Morphological and histochemical characterization of the pectoral fin muscle of batoids.

Batoids differ from other elasmobranch fishes in that they possess dorsoventrally flattened bodies with enlarged muscled pectoral fins. Most batoids also swim using either of two modes of locomotion: undulation or oscillation of the pectoral fins. In other elasmobranchs (e.g., sharks), the main locomotory muscle is located in the axial myotome; in contrast, the main locomotory muscle in batoids is found in the enlarged pectoral fins. The pectoral fin muscles of sharks have a simple structure, confined to the base of the fin; however, little to no data are available on the more complex musculature within the pectoral fins of batoids. Understanding the types of fibers and their arrangement within the pectoral fins may elucidate how batoid fishes are able to utilize such unique swimming modes. In the present study, histochemical methods including succinate dehydrogenase (SDH) and immunofluoresence were used to determine the different fiber types comprising these muscles in three batoid species: Atlantic stingray (Dasyatis sabina), ocellate river stingray (Potamotrygon motoro) and cownose ray (Rhinoptera bonasus). All three species had muscles comprised of two muscle fiber types (slow-red and fast-white). The undulatory species, D. sabina and P. motoro, had a larger proportion of fast-white muscle fibers compared to the oscillatory species, R. bonasus. The muscle fiber sizes were similar between each species, though generally smaller compared to the axial musculature in other elasmobranch fishes. These results suggest that batoid locomotion can be distinguished using muscle fiber type proportions. Undulatory species are more benthic with fast-white fibers allowing them to contract their muscles quickly, as a possible means of escape from potential predators. Oscillatory species are pelagic and are known to migrate long distances with muscles using slow-red fibers to aid in sustained swimming.

Microvascular anatomy suggests varying aerobic activity levels in the adipose tissues of diving tetrapods.

Adipose tissue has many important functions including metabolic energy storage, endocrine functions, thermoregulation and structural support. Given these varied functions, the microvascular characteristics within the tissue will have important roles in determining rates/limits of exchange of nutrients, waste, gases and molecular signaling molecules between adipose tissue and blood. Studies on skeletal muscle have suggested that tissues with higher aerobic capacity contain higher microvascular density (MVD) with lower diffusion distances (DD) than less aerobically active tissues. However, little is known about MVD in adipose tissue of most vertebrates; therefore, we measured microvascular characteristics (MVD, DD, diameter and branching) and cell size to explore the comparative aerobic activity in the adipose tissue across diving tetrapods, a group of animals facing additional physiological and metabolic stresses associated with diving. Adipose tissues of 33 animals were examined, including seabirds, sea turtles, pinnipeds, baleen whales and toothed whales. MVD and DD varied significantly (P < 0.001) among the groups, with seabirds generally having high MVD, low DD and small adipocytes. These characteristics suggest that microvessel arrangement in short duration divers (seabirds) reflects rapid lipid turnover, compared to longer duration divers (beaked whales) which have relatively lower MVD and greater DD, perhaps reflecting the requirement for tissue with lower metabolic activity, minimizing energetic costs during diving. Across all groups, predictable scaling patterns in MVD and DD such as those observed in skeletal muscle did not emerge, likely reflecting the fact that unlike skeletal muscle, adipose tissue performs many different functions in marine organisms, often within the same tissue compartment.

Dermal Denticle Diversity in Sharks: Novel Patterns on the Interbranchial Skin.

Shark skin is covered in dermal denticles-tooth-like structures consisting of enameloid, dentine, and a central pulp cavity. Previous studies have demonstrated differences in denticle morphology both among species and across different body regions within a species, including one report of extreme morphological variation within a 1 cm distance on the skin covering the branchial pouches, a region termed "interbranchial skin." We used gel-based profilometry, histology, and scanning electron microscopy to quantify differences in denticle morphology and surface topography of interbranchial skin denticles among 13 species of sharks to better understand the surface structure of this region. We show that (1) interbranchial skin denticles differ across shark species, and (2) denticles on the leading edge of the skin covering each gill pouch have different morphology and surface topography compared with denticles on the trailing edge. Across all species studied, there were significant differences in denticle length (P = 0.01) and width (P = 0.002), with shorter and wider leading edge denticles compared with trailing edge denticles. Surface skew was also higher in leading edge denticles (P = 0.009), though most values were still negative, indicating a surface texture more dominated by valleys than peaks. Overall, leading edge denticles were smoother-edged than trailing edge denticles in all of the species studied. These data suggest two hypotheses: (1) smoother-edged leading edge denticles protect the previous gill flap from abrasion during respiration, and (2) ridged denticle morphology at the trailing edge might alter water turbulence exiting branchial pouches after passing over the gills. Future studies will focus on determining the relationship between denticle morphology and water flow by visualizing fluid motion over interbranchial denticles during in vivo respiration.

La piel de los tiburones está cubierta de dentículos dérmicos, estructuras similares a los dientes que constan de un tejido esmaltado, una dentina y una cavidad pulpar central. Estudios anteriores han demostrado diferencias en la morfología de los dentículos tanto entre especies como entre diferentes regiones del cuerpo dentro de una misma especie, incluyendo un informe sobre la extrema variación morfológica dentro de una distancia de 1 cm en la piel que cubre las bolsas branquiales, una región denominada "piel interbranquial." Hemos utilizado perfilometría basada en gel, histología y microscopía electrónica de barrido, para cuantificar las diferencias en la morfología de los dentículos y la topografía de la superficie de la piel interbranquial de los dentículos en 13 especies de tiburones, para comprender mejor la estructura de la superficie de esta región. Demostramos que (1) los dentículos de la piel interbranquial difieren entre las especies de tiburones, y (2) los dentículos del borde anterior de la piel que cubre cada bolsa branquial tienen una morfología y una topografía superficial diferentes en comparación con los dentículos del borde posterior. En todas las especies estudiadas, hubo diferencias significativas en la longitud (P = 0.01) y en el ancho (P = 0.002), con dentículos del borde anterior más cortos y anchos que los del borde posterior. La inclinación de la superficie también era mayor en los dentículos del borde anterior (P = 0.009) aunque la mayoría de los valores seguían siendo negativos, lo que indicaba más valles que picos. En general, los dentículos de la parte anterior tenian los bordes mas lisos que los de la parte posterior en todas las especies estudiadas. Estos datos sugieren dos hipótesis: (1) los dentículos del borde anterior con bordes más lisos protegen la aleta branquial previa de la abrasión durante la respiración, y (2) la morfología de los dentículos con crestas en el borde posterior podría alterar la turbulencia del agua que sale de las bolsas branquiales después de pasar por las branquias. Futuros estudios se centrarán en determinar la relación entre la morfología de los dentículos y el flujo de agua mediante la visualización del movimiento del fluido sobre los dentículos interbranquiales durante la respiración in vivo.Translated by Laura Paez, Ph.D. student­Swiss Federal Institute of Technology Lausanne.

 ìƒì–´ì˜ 비늘은 유법랑질, 상아질, 치수강으로 이루어졌으며, 낱개로 ë³´ë©´ 생김새가 이빨을 닮았다. 기존의 연구에서는 상어 비늘의 형태학적 구조를 종 간 및 동종 내 다른 어체 부위의 다양한 각도에서 분석했는데, ê·¸ 중에서도 아가미구멍 사이의 1cm에 불과한 범위에서 예상 외의 표면미세구조 다양성을 발견한 연구가 주목된다. 상어 비늘의 형태학적 이해를 돕기 위하여, 본 연구에서는 젤을 사용한 프로필로메트리(gel-based profilometry), 조직학적 기법 및 주사전자현미경법을 통하여, 상어류 13종에서 아가미구멍 사이 피부의 비늘 형태와 표면미세구조를 분석했다. 본 연구의 결과는 (1) 아가미구멍 사이 피부의 표면형태에는 종 간 차이가 있고, (2) 아가미구멍 사이 피부의 전방 (머리 방향) 비늘은 후방 (꼬리 방향) 의 비늘에 비하여 훨씬 더 큰 수준의 다양성을 보였다는 것이다. 분석한 13종 모두를 통틀어, 아가미구멍 사이 피부의 전방 비늘은 후방의 비늘 보다 훨씬 넓고 (P = 0.01) ê¸¸ì—ˆë‹¤ (P = 0.002). 본 연구에서는 (1) 부드러운 머리 쪽 비늘이 상어가 숨 쉴 때마다 아가미구멍을 통해 빠져나온 물 흐름의 저항을 줄여준다는 것, 그리고 (2) 꼬리 쪽 가장자리의 비늘에서 두드러지는 들쭉날쭉한 가장자리는 아마도 비슷한 원리에서 아가미구멍을 통해 빠져나온 물의 소용돌이를 줄여 준다는 가설을 세울 수 있었다. 미래의 연구에서는 실험실 내의 조건에서 아가미구멍을 통해 흐르는 물의 역학적인 측면을 상어 비늘의 형태학적 측면과 연관지어 접근해야 할 것이다.Translated by Daemin Kim, Ph.D. student­Yale University.

Die Haut von Haien ist mit dermalen Dentikeln bedeckt - zahnähnlichen Strukturen, die aus Schmelz, Dentin und einer zentralen Pulpahöhle bestehen. Vorhergehende Studien haben Unterschiede in der Morphologie der Dentikel sowohl zwischen den Arten als auch zwischen verschiedenen Körperregionen innerhalb einer Art gezeigt, einschließlich eines Berichts über extreme morphologische Variationen innerhalb eines Abstands von 1 cm auf der Haut, die die Kiementaschen bedeckt, eine Region, die als "Interbranchialhaut" bezeichnet wird. Um die Oberflächenstruktur dieser Region besser zu versteshen, haben wir die Unterschiede in der Morphologie und Oberflächentopographie der Dentikel der Interbranchialhaut in 13 Haiarten mit Hilfe von Gel-Profilometrie, Histologie und Rasterelektronenmikroskopie quantifiziert. Wir konnten zeigen, dass (1) sich die Dentikel der Interbranchialhaut zwischen den Haiarten unterscheiden und (2) die Dentikel an der Vorderkante der Haut, die jede Kiementasche bedeckt, eine andere Morphologie und Oberflächentopographie aufweisen als die Dentikel an der Hinterkante. Bei allen untersuchten Arten gab es signifikante Unterschiede in der Länge (P = 0.01) und Breite (P = 0.002) der Dentikel, wobei die Dentikel an der Vorderkante kürzer und breiter waren als die Dentikel an der Hinterkante. Auch die Oberflächenschiefe war bei den Dentikeln der Vorderkante höher (P = 0.009), obwohl die meisten Werte immer noch negativ waren, was auf mehr Täler als Spitzen hinweist. Insgesamt waren die Vorderkanten-Dentikel bei allen untersuchten Arten glatter als die Hinterkanten-Dentikel. Diese Daten legen zwei Hypothesen nahe: (1) Glattere Vorderkantenzähne schützen den vorhergehenden Kiemenlappen vor Abrieb während der Atmung, und (2) die Morphologie der gezackten Zähne an der Hinterkante könnte die Wasserturbulenz beim Austritt aus den Kiementaschen nach dem Passieren der Kiemen verändern. Zukünftige Studien werden sich darauf konzentrieren, die Beziehung zwischen der Morphologie der Dentikel und der Wasserströmung zu bestimmen, indem die Flüssigkeitsbewegung über die Interbranchialdentikel während der In-vivo-Atmung sichtbar gemacht wird.Translated by Robin Thandiackal, postdoctoral fellow­Harvard University.

La peau des requins est recouverte de denticules dermiques - des structures semblables à des dents composées d'émail, de dentine et d'une cavité pulpaire centrale. Des études précédentes ont démontré que la morphologie des denticules diffère entre les espèces, mais également entre les différentes régions du corps au sein d'une même espèce. Il existe notamment une variation morphologique extrême sur une distance de 1 cm dans la région appelée "peau interbranchiale," soit la peau peau couvrant les poches branchiales. Nous avons utilisé la profilométrie à base de gel, l'histologie et la microscopie électronique à balayage pour quantifier les différences morphologiques et topographiques des denticules de la peau interbranchiale chez 13 espèces de requins, ceci afin de mieux comprendre la structure de la surface de cette région. Nos résultats montrent que (1) les denticules de la peau interbranchiale diffèrent selon les espèces de requins, et (2) les denticules situées sur le bord d'attaque de la peau couvrant chaque poche branchiale ont une morphologie et une topographie de surface différentes de celles des denticules situées sur le bord de fuite. Chez toutes les espèces étudiées, il y avait des différences significatives dans la longueur (P = 0.01) et la largeur (P = 0.002) des denticules, avec les denticules du bord antérieur plus courtes et plus larges que celles du bord postérieur. L'asymétrie de la surface était également plus élevée dans les denticules antérieures (P = 0.009), bien que la plupart des valeurs soient négatives, indiquant plus de vallées que de sommets.Par ailleurs, , les denticules du bord antérieur étaient plus lisses que celles du bord postérieur. Dans l'ensemble, ces données suggèrent deux hypothèses: (1) les denticules situées sur le bord d'attaque et possédant une surface plus lisse protègent le volet branchial précédent de l'abrasion pendant la respiration, et (2) la morphologie plutôt striée des denticules situées sur le bord de fuite pourrait modifier les caractéristiques turbulentes de l'écoulement sortant des poches branchiales après être passé sur les branchies. Les études futures se concentreront sur la détermination de la relation entre la morphologie des denticules et l'écoulement de l'eau en visualisant le mouvement du fluide sur les denticules interbranchiaux pendant la respiration in vivo.Translated by Elsa Goerig, postdoctoral fellow­Harvard University.

Microvessel density, lipid chemistry and N2 solubility in human and pig adipose tissue.

Decompression sickness (DCS) occurs when nitrogen gas (N2) comes out of solution too quickly, forming bubbles in the blood and tissues. These bubbles can be a serious condition; thus it is of extreme interest in the dive community to model DCS risk. Diving models use tissue compartments to calculate tissue partial pressures, often using data obtained from other mammalian species (i.e., pigs). Adipose tissue is an important compartment in these models because N2 is five times more soluble in fat than in blood; at any blood/tissue interface N2 will diffuse into the fat and can lead to bubble formation on ascent. Little is known about many characteristics of adipose tissue relevant to diving physiology. Therefore, we measured microvessel density and morphology, lipid composition, and N2 solubility in adipose tissue from humans and pigs. Human adipose tissue has significantly higher microvascular density (1.79 ± 0.04 vs. 1.21 ± 0.30%), vessel diameter (10.25 ± 0.28 vs. 6.72 ± 0.60 µm), total monounsaturated fatty acids (50.1 vs. 41.2 mol%) and N2 solubility (0.061 ± 0.003 vs. 0.054 ± 0.004 mL N2 mL⁻ ¹ oil) compared to pig tissue. Pig adipose tissue has significantly higher lipid content (76.1 ± 4.9 vs. 64.6 ± 5.1%) and total saturated fatty acids (38.8 vs. 29.5 mol%). Though two important components in gas kinetics within adipose tissue during diving (blood flow rates and degree of perfusion) are not well understood, our results indicate differences between the adipose tissue of humans and pigs. This suggests data from swine may not exactly predict gas dynamics for estimating DCS in humans.

Fatty acid composition and N2 solubility in triacylglycerol-rich adipose tissue: the likely importance of intact molecular structure.

Diving tetrapods (sea turtles, seabirds and marine mammals) are a biologically diverse group, yet all are under similar constraints: oxygen limitation and increased hydrostatic pressure at depth. Adipose tissue is important in the context of diving because nitrogen gas (N2) is five times more soluble in fat than in blood, creating a potential N2 sink in diving animals. Previous research demonstrates that unusual lipid composition [waxes and short-chained fatty acids (FA)] in adipose tissue of some whales leads to increased N2 solubility. We evaluated the N2 solubility of adipose tissue from 12 species of diving tetrapods lacking these unusual lipids to explore whether solubility in this tissue can be linked to lipid structure. Across all taxonomic groups, the same eight FA accounted for 70-80% of the entire lipid profile; almost all adipose tissues were dominated by monounsaturated FA (40.2-67.4 mol%). However, even with consistent FA profiles, there was considerable variability in N2 solubility, ranging from 0.051±0.003 to 0.073±0.004 ml N2 ml-1 oil. Interestingly, differences in N2 solubility could not be attributed to taxonomic group (P=0.06) or FA composition (P>0.10). These results lead to two main conclusions: (1) in triacylglycerol-only adipose tissues, the FA pool itself may not have a strong influence on N2 solubility; and (2) samples with similar FA profiles can have different N2 solubility values, suggesting that 3D arrangement of individual FA within a triacylglycerol molecule may have important roles in determining N2 solubility.

Use of abdominal wall allotransplantation as an alternative for the management of end stage abdominal wall failure in a porcine model.

BACKGROUND: We describe a novel approach for treating end stage abdominal wall failure using isolated abdominal wall allotransplantation in a porcine model. METHODS: Full thickness abdominal wall transplants were performed in 13 pairs of genetically mismatched pigs. All recipients received daily immunosuppresion after transplantation. Rejection was assessed by visual inspection and skin biopsies. At the end of the 28 d study period, thickness, stiffness, and tensile strength of the transplanted rectus muscle was measured and compared with native rectus muscle. RESULTS: Eight grafts were viable and showed no signs of herniation. Four grafts failed within the first week secondary to vascular thrombosis. One animal had viable graft but was euthanized due to an incarcerated inguinal hernia. Rejection was minimal in six of the eight recipients. At necropsy, the gross thickness of the transplanted muscle flap was reduced compared with the native muscle (4.3 mm versus 7.7 mm, P < 0.001). Histologically, the diameter of the muscle fiber decreased from 0.15 mm to 0.09 mm (P < 0.0001). While the stiffness measurements between the transplanted and native muscles were comparable, the transplanted muscles had significantly lower tensile strength than the native muscles. CONCLUSION: This study demonstrates the feasibility of isolated abdominal wall allotransplantation to provide a potential solution for end stage abdominal wall failure. Based on the model set forth, future work will evaluate the biomechanical properties of the composite allograft to provide a suitable dynamic abdominal wall replacement.

Isolated benign primary cutaneous plasmacytosis in children: two illustrative cases.

BACKGROUND: Plasma cells are normally found in bone marrow and the intestinal tract. They appear in the skin in malignant conditions, autoimmune diseases, infection, and idiopathic and poorly understood disorders such as primary nodular amyloidosis. It is uncommon to find collections of plasma cells in the skin in the absence of these conditions. OBSERVATIONS: We present 2 cases of cutaneous plasmacytosis, one in a white, female adolescent aged 15 years with an 11-year history of a solitary, asymptomatic, violaceous plaque on the left anterior tibia and the other in a white, male child aged 7 years with a 2-year history of a solitary erythematous plaque on the right anterior tibia. In both patients, infiltration of mature polyclonal plasma cells was confined to an area on the skin with papulonodules. There was no history of previous trauma, malignant conditions, autoimmune disease, or infection in either child. CONCLUSION: Although incipient or occult systemic disease cannot be definitively ruled out, the course of these 2 individuals suggests that isolated primary cutaneous plasmacytosis in children is a benign chronic process with no adverse sequelae.

Inactivation of the vitamin D receptor enhances susceptibility of murine skin to UV-induced tumorigenesis.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is the biologically active ligand for the vitamin D receptor (VDR). VDR(-/-) mice have a hair follicle-cycling defect resulting in alopecia. However, mice lacking 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1(-/-)), and having no circulating 1,25(OH)(2)D(3), have normal follicular function. These mouse models indicate that VDR functions independently of 1,25(OH)(2)D(3) in regulating hair-follicle cycling. Here, we show that VDR(-/-) mice rapidly develop chemically induced skin tumors, whereas CYP27B1(-/-) and wild-type mice do not, indicating that VDR, and not the 1,25(OH)(2)D(3) ligand, is essential for protection against skin tumorigenesis. Because the majority of human skin cancer results from exposure to UV, the susceptibility of VDR(-/-) mice to this carcinogen was also evaluated. VDR(-/-) mice developed UV-induced tumors more rapidly and with greater penetrance than did VDR(+/+) mice. p53 protein levels were upregulated at similar rates in UV-treated keratinocytes of VDR(-/-) and VDR(+/+) mice. However, rates of thymine-dimer repair and UV-induced apoptosis were significantly lower in VDR(-/-) epidermis compared with the wild type epidermis. UV-induced epidermal thickening was also attenuated in VDR(-/-) skin, indicating that VDR plays a critical role in the repair and removal of severely damaged keratinocytes and adaptation of the skin to chronic UV exposure.

Hypoxia leads to necrotic hepatocyte death.

Hepatocyte transplantation is being investigated as a therapy for liver disease; however, its success has been limited by rapid death of the cells following transplantation. This study was dedicated to elucidating the mode of death responsible for loss of transplanted hepatocytes in order to guide future strategies for promoting their survival. Using a tissue engineering model, it was found that the environment within polymer scaffolds containing transplanted cells was hypoxic after 5 days in vivo, with (90 +/- 3)% of hepatocytes existing at pO(2) < 10 mmHg. The primary mode of hepatocyte death in response to hypoxic conditions of 0 or 2 vol % oxygen was then determined in vitro. Several assays for features of apoptosis and necrosis demonstrated that hepatocytes cultured in an anoxic environment died via necrosis, while culture at 2% oxygen inhibited proliferation. These results suggest it will not be possible to prevent hepatocyte death by interfering with the apoptotic process, and hypoxic conditions in the transplants must instead be addressed. The finding that the environment within cell transplantation scaffolds is hypoxic is likely applicable to many cell-based therapies, and a similar analysis of the primary mode of death for other cell types in response to hypoxia may be valuable in guiding future strategies for their transplantation.

Azelaic acid in the treatment of papulopustular rosacea: a systematic review of randomized controlled trials.

OBJECTIVE: To evaluate the clinical efficacy of topical 20% azelaic acid cream and 15% azelaic acid gel compared with their respective vehicles and metronidazole gel in the treatment of papulopustular rosacea. DATA SOURCES: Electronic searches of MEDLINE, EMBASE, BIOSIS, and SciSearch through July or August 2004 and the Cochrane Central Register of Controlled Trials through 2004 (issue 3). We performed hand searches of reference lists, conference proceedings, and clinical trial databases. Experts in rosacea and azelaic acid were contacted. STUDY SELECTION: Randomized controlled trials involving topical azelaic acid (cream or gel) for the treatment of rosacea compared with placebo or other topical treatments. Two authors independently examined the studies identified by the searches. Ten studies were identified, of which 5 were included (873 patients). DATA EXTRACTION: Two authors independently extracted data from the included studies, then jointly assessed methodological quality using a quality assessment scale. DATA SYNTHESIS: Because standard deviation data were not available for 4 of the 5 studies, a meta-analysis could not be conducted. Four of the 5 studies demonstrated significant decreases in mean inflammatory lesion count and erythema severity after treatment with azelaic acid compared with vehicle. None of the studies showed any significant decrease in telangiectasia severity. CONCLUSIONS: Azelaic acid in 20% cream and 15% gel formulations appears to be effective in the treatment of papulopustular rosacea, particularly in regard to decreases in mean inflammatory lesion count and erythema severity. Compared with metronidazole, azelaic acid appears to be an equally effective, if not better, treatment option.

Delivery of hepatotrophic factors fails to enhance longer-term survival of subcutaneously transplanted hepatocytes.

Tissue engineering approaches have been investigated as a strategy for hepatocyte transplantation; however the death of a majority of transplanted cells critically limits success of these approaches. In a previous study, a transient increase in hepatocyte survival was achieved through delivery of vascular endothelial growth factor (VEGF) from the porous polymer scaffold utilized for cell delivery. To enhance longer-term survival of the hepatocytes, this delivery system was modified to additionally deliver epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in a sustained manner. Hepatocytes were subcutaneously implanted in SCID mice on scaffolds containing EGF and/or HGF, in addition to VEGF, and survival was monitored for two weeks. A short-term enhancement of hepatocyte survival was observed after one week and is attributed to VEGF-enhanced vascularization, which was not altered by EGF or HGF. Surprisingly, long-term hepatocyte engraftment was not improved, as survival declined to the level of control conditions for all growth factor combinations after two weeks. This investigation indicates that the survival of hepatocytes transplanted into heterotopic locations is dependent on multiple signals. The delivery system developed for the current study may be useful in elucidating the specific factors controlling this process, and bring therapeutic transplantation of hepatocytes closer to implementation.

Proliferative nodules with balloon-cell change in a large congenital melanocytic nevus.

The balloon-cell nevus was first described over 100 years ago. Since then, balloon-cell changes of melanocytes have been noted in numerous tumors, including melanoma, blue nevus, and Spitz nevus. Whether these changes reflect cellular deterioration or proliferative changes is a matter of debate. We report a case in which balloon-cell changes were found within proliferative nodules occurring in a large congenital melanocytic nevus.

Locally enhanced angiogenesis promotes transplanted cell survival.

A developing therapy for complete or partial loss of function in various tissues and organs involves transplanting an appropriate cell population, capable of compensating for the existing deficiencies. Clinical application of this type of strategy is currently limited by the death or dedifferentiation of the transplanted cells after delivery to the recipient. A delay in thorough vascularization of the implant area creates an environment low in oxygen and other nutrients, and likely contributes to the initial death of transplanted cells. We have addressed this problem by sustained delivery of vascular endothelial growth factor (VEGF), an initiator of angiogenesis, from a porous polymer matrix utilized simultaneously for cell delivery. As expected from previous studies, VEGF delivered from these constructs elicited an enhanced angiogenic response over a 2-week period when implanted subcutaneously in SCID mice. Hepatocytes implanted using VEGF-containing matrices demonstrated significantly greater survival after 1 week in vivo as compared with cells implanted on matrices without growth factor. The results of this study therefore indicate that enhancing vascularization in the location of transplanted cells promotes their survival. In addition, this delivery system may be used in future studies to directly promote cell survival and function by also providing growth factors specific to the transplanted cells.

Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

Designing alginate hydrogels to maintain viability of immobilized cells.

Hydrogel-forming materials have been widely utilized as an immobilization matrix and transport vehicle for cells. Success in these applications is dependent upon maintaining cell viability through the gel preparation process. We hypothesized that the high viscosity of pre-gelled solutions typically used in these applications may decrease cell viability due to the high shear forces required to mix cells with these solutions. Further, we proposed this harmful effect could be mediated by decreasing the molecular weight (Mw) of the polymer used to form the gel, while maintaining its gel-forming ability. To investigate this hypothesis, alginate was used as model system, as this copolymer consists of cross-linkable guluronic acid (G) blocks and non-cross-linkable blocks. Decreasing the Mw of alginate using irradiation (e.g., irradiating at dose of 2 Mrad) decreased the low shear viscosity of 2% (w/w) pre-gelled solutions from 1000 to 4 cP, while maintaining high elastic moduli, once cross-linked to form a gel. Importantly, the immobilization of cells with these polymer hydrogels increased cell viability from 40% to 70%, as compared to using high Mw polymer chains to form the gels. Furthermore, the solids concentration of gels formed with the low Mw alginate could be raised to further increase the moduli of gels without significantly deteriorating the viability of immobilized cells. This was likely due to the limited increase in the viscosity of these solutions. This material design approach may be useful with a variety of synthetic or naturally occurring block copolymers used to immobilize cells.