: The mean (SD) patient age was 56.9 (10.9) years and 87.5% of participants were male. At 6 months, there was no significant increase in LVEF for the BMC group (45.2% [95% CI, 42.8% to 47.6%] to 48.3% [95% CI, 45.3% to 51.3%) vs the placebo group (44.5% [95% CI, 41.0% to 48.0%] to 47.8% [95% CI, 43.4% to 52.2%]) (P = .96). There was no significant treatment effect on regional left ventricular function observed in either infarct or border zones. There were no significant differences in change in global left ventricular function for patients treated at day 3 (−0.9% [95% CI, −6.6% to 4.9%], P = .76) or day 7 (1.1% [95% CI, −4.7% to 6.9%], P = .70). The timing of treatment had no significant effect on regional left ventricular function recovery. Major adverse events were rare among all treatment groups. CONCLUSION: Among patients with STEMI treated with primary PCI, the administration of intracoronary BMCs at either 3 days or 7 days after the event had no significant effect on recovery of global or regional left ventricular function compared with placebo. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00684021.

An in vivo method to quantify lymphangiogenesis in zebrafish.

BACKGROUND: Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo. METHODS AND RESULTS: Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth. CONCLUSION: Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.

Anatomic features of the left main coronary artery and factors associated with its bifurcation angle: a 3-dimensional quantitative coronary angiographic study.

OBJECTIVE: To assess the anatomic characteristics of the left main coronary artery (LM), and the relation between anatomic and clinical factors and the LM bifurcation angle (BA) using a novel, three dimensional quantitative coronary angiography (3D QCA) software. BACKGROUND: Percutaneous intervention of the LM is a therapeutic option in selected patients with coronary artery disease (CAD). The anatomic features of the LM and its BA are determinants of procedural success and clinical outcome. However, those features and the factors that may affect the LM BA have not been fully described. METHODS: The LM anatomy was evaluated from angiograms of 203 patients (age = 66 ± 11 years, 31% female) with and without LM CAD using 3D QCA analysis (IC-PRO, Paieon, Israel). LM size as well as the proximal BA (between LM and LCX) and the distal BA (between left anterior descending coronary artery (LAD) and left circumflex coronary artery (LCX)) were measured in end-diastole. Angiographic and clinical findings were also recorded. RESULTS: 133/203 patients (65%) had no LM CAD. 3D QCA analysis demonstrated significant variability in the anatomy of the normal LM, including the LM branch vessels (LAD, LCX) diameter, and the LM BA. Among the 70 patients with LM CAD, 44 had distal LM disease. Importantly, patients with distal LM CAD had narrower proximal BA and a wider distal BA. Multivariate analysis (adjusted for clinical and anatomic variables) identified female sex (P = 0.02), trifurcation anatomy (P = 0.009), age > 75 years (P = 0.0009), and LM length > 12 mm (P = 0.001) as independent associates of the proximal BA. Independent associates of the distal BA were: trifurcation anatomy (P = 0.001), LM length > 12 mm (P < 0.0001), age > 75 years (P = 0.004), and a history of coronary bypass surgery (P = 0.04). CONCLUSIONS: The current study demonstrates significant variability in the anatomy of the LM. The LM BA differs between patients with and without distal LM CAD, and both anatomic and clinical factors may affect the LM BA. Our findings also emphasize the possible usefulness of 3D QCA in the assessment of the LM.

Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta.

BACKGROUND: Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. METHODS AND RESULTS: Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E-null (ApoE(-/-)) mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE(-/-) mice and localized primarily to stem cell antigen-1-positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (1 per 4 000 000 cells) capable of self-renewal and durable, low-level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of green fluorescent protein-positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. CONCLUSIONS: The postnatal murine aorta contains rare multipotent hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1-positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Predictive factors in identifying subspecialty fellowship applicants who will have academic practices.

BACKGROUND: The challenge of subspecialty fellowship directors is to recruit surgeons who are motivated to continue the tradition of teaching by entering academic medicine. The authors looked for predictive factors to help with more accurate selection of applicants. METHODS: Application and follow-up data from plastic surgery subspecialty fellows in craniofacial surgery, hand surgery, and microsurgery from the University of California, Los Angeles were reviewed for the years 1987 through 2002 (n = 62). Fellows were divided into three groups as follows: group 1, full-time academic; group 2, part-time clinical faculty; and group 3, private practice at 1 year and 5 years after fellowship. Common factors of fellows within the three groups were listed. RESULTS: Although a majority of applicants (95 percent) indicated an aspiration to practice academic medicine, only one-third remained in full-time academics 5 years after their subspecialty training. There was a trend toward leaving academic practice: the rates at 1 year were 74 percent for group 1 (academic) and 5 percent for group 3 (private practice), but by 5 years this had equalized (group 1, 34 percent; group 3, 32 percent). Group 1 (academic) showed more academic productivity publications per year, academic titles, editorial boards, and active participation in medical societies compared with group 3 (private practice). The factors that were more common to group 1 were married or married with children, five or more publications, one or more years of research, and 7 or more years of training. CONCLUSION: Plastic surgery fellowship directors may look at the following predictive factors of applicants if they would like their graduates to carry on the tradition of teaching future plastic surgeons: (1) previous dedicated research training, (2) more years of clinical training, and (3) more scientific publications.

Correction of massive hydrocephalus and brain wound in holoprosencephaly.

Holoprosencephaly is a rare disorder of embryologic development that denotes an incomplete or absent division of the embryonic forebrain (prosencephalon) into distinct lateral cerebral hemispheres. In most cases of holoprosencephaly, the fetus fails to survive to term, and those that do survive have severe functional limitations, including mental retardation. We present a case report of cranial vault remodeling in a 20-month-old female who was born with holoprosencephaly and a severe number 0/14 cleft. The patient developed chronic nonhealing ulcers secondary to hydrocephalus, ventriculoperitoneal shunts, and the underlying architecture of her cranial vault.

Osteogenic potentiation of human adipose-derived stem cells in a 3-dimensional matrix.

Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n = 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n = 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen I, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group 1 (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase [P < 0.05], 8.4-fold greater expression of osteonectin [P < 0.05], 6.4-fold greater expression of osteopontin [P < 0.05], 2.9-fold greater expression of collagen I [P < 0.05], and 2.5-fold greater expression of JNK2 [P < 0.05]). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer.

Noggin underexpression and runx-2 overexpression in a craniosynostosis rabbit model.

INTRODUCTION: Normal suture fusion has been shown to be driven by the molecular signals elucidated by the underlying dura. However, the pathogenesis of suture fusion in craniosynostosis is not well described. The purpose of our study was to examine the expression patterns of 2 important molecular signals (Noggin and Runx-2) in a cohort of congenital craniosynostotic rabbits to gain a better understanding of suture behavior in craniosynostosis. METHODS: Coronal (fusing) and sagittal (patent) rabbit cranial sutures from a colony of congenitally synostosed rabbits and wild-type (control) rabbits were harvested at a neonatal time point. These sections were then grown in organ culture and harvested for histology at 0, 7, or 14 days of culture. Fusion percentage was then assessed and an overall fusion score was calculated. Expression of Noggin and Runx-2 was then localized by immunohistochemistry and quantified by Western blot analysis. RESULTS: Histology of the wild-type cranial sutures (control) showed suture patency (score of 0%) for all coronal and sagittal sutures at 0 days, 7 days, and 14 days of organ culture. Sagittal sutures of craniosynostotic animals also showed suture patency (score of 0%) at all culture times (0, 7, and 14 days). Of the 18 coronal sutures from the craniosynostotic animals, 8 remained patent and 10 fused. For the coronal sutures that fused, fusion scores of 14%, 41%, and 84% were documented at 0, 7, and 14 days of organ culture, respectively. With immunolocalization, Noggin was found to be expressed in both the dura and suture cells underlying patent sutures, but not in fusing sutures in vitro. Runx-2 was found to be expressed in the dura beneath the suture and suture cells of fusing sutures, not patent sutures. Western blot densitometry confirmed these findings. CONCLUSIONS: Our results suggest that pathologic rabbit coronal sutures progressed toward complete suture fusion in vitro, and expression patterns of Noggin and Runx-2 paralleled that of a well-studied normal suture fusion model.