Techniques to investigate cellular and molecular interactions in the host response to implanted biomaterials

Evaluation of the host response to implanted materials requires systematic, objective investigations of responses at both the cellular and molecular levels. This article explains the basis behind two technologies: antibody and molecular techniques, which will give valuable information when applied to investigations of cells and molecules involved in the host biomaterial interaction. Such investigations are well underway, and a number of groups are now studying well characterised cell markers or molecules to evaluate the host response to biomaterials. Here we outline current technologies for the development of antibodies as tools to study cell markers or molecules, including those for which reagents are not yet available and DNA based technologies, whose continued application should prove an invaluable adjunct to existing approaches. These technologies may be particularly valuable to investigations focusing on newly characterised cytokines, receptors or cell adhesion molecules and subsequently provide a way forward for the production of advanced biomaterials.     

Mast cells

Mast cells are found resident in tissues throughout the body, particularly in association with structures such as blood vessels and nerves, and in proximity to surfaces that interface the external environment. Mast cells are bone marrow-derived and particularly depend upon stem cell factor for their survival. Mast cells express a variety of phenotypic features within tissues as determined by the local environment. Withdrawal of required growth factors results in mast cell apoptosis. Mast cells appear to be highly engineered cells with multiple critical biological functions. They may be activated by a number of stimuli that are both Fc epsilon RI dependent and Fc epsilon RI independent. Activation through various receptors leads to distinct signaling pathways. After activation, mast cells may immediately extrude granule-associated mediators and generate lipid-derived substances that induce immediate allergic inflammation. Mast cell activation may also be followed by the synthesis of chemokines and cytokines. Cytokine and chemokine secretion, which occurs hours later, may contribute to chronic inflammation. Biological functions of mast cells appear to include a role in innate immunity, involvement in host defense mechanisms against parasitic infestations, immunomodulation of the immune system, and tissue repair and angiogenesis.     

Mast cells and pterygium

A total of thirty pterygia were studied for mast cells using metachromatic dye (toluidine blue) together with routine histological stains. On microscopic examination, the lesion was differentiated and classified into angiomatous, fibrous and mixed varieties based on the vascular and collagenous components. The mast cell counts were undertaken in all types and were found to have significant correlation with the morphological types when compared to normal conjunctiva. 15.1 +/- 3.1, 22.7 +/- 3.8, 9.5 +/- 3.2 mast cell per mm2 were found in the angiomatous, mixed and fibrous types respectively. In the control material, the mast cell count was 12.4 +/- 2.3 per mm2. The results are discussed. It is believed that mast cells are actively involved in the genesis and progress of pterygium.     

Ultrastructural analyses of the attachment (bonding) zone between bone and implanted biomaterials

This report presents transmission electron and high voltage transmission electron microscopic observations of bone and associated remodeling tissues directly interfacing with endosteal dental implants. Undecalcified interfacial tissues were serially sectioned from mandibular samples encasing 60 implants placed into 30 dogs. Two-dimensional ultrastructural analyses and three-dimensional stereology showed that osteogenesis adjacent to dental implants is a dynamic interaction of osseous cells and a collagenous fiber matrix. This study showed that the interfacial bone consists of a mineralized collagen fiber matrix associated with an inorganic (hydroxylapatite) matrix. This study suggested that an unmineralized collagen fiber matrix initially is laid down directly at the implant surface, and that this matrix then is mineralized. Osteoblasts interacted with this matrix, eventually becoming encased within developing lacunae during the remodeling process. This process formed the cellular (osteocyte) aspects of the developed bone. Osteocyte processes extended through canaliculi directly to the implant surface. Apparently, these processes also were entrapped within canaliculi during the mineralization events. At times, these processes paralleled the implant surface. The bone-implant interfacial zone was primarily fibrillar (both mineralized and unmineralized) in morphology, with an electron-dense, ruthenium positive deposition. This electron-dense material was approximately 20 to 50 nanometers in thickness, and only this thin layer separated the remodeled mineralized bone from the implant.     

Genetic dissection of inflammatory responses

Recent advances in classical genetics, molecular biology, and genomics herald a renaissance of genetic analysis of hereditary disease in both humans and animal models. It is increasingly feasible to analyze multifactorial traits both genetically and functionally. These advances parallel research aimed at unravelling the genetic code of humans and model species. Only the integration of these two approaches will secure the functionally significant conclusion of the genomic exercise.     

Periapical inflammatory responses and their modulation

Periapical inflammatory responses occur as a consequence of bacterial infection of the dental pulp, as a result of caries, trauma, or iatrogenic insult. Periapical inflammation stimulates the formation of granulomas and cysts, with the destruction of bone. These inflammatory responses are complex and consist of diverse elements. Immediate-type responses--including vasodilatation, increased vascular permeability, and leukocyte extravasation--are mediated by endogenous mediators, including prostanoids, kinins, and neuropeptides. Non-specific immune responses--including polymorphonuclear leukocyte and monocyte migration and activation, and cytokine production--are elicited in response to bacteria and their products. Interleukin-1 and prostaglandins in particular have been implicated as central mediators of periapical bone resorption. Chronic periapical inflammation further involves specific T- and B-cell-mediated anti-bacterial responses, and activates a network of regulatory cytokines which are produced by Th1- and Th2-type T-lymphocytes. Various naturally occurring and genetically engineered models of immunodeficiency are beginning to help elucidate those components of the immune system which protect the pulpal/periapical complex. Both specific and non-specific responses interface with and are regulated by the neural system. The modulation of these responses by immune response modifies, cytokine antagonists, and other novel therapeutic agents is discussed. As an experimental model, periapical inflammation has many advantages which permit it to be used in studies of microbial ecology and pathogenesis, host response, neuroimmunology, and bone resorption and regeneration.     

A study of structure and degradation of nonpolymeric biomaterials implanted in bone using reflected and transmitted light microscopy

Headaches associated with exercise, cough, and sexual activity may present a diagnostic challenge in the emergency room or primary care provider's office. The majority of these patients have no underlying intracranial pathologic condition and have a good prognosis. However, new onset of these types of headache, with or without accompanying neurologic deficit, is an indication to exclude life-threatening conditions such as subarachnoid bleeding with neuroimaging and CSF examination. Since the pathophysiology is poorly understood, treatment choices are limited. Further research is needed to elucidate the pathophysiologic mechanisms of these uncommon headaches and to assess the cost-effectiveness of various diagnostic and follow-up strategies.     

Human immune cells mediate catecholamine secretion from adrenal chromaffin cells

OBJECTIVES: To determine the ability of human mononuclear cells to produce factors that cause catecholamine secretion from adrenomedullary chromaffin cells; to determine conditions that stimulate mononuclear cells to produce such factors; and to compare these results with catecholamine secretion in response to the cytokines interleukin (IL)-1 and IL-2. DESIGN: Randomized, controlled, prospective study using in vitro conditions. SETTING: University research laboratory. SUBJECTS: Human mononuclear cells and porcine chromaffin cells. INTERVENTIONS: Circulating human mononuclear cells were isolated and cultured overnight in RPMI media. Cell-free media from these cultures (conditioned media) were then tested for the ability to cause epinephrine secretion from porcine chromaffin cells. Mononuclear cells were stimulated with phytohemagglutinin or by mixing cells from two different individuals while suppression was tested with dexamethasone. Catecholamine secretion in response to IL-1 and IL-2 (50 and 500 units/well, respectively), or nicotinic agonist dimethylphenylpiperazinium (10 microM, which mimics the action of acetylcholine), was tested for comparison. MEASUREMENTS AND MAIN RESULTS: Isolated porcine chromaffin cells had stable catecholamine content at the time of secretion measurements, and catecholamine release from cells into the media was measured using electrochemical detection after high-performance liquid chromatography separation. Catecholamine secretion was expressed as a percentage of the total cellular content. Epinephrine secretion due to human conditioned media was 6.9 +/- 1.0% compared with 1.4 +/- 0.6% for control media (p < .05) and 14.6 +/- 3.3% for dimethylphenylpiperazinium (p < .05). Epinephrine secretion with conditioned media from mixed cells (mixed leukocyte reaction) was 16.6 +/- 1.2%, which was higher than the epinephrine secretion caused by media from a single donor (6.9% +/- 1.0, p < .001). Pretreatment with dexamethasone inhibited the formation of bioactive products from mixed mononuclear cell preparations. Cytokines IL-1 and IL-2 did not stimulate chromaffin cell epinephrine secretion above background release with control media incubation. In all cases, norepinephrine secretion was similar to that of epinephrine, and results are included in all figures. CONCLUSIONS: Factors released from human immune cells can mediate epinephrine and norepinephrine release from adrenomedullary cells through a nonneural mechanism. Such immune cell factor release can be modulated by immunostimulation and steroid suppression. Release of such factors in vivo may contribute to increased circulating epinephrine in response to infectious challenge and may be an important factor in the critically ill patient.     

Mast cells in elastic cartilage of the human epiglottis

Light and transmission electron microscopy revealed in the elastic cartilage of the human epiglottis the presence of mast cells. Cytoplasmic granules of these cells contain characteristic cylindrical bodies, whose texture is different from that found in cytoplasmic granules of mast cells from other sites. The presence of mast cells in elastic cartilage, described for the first time, and the different substructure of their cytoplasmic granules, is considered as another proof of the heterogeneity of that cell type.     

Cell viability and inflammatory response in hydrogel sponges implanted in the rabbit cornea

In the quest for the development of a functional keratoprosthesis, the biocompatibility of the porous skirt material in the Chirila keratoprosthesis (KPro) was investigated. The population of live and dead cells within, and the inflammatory response to, a tissue-integrating poly(2-hydroxyethyl methacrylate) (PHEMA) sponge were studied. Samples of the hydrogel sponge were implanted in rabbit corneas and explanted at predetermined time points up to 12 weeks. The explanted sponges were subjected to cell viability assay using two types of fluoroprobes, 5-chloromethylfluorescein diacetate and ethidium homodimer-1. A semiquantitative analysis was performed to assess the number of dead cells within the sponge and in the area of corneal stroma proximal to the sponge. Five rabbits were used for each end point (2, 4 and 12 weeks). To investigate the inflammatory response to the sponge, immunocytochemistry, using specific antibodies to rabbit macrophages, enzyme histochemistry of chloroacetate esterase (to detect neutrophils) and transmission electron microscopy (TEM) were also employed at 24 h, 2, 4 and 12 weeks after implantation. Four weeks after implantation, fewer viable cells were observed in the sponge when compared to the 2-week implant. However, the proportion of viable cells increased dramatically by 12 weeks. The proportion of nonviable cells decreased gradually with time; central sponge contained 34+/-11 % dead cells after 2 weeks, and 15+/-4.3% after 12 weeks. The staining of inflammatory cells demonstrated the presence of macrophages and neutrophils up to 12 weeks after implantation. TEM confirmed the presence of these cell types and others. including eosinophils and myofibroblasts, as well as blood capillaries. The presence of a significant number of viable cells at each time point and the uniform reduction of the nonviable cell proportion with time suggests that the sponge is a conducive environment supporting a prolific, viable cellular colonization. Dead cells observed in the first instance indicate a normal injury pattern. However, the presence of a small but significant proportion of invading inflammatory cells 12 weeks after implantation confirms a characteristic pattern of wound healing within the sponges.     

Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor-alpha or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.     

Mast cells and biogenic amines in radiation-induced pulmonary fibrosis

OBJECTIVE: To determine womens' beliefs regarding the risks and health benefits of oral contraceptives (OCs). METHODS: Between April 26 and June 7, 1991, 247 women completed a self-administered questionnaire at the Yale University Health Services evaluating their perceptions of the risks and benefits of OC agents. RESULTS: The mean age of the study population was 30.2 years (range 16-68), and more than 90% of the study group had at least 1 year of college education. Forty-nine percent of the study group believed there are substantial risks to OC use. Between 80-95% of women were unaware of the following health benefits of OCs: decreased risks of endometrial cancer, ovarian cancer, pelvic inflammatory disease, ectopic pregnancy, anemia, and benign breast disease. CONCLUSIONS: Perceptions of the risks of OCs are exaggerated and there is a clear knowledge deficit with regard to their health benefits. Increased educational efforts by health care providers should emphasize the health benefits of OCs and attempt to dispel the common misconceptions.     

Intercellular adhesion molecule-3 on endothelial cells. Expression in tumors but not in inflammatory responses

Intercellular adhesion molecule-3 (ICAM-3) was identified as the third counter-receptor for lymphocyte function-associated antigen-1. ICAM-3 is absent on endothelial cells in normal tissues but found on endothelial cells in lymphomas. Here, we examined ICAM-3 expression on vascular endothelial cells in lymphomas, nonlymphoid malignancies, benign tumors, and inflammatory diseases. We compared the expression of ICAM-3 on endothelial cells with the severity of inflammatory infiltrates and with the presence of E-selectin and VCAM-1. We found that ICAM-3 expression on endothelial cells was high on both benign and malignant tumors whereas it was low in inflammatory diseases. In contrast to E-selectin, ICAM-3 expression on endothelial cells was not correlated to the severity of inflammatory infiltrates. In hemangiomas, we showed by Northern blot analysis and immunocytochemistry that ICAM-3 expression was induced and that it was localized in immature areas that sustain the early stages of angiogenesis. Therefore, expression of ICAM-3 on blood vessels does not seem to play a role in the recruitment of leukocytes during inflammation but rather is correlated with angiogenesis and tumor development.     

Effects of acute progesterone administration upon responses to acute psychosocial stress in men.

Animal studies suggest that neuroactive steroids, in particular progesterone and its metabolites, have stress-dampening effects. However, few studies have explored these effects in humans. In this study, we investigated the effects of acute progesterone administration on responses to the Trier Social Stress Test (TSST). Healthy men participated in the TSST 3.5 hrs after intramuscular injection of 0, 50, or 100 mg progesterone (N = 16, 14, and 14). We measured cardiovascular (heart rate, blood pressure), hormonal (plasma adrenocorticotrophic hormone, cortisol, and noradrenaline), and subjective (e.g., anxiety, arousal) responses to stress in the three groups. Before the TSST, progesterone injections increased plasma levels without altering physiological or subjective states. Stress produced its expected physiological and subjective effects among placebo-treated individuals. Progesterone 50 mg attenuated peak increases in plasma cortisol and reduced changes in negative mood and alertness after stress, yet it increased plasma noradrenaline and systolic blood pressure. Progesterone 100 mg also attenuated stress-induced increases in alertness and arousal, yet it potentiated stress-induced increases in diastolic pressure. Thus, progesterone dampened some of the psychological effects of stress but produced inconsistent effects on physiological stress responses. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Thyroxine administration prevents streptococcal cell wall-induced inflammatory responses

Administration of streptococcal cell wall (SCW) preparation induces an inflammatory response in susceptible animals that is a model frequently used for rheumatoid arthritis. The degree of inflammation produced by SCW is greatly enhanced by low endogenous levels of glucocorticoids due to diminished hypothalamic-pituitary-adrenal activity. Because decreased glucocorticoid production is known to occur in the hypothyroid state, we tested whether hypothyroidism would increase, and conversely, whether hyperthyroidism would decrease, the inflammatory responses to SCW. Adult female Sprague Dawley rats were fed a regular diet (control), L-T4 (T4; hyperthyroid), or 6-propyl-thiouracil (hypothyroid) in drinking water for 7 weeks. Hypothyroidism resulted in elevated plasma levels of TSH and hypothalamic preproTRH messenger RNA (mRNA) while reducing anterior pituitary POMC mRNA and plasma ACTH and corticosterone levels. In contrast, hyperthyroid rats produced opposite results: decreased measures of central thyroid function but increased pituitary-adrenal function. Three days after administration of SCW, macrophage inflammatory protein-1alpha and interleukin-1beta mRNA expression increased dramatically in controls and even further in hypothyroid animals, as measured by Northern blot analysis. In contrast, T4-treated rats showed significant inhibition of these inflammatory markers. Thus, the hyperthyroid state combined with increased endogenous glucocorticoid levels is protective against inflammatory challenges. The inverse relationship between preproTRH expression and pituitary-adrenal function suggests the possibility of a direct inhibitory link connecting the hypothalamic-pituitary-adrenal and thyroid axes, and suggests alternative sites of therapeutic intervention for rheumatoid arthritis and other inflammatory associated disorders.