Third ventriculostomy in a single pediatric surgical unit

Purpose: Endoscopic third ventriculostomy (ETV) is a successful method of treatment for obstructive hydrocephalus that has become popular over the last 20years. The purpose of this paper is to study the outcome of infants with obstructive hydrocephalus treated by ETV by a single surgeon and to evaluate the safety, reliability, and efficacy of this treatment. Methods: All data were collected retrospectively. Between July 1999 and June 2005, 14 children underwent an ETV. In one child, a second ETV was performed. The age of the eight female and six male patients at the time of ETV ranged from less than 1month up to 13years and 11months. The indication for an ETV was an obstructive hydrocephalus. Median follow-up period was 5years and 9months. The need of a further operation after ETV was defined as a failure of ETV. Results: In six patients, the first ETV was successful. In the remaining eight patients, there was a need for further treatment (ventriculoperitoneal shunt). Although the follow-up shunt failed in one patient, he was successfully treated by a second ETV. Conclusion: Our study suggests that ETV can be successfully done in a small pediatric unit, but with a lower success rate because of small caseload, and therefore, lower experience and routine of the surgeon. Therefore, we propose a centralization of patients to obtain a higher number of cases. We confirm that ETV is a safe, reliable, and efficient method with a better outcome in children than infant

The silent epidemic of falls from buildings: analysis of risk factors

This study wanted to search for potential risk factors associated with falls from windows and balconies in order to eventually improve prevention. All children under the age of 16years suffering from head injuries/multiple trauma due to falls from windows or balconies treated over the last 7years at the intensive care unit (ICU) of the University Children's Hospital Zürich were analysed retrospectively (group A). Fifty patients out of all children suffering from head injuries/multiple trauma due to other types of accidents in the same period were selected at random as controls (group B). Out of a total of 241 children with head injury and/or multiple trauma, 31 (13%) fell out of a building. Twenty-seven of these victims (87%) fell from the third floor or lower. Twenty-one of the falls (68%) occurred at home. Fifteen children (49%) climbed on a piece of furniture before falling. In almost 20% of the accidents dangerous balcony or house constructions led to the fall. Parents did not witness the fall, except for three cases (10%) with direct parental involvement (one mother jumped out with her child, two mothers threw their child out of the window). Two children (6%) attempted suicide. Children aged 0-5years were predominantly represented (84%), and all six children who died were in this age group. There were significantly more patients with foreign nationalities and lower socio-professional categories in group A than in group B. In both groups, the accidents concerned the youngest child of the family in approximately 50% and happened mostly during summer evenings. There were no significant differences in injured systems and in injury severity between the two groups. This study identified young age, an immigrant family setting, low socio-professional category of the parents, dangerous house constructions, inappropriate furniture placement, and summertime evenings as risk factors for serious building falls in children. This information may foster focused preventio

"Trooping the color”: restoring the original donor skin color by addition of melanocytes to bioengineered skin analogs

Purpose: Autologous skin substitutes to cover large skin defects are used since several years. Melanocytes, although essential for solar protection and pigmentation of skin, are not yet systematically added to such substitutes. In this experimental study, we reconstructed melanocyte-containing dermo-epidermal skin substitutes from donor skins of different skin pigmentation types and studied them in an animal model. Features pertinent to skin color were analyzed and compared in both skin substitutes and original donor skin. Methods: Keratinocytes, melanocytes, and fibroblast were isolated, cultured, and expanded from skin biopsies of light- and dark-pigmented patients. For each donor, melanocytes and keratinocytes were seeded in different ratios (1:1, 1:5, 1:10) onto collagen gels previously populated with autologous fibroblasts. Skin substitutes were then transplanted onto full-thickness wounds of immuno-incompetent rats. After 8weeks, macroscopic and microscopic analyses were conducted with regard to skin color and architecture. Results: Chromameter evaluation revealed that skin color of reconstructed light- and dark-pigmented skin was very similar to donor skin, independent of which melanocyte/keratinocyte ratio was added. Histological analyses of the skin analogs confirmed these findings. Conclusion: These data suggest that adding autologous melanocytes to bioengineered dermo-epidermal skin analogs can sustainably restore the patients' native skin colo

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

Purpose: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. Methods: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. Results: After 3weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. Conclusion: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivit

A new model for preclinical testing of dermal substitutes for human skin reconstruction

Background: Currently, acellular dermal substitutes used for skin reconstruction are usually covered with split-thickness skin grafts. The goal of this study was to develop an animal model in which such dermal substitutes can be tested under standardized conditions using a bioengineered dermo-epidermal skin graft for coverage. Methods: Bioengineered grafts consisting of collagen type I hydrogels with incorporated human fibroblasts and human keratinocytes seeded on these gels were produced. Two different dermal substitutes, namely Matriderm®, and an acellular collagen type I hydrogel, were applied onto full-thickness skin wounds created on the back of immuno-incompetent rats. As control, no dermal substitute was used. As coverage for the dermal substitutes either the bioengineered grafts were used, or, as controls, human split-thickness skin or neonatal rat epidermis were used. Grafts were excised 21days post-transplantation. Histology and immunofluorescence was performed to investigate survival, epidermis formation, and vascularization of the grafts. Results: The bioengineered grafts survived on all tested dermal substitutes. Epidermis formation and vascularization were comparable to the controls. Conclusion: We could successfully use human bioengineered grafts to test different dermal substitutes. This novel model can be used to investigate newly designed dermal substitutes in detail and in a standardized wa

Immune cell populations and cytokine production in spleen and mesenteric lymph nodes after laparoscopic surgery versus conventional laparotomy in mice

Purpose: There is evidence that open as well as minimally invasive abdominal surgery impair post-operative innate and acquired immune function. To compare the impact of these approaches as well as the one of different peritoneal gas exposures on immune function, we investigated cellular as well as cytokine-based immune parameters in mesenteric lymph nodes and the spleen postoperatively. Methods: Mice (n=26) were randomly assigned to the 4 study groups: (1) sham controls undergoing anesthesia alone, (2) laparotomy, and (3) air, or (4) carbon dioxide pneumoperitoneum. Mice were sacrificed 48h after the intervention, and their spleens and mesenteric lymph nodes were harvested. Cytokine production (TNF-α, IL-6, IL-10, and IFN-γ), splenic T cell subpopulations (cytotoxic T cells, T helper cells, and regulatory T cells) were analyzed. Results: TNF-α production of splenocytes 16h after ex vivo lipopolysaccharides (LPS) stimulation was significantly increased in the laparotomy group compared to all other groups. In contrast, TNF-α production of lymph node cells and IL-6 production of splenocytes after ex vivo LPS stimulation did not differ significantly between the groups. The numbers of regulatory T cells (Treg) in the spleen differed between groups. A significant reduction in Treg cell frequency was detected in the CO2 insufflation group compared to the laparotomy and the air insufflation group. Conclusion: Our findings demonstrate a distinct difference in immune effector functions and cellular composition of the spleen with regard to splenic TNF-α production and increased numbers of Treg cells in the spleen. These findings are in line with a higher peritoneal inflammatory status consequent to peritoneal air rather than CO2 exposure. Treg turned out to be key modulators of postoperative dysfunction of acquired immunit

Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

Purpose: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. Methods: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thickness skin wounds on the back of immuno-incompetent rats were covered with skin grafts with (1) amniocytes in the dermis, (2) fibroblasts in the dermis, or, (3) acellular dermis. Grafts were excised 7 and 21days post transplantation. Histology and immunofluorescence were performed to investigate epidermis formation, stratification, and expression of established skin markers. Results: The epidermis of skin grafts engineered with amniocytes showed near-normal anatomy, a continuous basal lamina, and a stratum corneum. Expression patterns for keratin 15, keratin 16, and Ki67 were similar to grafts with fibroblasts; keratin 1 expression was not yet fully established in all suprabasal cell layers, expression of keratin 19 was increased and not only restricted to the basal cell layer as seen in grafts with fibroblasts. In grafts with acellular dermis, keratinocytes did not survive. Conclusion: Dermo-epidermal skin grafts with amniocytes show near-normal physiological behavior suggesting that amniocytes substitute fibroblast function to support the essential cross-talk between mesenchyme and epithelia needed for epidermal stratification. This novel finding has considerable implications regarding tissue engineerin

Determining the origin of cells in tissue engineered skin substitutes: a pilot study employing in situ hybridization

Background: Definitive and high-quality coverage of large and, in particular, massive skin defects remains a significant challenge in burn as well as plastic and reconstructive surgery because of donor site shortage. A novel and promising approach to overcome these problems is tissue engineering of skin. Clearly, before eventual clinical application, engineered skin substitutes of human origin must be grafted and then evaluated in animal models. For the various tests to be conducted it is indispensable to be able to identify human cells as such in culture and also to distinguish between graft and recipient tissue after transplantation. Here we describe a tool to identify human cells in vitro and in vivo. Methods: In situ hybridization allows for the detection and localization of specific DNA or RNA sequences in morphologically preserved cells in culture or tissue sections, respectively. We used digoxigenin-labeled DNA probes corresponding to human-specific Alu repeats in order to identify human keratinocytes grown in culture together with rat cells, and also to label split and full thickness skin grafts of human origin after transplantation on immuno-incompetent rats. Results: Digoxigenin-labeled DNA probing resulted in an intensive nuclear staining of human cells, both in culture and after transplantation onto recipient animals, while recipient animal cells (rat cells) did not stain. Conclusion: In situ hybridization using primate-specific Alu probes reliably allows distinguishing between cells of human and non-human origin both in culture as well as in histological sections. This method is an essential tool for those preclinical experiments (performed on non-primate animals) that must be conducted before novel tissue engineered skin substitutes might be introduced into clinical practic

Optimizing in vitro culture conditions leads to a significantly shorter production time of human dermo-epidermal skin substitutes

Introduction: Autologous dermo-epidermal skin substitutes (DESS) generated in vitro represent a promising therapeutic means to treat full-thickness skin defects in clinical practice. A serious drawback with regard to acute patients is the relatively long production time of 3-4weeks. With this experimental study we aimed to decrease the production time of DESS without compromising their quality. Methods: Two in vitro steps of DESS construction were varied: the pre-cultivation time of fibroblasts in hydrogels (1, 3, and 6days), and the culture time of keratinocytes (3, 6, and 12days) before transplantation of DESS on nude rats. Additionally, the impact of the air-liquid interface culture during 3days before transplantation was investigated. 3weeks after transplantation, the macroscopic appearance was evaluated and histological sections were produced to analyze structure and thickness of epidermis and dermis, the stratification of the epidermis, and the presence of a basal lamina. Results: Optimal DESS formation was obtained with a fibroblast pre-cultivation time of 6days. The minimal culture time of keratinocytes on hydrogels was also 6days. The air-liquid interface culture did not improve graft quality. Conclusion: By optimizing our in vitro culture conditions, it was possible to very substantially reduce the production time for DESS from 21 to 12days. However, pre-cultivation of fibroblasts in the dermal equivalent and proliferation of keratinocytes before transplantation remain crucial for an equilibrated maturation of the epidermis and cannot be completely skippe

Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells

Background/Purpose: In a multi-project research line, we are currently testing whether a morphologically and functionally near normal epidermis can be cultured from human sweat gland (SG) cells and be used as a skin substitute. The present study focuses on the stratum corneum of the epidermis that assumes a vital barrier function for the skin. The main process in the formation of the cornified cell envelope in human epidermis, i.e. crosslinking of proteins and lipids, is catalyzed by several transglutaminases (TG). Therefore, we compared the expression patterns of various TG and their substrates in SG-derived versus keratinocyte-derived epidermal substitutes. Methods: Sweat gland cells, keratinocytes, and fibroblasts were isolated from human skin samples and cultivated separately to generate epidermal substitutes. These were transplanted onto the back of athymic rats. After 2weeks, the transplants were excised and analyzed histologically as well as by indirect immunofluorescence. We looked at the expression of TG1, 3, 5, and their substrates involucrin and loricrin (=markers of epidermal differentiation) in SG-derived and keratinocyte-derived skin substitutes as well as in normal skin. Results: The SG cell-derived epidermis was near normal anatomically, formed a cornified cell envelope and demonstrated TG1, 3, and 5 as well as involucrin and loricrin expression patterns similar to those found in keratinocyte-derived epidermis and normal control skin. Conclusion: These findings support the thesis that SG cells have the potential to form a near normal stratified epidermal analog that might be used as a skin substitute. The expression of TG1 and 3, not normally expressed in human SG, suggests the presence of re-programmed SG cells and/or stem cells capable of both de novo generating and maintaining an epidermi