Noninflammatory Stress-Induced Remodeling of Mandibular Bone: Impact of Age and Pregnancy.

PURPOSE: The impact of noninflammatory stress, such as aging and pregnancy, on human long bone remodeling is well-established, but little is known about the impact of these stressors on oral bones, including the mandibular bone. To begin to fill this gap in our knowledge, we utilized a mouse mandibular model to evaluate the impact of noninflammatory simple stressors, ie, aging and pregnancy, on bone mandibular architecture and bone density in the mandible using micro-CT. MATERIALS AND METHODS: For the present study, mandibles were obtained from both aged and pregnant C57BL/6 mice and analyzed using micro-CT technology. Micro-CT metrics included bone volume fraction (BVF), total volume (TV), tissue density, and apparent density in the mandible on the mandibular area of compact and trabecular bone, in which the teeth are embedded. All bone-related metrics data from aged and pregnant mice were analyzed using ANOVA analysis and visualized in boxplots. RESULTS: Age-dependent bone remodeling occurred over 4 to 18 weeks of age, ie, increases in BVF, TV, BV/TV, as well as tissue and bone density. Evaluation of bone remodeling in breeder mice (repeated pregnancy model) and virgin mice (age-matched controls) at 37 weeks of age demonstrated that breeder mice had a dramatic decline in all bone metrics measured. CONCLUSIONS: This study underscores the need for more research on noninflammatory stress-related mandibular bone remodeling (eg, age and pregnancy), which compromises bone strength and tooth anchoring. The data also underscores loss of alveolar bone height, as in periodontitis, is an important metric for a more complete assessment of bone loss. This report on mice provides essential data that can be applied for oral-maxillofacial surgeons and periodontists when planning for dental implants in patients with such stressors. Periodontitis related bone loss occurs independent of skeletal homeostasis, although osteoporosis may adversely affect alveolar bone height in humans.

Insulin Derived Fibrils Induce Cytotoxicity in vitro and Trigger Inflammation in Murine Models.

BACKGROUND: Effective exogenous insulin delivery is the cornerstone of insulin dependent diabetes mellitus management. Recent literature indicates that commercial insulin-induced tissue reaction and cellular cytotoxicity may contribute to variability in blood glucose as well as permanent loss of injection or infusion site architecture and function. It is well accepted that insulin formulations are susceptible to mechanical and chemical stresses that lead to insulin fibril formation. This study aims to characterize in vitro and in vivo toxicity, as well as pro-inflammatory activity of insulin fibrils. METHOD: In vitro cell culture evaluated cytotoxicity and fibril uptake by macrophages and our modified murine air-pouch model quantified inflammatory activity. The latter employed FLOW cytometry and histopathology to characterize fibril-induced inflammation in vivo, which included fibril uptake by inflammatory phagocytes. RESULTS: These studies demonstrated that insulin derived fibrils are cytotoxic to cells in vitro. Furthermore, inflammation is induced in the murine air-pouch model in vivo and in response, macrophages uptake fibrils both in vitro and in vivo. CONCLUSIONS: Administration of insulin fibrils can lead to cytotoxicity in macrophages. In vivo data demonstrate insulin fibrils to be pro-inflammatory which over time can lead to cumulative cell/tissue toxicity, inflammation, and destructive wound healing. Long term, these tissue reactions could contribute to loss of insulin injection site architecture and function.

In vitro activation of human peripheral blood mononuclear cells induced by human biologic meshes.

BACKGROUND: Inflammation and wound healing play critical roles in the integration of biologic meshes (BMs) at sites of hernia repair. Monocytes/macrophages (M/MQs) are key cells involved in mesh integration. Interleukin-1beta (IL-1beta) is one of the major M/MQ-derived cytokines, and its expression is a reflection of the degree of M/MQ activation. We hypothesized that BMs induce M/MQ activation in vitro and that IL-1beta expression by M/MQ varies among various BMs. MATERIALS AND METHODS: Acellular human dermis-derived BM samples (AlloDerm, AlloMax, FlexHD) were placed in 48-well plates and cultured with peripheral blood mononuclear cells (PBMCs) from three healthy human subjects for 7 d. The resulting supernatants were assayed for IL-1beta levels by enzyme-linked immunosorbent assay (ELISA), and the BMs were evaluated histologically. RESULTS: IL-1beta expression varied among donors as well as the BMs [AlloDerm (2.11-38.25pg/10(6) PBMCs); AlloMax (13.12-715.40pg/10(6) PBMCs); and FlexHD (116.69-665.40pg/10(6) PBMCs)]. Analysis of this data indicated that AlloMax and FlexHD induced significantly more M/MQ activation compared with AlloDerm (P<0.05). Histologic evaluation of the BMs indicated adherence of M/MQs on BM surface, however no degradation was detected. CONCLUSION: For the first time, we have demonstrated that M/MQs are activated to varying levels by human BMs in vitro. These differences may be related to BM processing technologies and/or the biologic variation between donors. Our results raise the possibility that these differences in M/MQ activation could result in varying intensity of inflammation and wound healing that control the integration of BMs at sites of hernia repair.

Human monocyte activation by biologic and biodegradable meshes in vitro.

BACKGROUND: Inflammation and wound healing play critical roles in the integration of biologic and biodegradable meshes (BMs) at hernia repair sites. Monocytes/macrophages (M/MØs) are key cells controlling inflammation and wound healing. These cells release inflammatory cytokines and growth factors such as interleukin (IL)-1beta, IL-6, IL-8, and vascular endothelial growth factor (VEGF) upon activation. Although BMs have been increasingly used in hernia repairs worldwide, to date, investigations of inflammatory responses to various BMs have been limited. METHODS: Mesh samples of three acellular human dermis-derived biologic meshes (AlloDerm, AlloMax, FlexHD) and one biodegradable synthetic mesh (Bio-A) were placed in 96-well plates. Human peripheral blood mononuclear cells (PBMCs) were isolated from six healthy subjects, added to each well, and incubated for 7 days. Culture supernatants were assayed for IL-1beta, IL-6, IL-8, and VEGF levels using a multiplex bead-base immunoassay system (Bio-Plex). RESULTS: All four meshes induced cytokine expression from activated M/MØs to varying degrees in vitro. FlexHD induced significantly more IL-1beta (2,591 pg/ml) than AlloMax (517 pg/ml), AlloDerm (48 pg/ml), or Bio-A (28 pg/ml) (p < 0.001). AlloMax stimulated a significantly greater quantity of IL-6 (38,343 pg/ml) than FlexHD (19,317 pg/ml), Bio-A (191 pg/ml), or AlloDerm (103 pg/ml) (p < 0.05). Interleukin-8 and VEGF displayed trends similar to that of IL-6. There were no significant differences in cytokine production between AlloDerm and Bio-A. CONCLUSION: This study demonstrated that human macrophages are activated by human dermis-derived biologic and biodegradable meshes in vitro. A wide range of cytokine and growth factor induction was seen among the different mesh products. These differences in M/MØ activation may be related to the proprietary processing technologies of the studied meshes. The study results raise the possibility that these differences in M/MØ activation could indicate varying intensities of inflammation that control integration of different biologic meshes at the sites of hernia repair.

Continuous glucose monitoring in normal mice and mice with prediabetes and diabetes.

It is well established that the key to minimizing diabetes-associated complications, in both type 1 and type 2 diabetes, is tight regulation of blood glucose levels. Currently the major approach to regulating blood glucose levels in patients with diabetes relies on external blood glucose monitors. However, poor patient compliance usually results in limited insights into the dynamic range of blood glucose levels (i.e., hyperglycemia vs. hypoglycemia), and inadequate prediction and control of blood glucose levels in these patients. Implantable glucose sensors hold promise for controlling blood glucose levels, but currently these sensors have only limited in vivo life span. Recently we have developed an extremely robust murine model for implantable glucose sensors. In the present study, we have extended this model by developing a complete system for real-time continuous glucose monitoring in normal mice and mice with prediabetes and diabetes (type 1). These studies demonstrated that (1) glucose sensors can be implanted and maintained subcutaneously in the mice; (2) continuous glucose sensor data can be obtained for at least 5 days; and (3) subcutaneous blood glucose sensing paralleled blood glucose levels in normal mice and mice with prediabetes and diabetes. Subcutaneous blood glucose sensing also successfully tracked changes in blood glucose levels induced in the mice with diabetes by administration of oral glucose or insulin. These results mirror the results for subcutaneous blood glucose sensing seen in both normal subjects and patients with diabetes, and therefore validate both our continuous glucose monitoring system in the mouse, and the use of the mouse as a model for implantable glucose sensing in vivo.

Enhancement of implantable glucose sensor function in vivo using gene transfer-induced neovascularization.

The in vivo failure of implantable glucose sensors is thought to be largely the result of inflammation and fibrosis-induced vessel regression at sites of sensor implantation. To determine whether increased vessel density at sites of sensor implantation would enhance sensor function, cells genetically engineered to over-express the angiogenic factor (AF) vascular endothelial cell growth factor (VEGF) were incorporated into an ex ova chicken embryo chorioallantoic membrane (CAM)-glucose sensor model. The VEGF-producing cells were delivered to sites of glucose sensor implantation on the CAM using a tissue-interactive fibrin bio-hydrogel as a cell support and activation matrix. This VEGF-cell-fibrin system induced significant neovascularization surrounding the implanted sensor, and significantly enhanced the glucose sensor function in vivo. This model system, for the first time, provides the "proof of principle" that increasing vessel density at the sites of implantation can enhance glucose sensor function in vivo, and demonstrates the potential of gene transfer and tissue interactive fibrin bio-hydrogels in the development of successful implants.

Ex ova chick chorioallantoic membrane as a novel model for evaluation of tissue responses to biomaterials and implants.

One of the major obstacles in developing rationale strategies to control inflammation and fibrosis surrounding implants is the lack of a simple and inexpensive in vivo model to screen tissue reactions to various biomaterials and implants. To begin to fill this gap, we have developed an ex ova model of the chick embryo chorioallantoic membrane (CAM) for testing of tissue reaction to biomaterials and implants. For these studies, we evaluated tissue reactions (inflammation and fibrosis) to two commonly used biomaterials (nylon and silastic) grossly and histologically in the ex ova CAM. Nylon mesh was incorporated within the CAM tissue 4 days postplacement. After 8 days postplacement, the nylon mesh was totally incorporated into the CAM. Histologically, little or no inflammation was seen associated with the incorporated nylon mesh at any time point. In the case of silastic tubing, significant incorporation of the CAM was seen grossly by 1-2 days postplacement. Incorporation of the tubing continued at day 8 postplacement of the silastic tubing, with ingrowth of the CAM into the lumen of the tubing. Histological evaluation of CAMs indicated that no significant tissue reactions (inflammation or fibrosis) occurred in the CAM tissue surrounding the silastic tubing or in the CAM tissue and vasculature that had grown into the silastic tubing. To our knowledge, this report represents the first investigation of the usage of the ex ova CAM model, a shell-less chick embryo model (ex ova), as an in vivo model to test the tissue reactions to biomaterials and implants.

Metabolic biofouling of glucose sensors in vivo: role of tissue microhemorrhages.

OBJECTIVE: Based on our in vitro study that demonstrated the adverse effects of blood clots on glucose sensor function, we hypothesized that in vivo local tissue hemorrhages, induced as a consequence of sensor implantation or sensor movement post-implantation, are responsible for unreliable readings or an unexplained loss of functionality shortly after implantation. RESEARCH DESIGN AND METHODS: To investigate this issue, we utilized real-time continuous monitoring of blood glucose levels in a mouse model. Direct injection of blood at the tissue site of sensor implantation was utilized to mimic sensor-induced local tissue hemorrhages. RESULTS: It was found that blood injections, proximal to the sensor, consistently caused lowered sensor glucose readings, designated temporary signal reduction, in vivo in our mouse model, while injections of plasma or saline did not have this effect. CONCLUSION: These results support our hypothesis that tissue hemorrhage and resulting blood clots near the sensor can result in lowered local blood glucose concentrations due to metabolism of glucose by the clot. The lowered local blood glucose concentration led to low glucose readings from the still functioning sensor that did not reflect the systemic glucose level.

Importance of interleukin-1 and interleukin-1 receptor antagonist in short-term glucose sensor function in vivo.

BACKGROUND: The importance of the interleukin (IL)-1 cytokine family in inflammation and immunity is well established as a result of extensive in vitro and in vivo studies. In fact, much of our understanding of the in vivo importance of interleukin-1beta (IL-1B) is the result of research utilizing transgenic mice, such as overexpression or deficiencies of the naturally occurring inhibitor of IL-1 known as interleukin-1 receptor antagonist (IL-1RA). For the present studies, we utilized these transgenic mice to determine the role of IL-1B in glucose sensor function in vivo. METHODS: To investigate the role of IL-1B in glucose sensor function in vivo, we compared glucose sensor function in trans-genic mice that (1) overexpressed IL-1RA [B6.Cg-Tg(II1rn)1Dih/J] and (2) are deficient in IL-1RA (B6.129S-Il1rn(tm1Dih)/J), with mice that have normal levels of IL-1RA (C57BL/6). RESULTS: Our studies demonstrated that, during the first 7 days post-sensor implantation (PSI), mice deficient in IL-1RA had extensive inflammation and decreased sensor function when compared to normal or IL-1RA-overexpressing mice. CONCLUSION: These data directly support our hypothesis that the IL-1 family of cytokines and antagonists play a critical role in controlling tissue reactions and thereby sensor function in vivo during the first 7 days PSI.

Blood-induced interference of glucose sensor function in vitro: implications for in vivo sensor function.

BACKGROUND: Although tissue hemorrhages, with resulting blood clots, are associated with glucose sensor implantation, virtually nothing known is about the impact of red blood cells and red blood cell clots on sensor function in vitro or in vivo. In these studies, we tested the hypothesis that blood can directly interfere with glucose sensor function in vitro. METHODS: To test this hypothesis, heparinized human whole blood (HWB) and nonheparinized human whole blood (WB) were obtained from normal individuals. Aliquots of HWB and WB samples were also fractionated into plasma, serum, and total leukocyte (TL) components. Resulting HWB, WB, and WB components were incubated in vitro with an amperometric glucose sensor for 24 hours at 37 degrees C. During incubation, blood glucose levels were determined periodically using a glucose monitor, and glucose sensor function (GSF) was monitored continuously as nanoampere output. RESULTS: Heparinized human whole blood had no significant effect on GSF in vitro, nor did TL, serum, or plasmaderived clots from WB. Sensors incubated with WB displayed a rapid signal loss associated with clot formation at 37 degrees C. The half-life was 0.8 +/- 0.2 hours (n = 16) for sensors incubated with WB compared to 3.2 +/- 0.5 (n = 12) for sensors incubated with HWB with a blood glucose level of approximately 100 mg/dl. CONCLUSIONS: These studies demonstrated that human whole blood interfered with GSF in vitro. These studies further demonstrated that this interference was related to blood clot formation, as HWB, serum, plasma-derived clots, or TL did not interfere with GSF in vitro in the same way that WB did. These in vitro studies supported the concept that the formation of blood clots at sites of glucose sensor implantation could have a negative impact on GSF in vivo.

Localization of the chemokine interleukin-8 and interleukin-8 receptors in human gingiva and cultured gingival keratinocytes.

Interleukin-8 (IL-8) has been implicated in a wide variety of diseases. Previous studies have demonstrated the expression of IL-8 in periodontal tissues, yet little is known about the exact source(s), mechanisms and factors involved in gingival expression of IL-8. Additionally, nothing is known about the presence and distribution of IL-8 receptors (IL-8R) in gingival cells. Therefore it was hypothesized that, in vivo, periodontal pathogens induce IL-8 expression from gingival keratinocytes (GK) which enhances leukocyte, microvascular endothelial cell (MVEC) and GK migration via specific IL-8 receptors present on these cells. The objective of the present study was to determine the distribution of IL-8 and IL-8R in gingival tissues and cultured human GK in vitro. Standard immunohistochemical and immunocytochemical techniques were utilized in order to localize IL-8 and its receptors CXCR-1 and CXCR-2 in archival gingival specimens (eight periodontitis and four non-inflamed controls) and in cultured gingival keratinocytes. It was demonstrated that, in vivo, IL-8 and IL-8R were present in gingival epithelium, MVEC and leukocytes. In vitro studies verified the above results, by showing expression of IL-8 and IL-8R in cultured gingival keratinocytes. It is concluded that IL-8 and IL-8 receptors are expressed in gingival epithelium both in vivo and in vitro. This new evidence indicates that epithelium plays a critical role in the host defense against invading pathogens and that keratinocytes can actively respond to IL-8 and other host cytokines, via specific receptors.