A Platform for Testing the Biocompatibility of Implants: Silicone Induces a Proinflammatory Response in a 3D Skin Equivalent.

Biocompatibility testing of materials is carried out in 2D cell cultures or animal models despite serious limitations. 3D skin equivalents are advanced in vitro models for human skin. Silicone has been shown to be noncytotoxic but capable of eliciting an immune response. Our aim was to (1) establish a 3D skin equivalent to (2) assess the proinflammatory properties of silicone. We developed a coculture of keratinocytes and fibroblasts resulting in a 3D skin equivalent with an implant using samples from a breast implant. Samples with and without the silicone implant were studied histologically and immunohistochemically in comparison to native human skin samples. Cytotoxicity was assessed via LDH-assay, and cytokine response was assessed via ELISA. Histologically, our 3D skin equivalents had a four-layered epidermal and a dermal component. The presence of tight junctions was demonstrated in immunofluorescence. The only difference in 3D skin equivalents with implants was an epidermal thinning. Implanting the silicone samples did not cause more cell death, however, an inflammatory cytokine response was triggered. We were able to establish an organotypical 3D skin equivalent with an implant, which can be utilised for studies on biocompatibility of materials. This first integration of silicone into a 3D skin equivalent confirmed previous findings on silicone being non-cell-toxic but capable of exerting a proinflammatory effect.

Organotypic 3D Co-Culture of Human Pleura as a Novel In Vitro Model of Staphylococcus aureus Infection and Biofilm Development.

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1ß) proved host-pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host-pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.

Novel 3D organotypic co-culture model of pleura.

Pleural mesothelial cells are the predominant cell type in the pleural cavity, but their role in the pathogenesis of pleural diseases needs to be further elucidated. 3D organotypic models are an encouraging approach for an in vivo understanding of molecular disease development. The aim of the present study was to develop a 3D organotypic model of the pleural mesothelium. Specimens of human pleura parietalis were obtained from patients undergoing surgery at the University Hospital Leipzig, Germany. 3D co-culture model of pleura was established from human pleural mesothelial cells and fibroblasts. The model was compared to human pleura tissue by phase-contrast and light microscopy, immunochemistry and -fluorescence as well as solute permeation test. Histological assessment of the 3D co-culture model displayed the presence of both cell types mimicking the morphology of the human pleura. Vimentin and Cytokeratin, PHD1 showed a similar expression pattern in pleural biopsies and 3D model. Expression of Ki-67 indicates the presence of proliferating cells. Tight junctional marker ZO-1 was found localized at contact zones between mesothelial cells. Each of these markers were expressed in both the 3D co-culture model and human biopsies. Permeability of 3D organotypic co-culture model of pleura was found to be higher for 70 kDa-Dextran and no significant difference was seen in the permeability for small dextran (4 kDa). In summary, the presented 3D organoid of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment.

Leptin promotes adipocytes survival in non-vascularized fat grafting via perfusion increase.

BACKGROUND: Though autologous fat transplantation is regularly and successfully performed in plastic surgery, little is known about the factors that contribute to the rise of preadipocytes and how the viability of adipocytes is regulated. As sufficient blood supply is a key parameter for the transplant's survival, we opted to analyse the development of preadipocytes within the fat transplant via stimulation of tissue perfusion with the angiogenesis enhancing hormone leptin. METHODS: In a murine (C57BL/6N) model inguinal fat was autologously transplanted into a dorsal skinfold chamber. In the intervention group the fat transplant was treated with local administration of leptin (3 µg/ml) at days 3, 7 and 10 after transplantation. Saline solution was administered respectively in the control group. On the postoperative days 3, 7, 10, and 15 intra vital microscopy was done to assess the functional vessel density, vessel diameter, adipocyte survival and preadipocyte development. The study was completed by histological tissue analysis on days 15 after transplantation. RESULTS: Leptin administration leads to an increase of angiogenesis, which starts from day 7 after implantation and elevates perfusion as well as functional vessel density FVD at days 10 and 15 after transplantation. Perfusion develops first from the border zones of the transplant. Histological evaluation showed that the percentage of perilipin positive adipocytes increased markedly in the study group of mice. Moreover, fat transplants of mice of the leptin group disclosed significantly higher Pref-1 positive cells than fat transplants of the control group. The findings reported in this study indicate that the leptin can enhance the survival and the quality of grafted fat tissue, which may be due to induction of angiogenesis. CONCLUSION: Leptin administration to fat transplants induced an increase in angiogenesis in the transplanted tissue and may play a role in reducing the resorption rate of lipoaspirates.

New model in diabetic mice to evaluate the effects of insulin therapy on biofilm development in wounds.

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7m +/+ Leprdb/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with Staphylococcus aureus ATCC 49230 (106 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence in situ hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistochemical staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19). Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and S. aureus-induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.

Full Length Interleukin 33 Aggravates Radiation-Induced Skin Reaction.

The interleukin (IL)-1 family member IL-33 has been described as intracellular alarmin with broad roles in wound healing, skin inflammation but also autoimmunity. Its dichotomy between full length (fl) IL-33 and the mature (m) form of IL-33 and its release by necrosis is still not fully understood. Here, we compare functional consequences of both forms in the skin in vivo, and therefore generated two lines of transgenic mice which selectively overexpress mmIL-33 and flmIL-33 in basal keratinocytes. Transgene mRNA was expressed at high level in skin of both lines but not in organs due to the specific K14 promoter. We could demonstrate that transgenic overexpression of mmIL-33 in murine keratinocytes leads to a spontaneous skin inflammation as opposed to flmIL-33. K14-mmIL-33 mice synthesize and secrete high amounts of mmIL-33 along with massive cutaneous manifestations, like increased epidermis and dermis thickness, infiltration of mast cells in the epidermis and dermis layers and marked hyperkeratosis. Using skin inflammation models such as IL-23 administration, imiquimod treatment, or mechanical irritation did not lead to exacerbated inflammation in the K14-flmIL-33 strain. As radiation induces a strong dermatitis due to apoptosis and necrosis, we determined the effect of fractionated radiation (12 Gy, 4 times). In comparison to wild-type mice, an increase in ear thickness in flmIL-33 transgenic mice was observed 25 days after irradiation. Macroscopic examination showed more severe skin symptoms in irradiated ears compared to controls. In summary, secreted mmIL-33 itself has a potent capacity in skin inflammation whereas fl IL-33 is limited due to its intracellular retention. During tissue damage, fl IL-33 exacerbated radiation-induced skin reaction.