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Craniofacial dysmorphism is associated with thousands of genetic and environmental disorders. Delineation of salient facial characteristics can guide clinicians towards a correct clinical diagnosis and understanding the pathogenesis of the disorder. Abnormal facial shape might require craniofacial surgical intervention, with the restoration of normal shape an important surgical outcome. Facial anthropometric growth curves or standards of single inter-landmark measurements have traditionally supported assessments of normal and abnormal facial shape, for both clinical and research applications. However, these fail to capture the full complexity of facial shape. With the increasing availability of 3D photographs, methods of assessment that take advantage of the rich information contained in such images are needed. In this article we derive and present open-source three-dimensional (3D) growth curves of the human face. These are sequences of age and sex-specific expected 3D facial shapes and statistical models of the variation around the expected shape, derived from 5443 3D images. We demonstrate the use of these growth curves for assessing patients and show that they identify normal and abnormal facial morphology independent from age-specific facial features. 3D growth curves can facilitate use of state-of-the-art 3D facial shape assessment by the broader clinical and biomedical research community. This advance in phenotype description will support clinical diagnosis and the understanding of disease pathogenesis including genotype–phenotype relations.

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that β-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

Many disorders present with characteristic abnormalities of the craniofacial complex. Precise descriptions of how and when these abnormalities emerge and change during childhood and adolescence can inform our understanding of their underlying pathology and facilitate diagnosis from craniofacial shape. In this paper we develop a framework for analysing how anatomical differences between populations emerge and change over time, and for binary group classification that adapts to the age of each participant. As a proxy for a disease-control comparison we use a database of 3D photographs of normally developing boys and girls to examine emerging sex-differences. Essentially we define 3D craniofacial ‘growth curves’ for each sex. Differences in the forehead, upper lip, chin and nose emerge primarily from different growth rates between the groups, whereas differences in the buccal region involve different growth directions. Differences in the forehead, buccal region and chin are evident before puberty, challenging the view that sex differences result from pubertal hormone levels. Classification accuracy was best for older children. This paper represents a significant methodological advance for the study of facial differences between growing populations and comprehensively describes developing craniofacial sex differences.

Tissue engineering of vascularized constructs has great utility in reconstructive surgery. While we have been successful in generating vascularized granulation-like tissue and adipose tissue in an in vivo tissue engineering chamber, production of other differentiated tissues in a stable construct remains a challenge. One approach is to utilize potent differentiation factors, which can influence the base tissue. Endothelial precursor cells (EPCs) have the ability to both carry differentiation factors and home to developing vasculature. In this study, proof-of-principle experiments demonstrate that such cells can be recruited from the circulation into an in vivo tissue engineering chamber. CXC chemokine ligand 12 (CXCL12)/stromal cell–derived factor 1 was infused into the chamber through Alzet osmotic pumps and chamber cannulation between days 0 and 7, and facilitated recruitment of systemically inoculated exogenous human EPCs injected on day 6. CXCL12 infusion resulted in an eightfold increase in EPC recruitment, 2 ( p  = 0.03) and 7 days postinfusion ( p  = 0.008). Delivery of chemotactic/proliferation and/or differentiation factors and appropriately timed introduction of effective cells may allow us to better exploit the regenerative potential of the established chamber construct.

Repair of substantial cranial defects in adults and children may be compromised due to limitations in donor bone stocks for autologous grafts. We evaluated the capability of autologous adipose-derived mesenchymal cells (ADCs) in combination with polylactic acid (PLA) scaffolds to regenerate bone in a critical-sized skull defect. Thirty adult New Zealand White rabbits were divided into six groups of five animals each: (1) PLA alone (control), (2) fibronectin-coated PLA, (3) PLA with ADCs, (4) fibronectin-coated PLA with ADCs, (5) PLA with osteogenically induced ADCs (osADCs), and (6) fibronectin-coated PLA with osADCs. All the animals were humanely killed after 6 weeks. X-ray, histology, and histomorphometric analysis were performed to evaluate the new bone formation inside the PLA scaffold. Radiographically and histomorphometrically, the groups in which the PLA was not fibronectin coated showed no bone formation in contrast to the fibronectin-coated groups (Gp1 vs. Gp2, p < 0.0005); the group treated with osteo-induced ADCs and fibronectin (Gp6) showed significantly more bone formation than the group treated with undifferentiated ADCs (Gp4) and the group treated without cells (Gp5, p < 0.0005, in both cases). These data indicate that the surface treatment with fibronectin promotes bone formation within the scaffold, and that autologous, osteo-induced adipose-derived cells enhance bone formation if seeded into a fibronectin-treated PLA scaffold.

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34 Neg CD31 Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34 Neg CD31 Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions ( P  < 0.0005 at 48 h, two-way ANOVA), increased migration ( P  < 0.001, two-way ANOVA) and formed fewer ( P  = 0.029, independent samples t test), shorter tubes ( P  = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplanin Pos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

Transplantation of islets into the portal vein of diabetic patients has emerged as a promising procedure for the treatment of type 1 diabetes. However, shortages of donors and adverse effects leading to graft impairment and/or rejection have prevented this procedure from achieving widespread clinical application. The aim of this study was to develop a method that could support the survival and function of transplanted islets using a prevascularized tissue engineering chamber. Islets were transplanted into tissue engineering chambers established on the epigastric pedicle in the groin of diabetic mice. Islets were transplanted at the time of chamber implantation or with 21 days prevascularization of the chamber. Transplantation of islets into prevascularized chambers into diabetic RIP-K b mice resulted in a significant reduction in blood glucose levels that became evident in the third week and improved glycemic control as measured by a glucose tolerance test. This study highlights that islet survival and function are potentiated by allowing a period of prevascularization within tissue engineering chambers before islet transplantation. This novel prevascularized chamber may be an improved method of islet transplantation. It can be easily accessed for islet seeding, easily retrieved, and transplanted to alternative anatomical sites by microvascular methods.

In in vivo tissue engineering, many implanted cells die because of hypoxic conditions immediately postimplantation. The aim of this study was to determine whether delayed myoblast implantation, at day 4 or 7, improves myoblast survival compared with implantation at day 0 in an in vivo arterio-venous loop (AB loop) chamber model. In adult inbred Sprague-Dawley rats, an AB loop was inserted into a plastic chamber (day 0). In Group I, day 0, two million DiI-labeled (neonatal inbred) myoblasts were implanted around the AB loop. In Groups II and III, day 0, the AB loop was created and inserted into a novel delayed cell seeding chamber, and 4 (Group II) or 7 days (Group III) later the delay chamber was seeded with 2 million DiI-labeled myoblasts. Constructs were harvested 7-day postmyoblast implantation, for morphometric determination of DiI/DAPI-positive myoblasts/mm 2 , and percent vascular volume on Griffonia simplicifolia lectin (endothelial cell marker)–labeled tissue sections. Control (nonmyoblast seeded) and experimental (myoblast seeded) constructs demonstrated similar capillary and tissue growth patterns. DiI/DAPI-labeled myoblasts/mm 2 appeared in similar numbers in constructs implanted at days 0 and 4, but increased markedly in day-7 implanted constructs. The percent vascular volume increased significantly ( p  = 0.03) over time. A positive correlation existed between myoblast survival and construct vascularity ( p  = 0.017). In conclusion, delaying myoblast implantation to 7-day postconstruct assembly, when new capillary growth is well established, significantly correlates with increased myoblast survival and indicates that cell seeding in regenerative procedures should always occur into an established vascular bed.

Endothelial cell protection against ischemia/reperfusion injury by lecithinized superoxide dismutase. Organs used for transplantation may experience long periods of cold ischemic preservation and consequently oxygen free radical-mediated damage following reperfusion. Lecithinized superoxide dismutase (lec-SOD) is a novel free radical scavenger that has been shown to bind with high affinity to cell membranes. The aim of this study was to determine whether lec-SOD bound to endothelial cells under organ preservation conditions to mediate direct antioxidant activity at the endothelial cell surface and thus offer protection against the harmful effects of ischemia/reperfusion injury. An in vitro study was performed on large vessel endothelial cells (HUVEC) and a human microvascular endothelial cell line HMEC-1, to investigate the potential therapeutic benefits of incorporating lec-SOD into organ preservation solution. A cold hypoxia/reoxygenation system was developed to examine lec-SOD binding affinity to endothelial cells, protection against hypoxia/reoxygenation-induced cell death, and neutrophil adhesion. Lec-SOD bound to endothelial cells with higher affinity than unmodified recombinant human superoxide dismutase (rhSOD) and significantly protected both HUVEC and HMEC-1 from cell death following 27 hours of cold hypoxia (P < 0.01). Furthermore, neutrophil adhesion to the endothelium stimulated by hypoxia and reoxygenation was significantly inhibited by treatment with lec-SOD but not by lecithin or rhSOD (P < 0.01). Analysis by flow cytometry demonstrated that E-selectin and ICAM-1 were up-regulated by hypoxia/reoxygenation that was inhibited in part by lec-SOD. The results from this study suggest that incorporation of lec-SOD into organ preservation solutions provides effective protection to endothelial cells against cold ischemia and reperfusion injury following transplantation.