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U.S. Department of Health and Human Services

About FDA

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FY 2001 Host Response

Biomaterials in contact with human tissues may have direct or indirect adverse (toxic) effects. The various types of toxicity may be time-limited and result in minimal and reversible effects, or may be longer-lasting resulting in serious tissue damage. The OST program in Host Response to Medical Devices consists of focused laboratory studies and risk assessment approaches to identifying causal agents, understanding the means and mechanisms underlying adverse biological effects, and developing approaches to minimizing their consequences. These studies make use of cutting edge technology and deal with serious emerging health issues. They encompass methods development, biomaterials safety evaluation, and forensic studies, as well as longer-term hypothesis testing to investigate unanticipated adverse effects of new materials and devices. The aim is to provide the Center with independent, timely and authoritative scientific support at various points in the Total Product Life Cycle.
I. Biomaterial Effects on Immune System

Development of a Standard Methodology for Testing for Classical Pathway Complement Activation in Serum by Solid Materials

Key words: complement activation, classical complement pathway, medical materials, immunotoxicology, standard

OST scientists conducted experiments to acquire data and develop procedures for a standard methodology capable of screening medical device materials for classical pathway complement activation in human serum. Complement activation is a potential hazard when a patient’s blood contacts medical device materials. Although the expected pathway for activation, should that occur, is via the alternative pathway (not requiring antibodies), some medical devices may directly or indirectly activate complement by the antibody-dependent classical pathway. OST scientists have previously developed two other related standard methodologies: 1) testing for complement activation by either pathway (ASTM F1984-99), and 2) testing for complement activation by only the alternative pathway (ASTM F2065). In the present methodology, C4-deficient guinea pig serum was used as an indicator to test for the presence of the classical-pathway specific component C4 following exposure of human serum to a device material. This standard methodology will be submitted to ASTM for acceptance as a voluntary standard useful to industry in testing materials for use in new blood-contacting medical devices. In addition, it will help CDRH reviewers determine validity of data submitted by applicants claiming lack of immunotoxicity of device materials.

Development of Assays for Determining Complement-mediated Immunotoxicity of Cardiovascular Devices

Key words: complement, restenosis, balloon angioplasty, porcine, immunohistochemistry, immunotoxicology, serology

OST scientists initiated immunohistochemical and serological investigations to evaluate complement activation in serum or deposition of complement components in coronary arteries of pigs during experimental angioplasty. Complement activation is an immediate result of tissue injury and can be amplified by materials present on medical devices. Neointimal hyperplasia (overgrowth of the attaching artery) is a common outcome and failure mode of cardiovascular device interventions. Activation products of complement may play an important role in regulating neointimal hyperplasia. Serological assays include the development of a porcine-specific C3a ELISA, and an ELISA for C5b-9 is currently being developed. In parallel, an immunohistochemical assay was established to detect porcine C3 in formalin-fixed preparations in arteries. Preliminary data suggest that porcine coronary arteries treated by balloon angioplasty may be subject to complement deposition, while arteries not injured by balloon overstretch are not. These data will be extended by appropriate time course studies in several pigs. Determining the immunotoxicological basis for adverse outcomes in the porcine model may provide a predictive model for adverse events in humans undergoing similar cardiac interventions. These studies may also assist in designing improved cardiovascular devices.

Autoantibody Responses Induced by Silicone Gel and Oil

Key words: autoantibodies, silicone gel/oil.

The goal of this project is to develop animal models for evaluating potential adverse immunological effects of implanted biomaterials. The initial study investigated the production of autoantibodies in female Sprague-Dawley and Dark Agouti rats injected with silicone gel from a breast implant. This study demonstrated the presence of anti-collagen antibodies in the serum of rats injected with dimethylsiloxane oil alone or a gel/oil mixture. To repeat and confirm these results, rats were injected at 5-8 weeks of age with an emulsion of silicone gel isolated from a breast implant and medical-grade 20 cs dimethylsiloxane oil, or 1000 cs dimethylsiloxane oil alone. Incomplete Freund’s adjuvant plus collagen I was used as the positive control and saline alone as the negative control. These injections, following the original protocol, were placed in the right mammary area of each rat. Serum samples are being collected from the tail vein of each rat at intervals of 2 weeks, 4 weeks, 8 weeks, and then every 28 days for 18 months. The samples will be analyzed for autoantibodies to rat collagen and to nuclear antigens.

Cytotoxicity of In Situ Polymerization of Medical Devices

Key words: in situ polymerization, cytotoxicity, standards

The current biocompatibility standards, ASTM (F748) and ISO (TC194-10993-1), describe methods for testing cytotoxicity of medical devices that are in a final polymerized form and not suitable for devices that polymerize in situ and have tissue contact. A method to make contact between the polymerization device and the test cells was devised. Cytotoxicity tests on a series of in situ polymers were performed and the information published. Application of the in vitro tests will aid ASTM and ISO in adapting current standards to assess the safety of these in situ polymerization devices.

Endocrine Disruption by Medical Device Materials

Key words: endocrine disruption, women's health, bisphenol A, hazard analysis, mode of action, development of standards

CDRH is concerned with the potential for certain medical device materials (including tissue engineering medical products) that adversely affect women’s health, by mimicking or interfering with endogenous hormone actions. OST scientists, in collaboration with researchers at George Washington University, are improving the current standard assay for estrogenic activity of materials (currently under revision by ICCVAM, to which OST contributes expert knowledge). OST scientists have amplified the current assay by including key biomarkers of exposure that make it more specific and sensitive, and they have applied this assay to evaluating bisphenol A, a controversial plasticizer. Results of these studies have been published in peer-reviewed journals. One paper was honored as the journal’s "Highlight Paper of the Month."

II. Genetic Technologies

OST is developing expertise in genomic and genetic technologies in preparation for the submission of devices based on these technologies. OST collaborated with external biotechnology laboratories to aid keeping pace with technological development. Two projects, one in genetics and one in genomics, are also providing new approaches to understanding host responses to medical devices.

A p53 Mutation Assay for Evaluating the Cancer-Inducing Potential of Medical Device Materials

Key words: genetic testing, p53 gene, cancer risk, microarrays, gene chips

Sequence changes in the p53 gene are the single genetic change most commonly found in cancer. An assay to directly measure these interactions, as a preclinical test for cancer risk assessment, is being developed. Such an assessment is a requirement for all new device approvals. Major progress was made in methods development to make a yeast reporter gene assay practical for screening of device materials. In particular, recovery of p53 mutants was enhanced from 2.5 times background (published data on original assay) to 100 times background in an OST laboratory. Collaborators on this project will aid in the genetic analysis of mutants by traditional sequencing, hybridization on microarrays, or analysis by electronic DNA chips.

Development of a Diagnostic Gene Expression Microarray for Type I Allergy Using Transcriptome Profiling

Key words: genomics, differential gene expression, latex allergy, microarrays

Genomic or gene expression technologies represent a new way to characterize the physiology of a cell or a person, most often in comparison with another condition. OST has established a genomics laboratory capable of differential gene expression technologies using several instruments. Funding was obtained from the Office of Women’s Health for a genomics project in which gene expression comparisons are made in men and women who are either allergic or not allergic to latex. The patterns may yield differences that could be used as a basis for a diagnostic test for latex allergy and provide information related to the cause of this adverse response. The laboratory has been set up to provide the capability for validating microarray technologies using more traditional differential display and real-time PCR measurements.

III. Tissue / Material Interactions

Development of an In vitro Test Method for Studying Human Endothelial Cell Monolayers Undergoing Immunotoxic Reactions Associated with the Use of Cardiovascular Devices

Key words: HUVEC, endothelial cells, immunotoxicity, cardiovascular devices

OST scientists have developed a fluorescence-based in vitro methodology for determining cell function and intracellular calcium signaling in monolayers of human umbilical cord endothelial cells (HUVEC). Increase in intracellular calcium can change normal endothelial cell function, affect communication with smooth muscle cells, and be indicative of lytic cell death (necrosis) or programmed cell death (apoptosis). HUVEC monolayers are used as a model of the endothelial cells lining the inside of coronary arteries. During cardiovascular procedures such as balloon angioplasty, endothelial cells may be damaged or have their functions perturbed. These effects may subsequently contribute to overgrowth of smooth muscle cells (restenosis), with re-blockage of the coronary artery within weeks or months. Conditions of the HUVEC monolayer assay were optimized, including culture conditions, labeling with the calcium-sensitive dye Fluo-4, best procedure for determining Fluo-4 fluorescence using a fluorescence 24-well plate reader, and the use of different culture media necessary to test immunotoxic components such as complement and cytokines. This assay may be used to determine the role of specific blood-borne factors in risks associated with the use of cardiovascular devices. The procedure should be readily adaptable for studying endothelial cells from human cardiac arteries plus associated smooth muscle cells central to the critical problem of restenosis.

Tissue Engineering

Key words: tissue engineering, tissue engineered combination products, standards, research/technology assessment

An OST scientist served as Chair of the FDA Inter-Center Tissue Engineering Working Group, providing leadership in programs designed to develop guidance and planning options to the Center and Agency. These encompass 1) monitoring and assessing technology, 2) evaluating applications in medical products, 3) developing standards for tissue-engineered medical products, and 4) education/training for Center/Agency review/research staff and the at-large scientific community. Several accomplishments have contributed to the knowledge base for the Center/Agency and have facilitated scientific and regulatory assessment of new biotechnology-derived and tissue-engineered combination medical products.

These projects have focused on addressing the scientific and regulatory considerations for new products, developing information for Center/Agency decision-making, and for developing policy regarding product safety and effectiveness issues. The focus has also been on analyzing products in review and under development; communicating information to the Center/Agency and scientific community through different mechanisms; educating Center/Agency staff and the research and development community through presentations, publications and short courses; and participating in the development of cooperative programs with other Federal agencies.

Standardization of Cell-Based Methods for Evaluating Natural Materials Used for Tissue Engineered Medical Products (TEMPs)

Key words: cell adhesion, histomorphometric

Cell adhesion assays are being developed which should assess the ability of cells to adhere to substrates (i.e., polymers and natural materials such as collagen), as well as to maintain the expected cell phenotype when cultured/grown on TEMPs substrates. Histomorphometric techniques, currently under development, will provide an additional quantitative assessment of cell characteristics on these substrates. Scientists will establish criteria to ensure the reproducibility of both the surface coating technique and the adhesion assay wash procedure, which use an in-house apparatus. To date, cell adhesion assay parameters have been established for cell concentration, dye concentration linearity range, and wash conditions. Preliminary data have shown a two-fold difference in cell adhesion to polymethylmethacrylate and ultra-high molecular weight polyethylene polymers.

This project has resulted in the formation of a Task Group within Division IV, Tissue Engineered Medical Products, to develop an ASTM standard for cell adhesion assays for tissue engineering.

Other work in the area of standards has resulted in a draft ASTM standard for characterizing Type I collagen, which is currently under ballot in the Division IV, and a publication in the New York Academy of Sciences summarizes two standards that have been previously approved by ASTM.

Medical Device Particles and Adverse Cellular Reactions

Key words: wear particles, implants, standard, titanium alloy

Wear particles may be released from failed devices or are formed at articulating surfaces of implanted devices. Particles may also be injected directly into the body for clinical purposes. A murine macrophage cell system has been used to study the inflammatory potential of particles. Data generated on the inflammatory potential of particles helped 1) serve as the basis of the voluntary consensus standard ASTM F1903-98 on evaluating the biological effects of particles, 2) provide good science for Health Hazard Evaluations, and 3) allow CDRH scientists to review Corrective Action Plan. Titanium alloy particles were examined in FY 2001, and the results were presented at two national meetings in 2001 (Society for Biomaterials; Gordon Research Conference on Biocompatibility) and at the FDA Science Forum.

Reducing Allergies associated with Latex-containing Products

Key words: latex, natural rubber latex, proteins, allergy, powder

Medical devices made of natural rubber latex (NRL) contain proteins that can potentially cause life threatening allergic reactions. The Division of Life Sciences in OST conducted research targeting reducing the sensitization-potential of these products. These efforts focused on 1) identifying proteins responsible for inducing sensitization, and 2) developing methods to measure proteins on finished NRL products. The new ASTM standard D6499 (ELISA Inhibition Test) was developed as a more sensitive assay for quantifying antigenic NRL proteins. In collaboration with other research laboratories, studies were conducted to correlate the ELISA Test with other known methods for NRL protein quantitation. OST continues to evaluate the role of glove powder and to quantitate the powder-bound protein. The OST research resulted in proposing a new regulation for maximum powder and protein level on medical gloves. Research in collaboration with the ASTM D11 working group is being conducted to develop an HPLC chromatography method for analyzing chemical accelerants used in the manufacturing of latex products.

Studies on Nanotechnology for Detecting Biofilms

Key words: biofilms, nanotechnology

The FDA Office of Science and Coordination funded an inter-Center project on the use of nanotechnology for assessment of biological hazards. The CDRH portion of this project was to assess the application of nanotechnology for the early events in biofilm formation on medical devices. This project included leveraging with Cornell University for the production of micro-cantilevers. While Cornell is fulfilling its obligations, cantilevers used in Atomic Force Microscopy (AFM) were used to obtain preliminary information. The results indicated that these probes detect the biofilm-related adhesion events occurring in the first 4 hours, whereas standard techniques cannot detect adherence until 8-12 hours.