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FY 2000 Tissue/Material Interactions


Key words: biocompatibility, medical device toxicology, risk assessment, standards development, methods development, laboratory research, materials toxicology, foreign body reactions

The Tissue/Material Interaction Program Area in OST encompasses an interconnected program of laboratory research, risk assessment, and standards development activities designed to provide a scientific basis for regulatory decision making in CDRH. OST remains at the forefront in developing methods for risk assessment and medical device toxicology. Information from laboratory-based research efforts are used to develop consensus standards, guidance documents, and risk assessments that form the basis of a knowledge management framework within CDRH. Specifically, OST serves as an independent source of data on medical device toxicology and risk assessment for risk managers in CDRH Offices (primarily ODE) with the primary responsibility for device evaluation.

The Division of Life Sciences laboratory research program performs experimental studies that generate independent data for use in developing standards and guidance documents. These documents provide a scientific basis for such ASTM standards as natural rubber latex protein content in gloves and condoms, for FDA guidance for the content of phthalate esters, medical grade plastics and for the assessment of the role of endotoxin in latex allergies.

Toxicity evaluation of medical device materials presents two challenges: compounds and chemicals that are released from device materials and foreign body reactions induced by medical devices and their materials. New methods are being developed to assess the adverse effects that may occur following exposure to compounds released from medical device materials and the interaction between medical device materials and cells or tissues. The scope of these studies range from short-term forensic studies (e.g., etiology of latex rubber sensitivity) to long-range mechanistic studies (e.g., effects of implant degradation and wear particles and development of more predictive molecular biomarkers). Laboratory research in this program area also addresses clinically important, high-profile issues, such as the gender differences in coronary artery response to interventional cardiology devices (e.g. balloon angioplasty catheters and cardiovascular stents) and the ability of compounds released from medical devices to have estrogenic effects. These projects underscore OST’s commitment to women’s health issues in this program area.

Supporting the development of standards, guidance documents, and regulations. The development of consensus standards for regulatory assessment is a core goal of the Office of Science and Technology. Both laboratory-based research and risk assessments are performed in the Tissue/Material Interaction Program to directly support the development of consensus standards, guidance documents, and regulations. For example, research conducted in OST’s Division of Life Sciences to develop a method for quantifying natural rubber latex proteins served as the basis for ASTM standard D6499. OST scientists also provided the leadership role in developing a proposed rule and guidance document for medical gloves. OST experience in latex research has positioned the Center to develop consensus from a wide and dissenting body of opinion among industry and consumers on the proper scientific and regulatory approach for tackling the problem of adverse effects associated with exposure to NRL-containing medical devices. OST’s tissue engineering program has resulted in the development of a cooperative program with other Federal agencies through the MATES working group of the subcommittee on Biotechnology. In support of ODE review efforts, scientists have contributed to the review of ASTM terminology relating to tissue engineered products. Scientists have also developed draft ASTM standards on alginates for tissue engineered products.

Addressing clinically important, high profile issues in CDRH. Restenosis, or re-narrowing of coronary arteries, is a clinically important problem that occurs following balloon angioplasty. OST scientists have developed a large animal model of vascular disease that can be used to investigate the role of factors such as gender and hormonal status on the response of coronary arteries to interventional devices. An issue that has received media attention recently is the ability of certain chemical compounds to interfere with the endocrine system. One such compound that can be released from medical device materials, bisphenol A, can interfere with endogenous endocrine and hormone homeostasis. OST scientists have conducted research to characterize the estrogenic effects of bisphenol A, and this information can be used to reduce uncertainties in assessing the risk of this compound. Another issue that has received considerable media attention recently is the potential for phthalate esters used as plasticizers in PVC medical devices. OST has been assigned the lead role in the Center for assessing the human health risks posed by patient exposure to these compounds.

Addressing emerging issues and supporting the science base in CDRH. Understanding the mechanism or mode of action by which device materials or compounds released from device materials produce adverse effects is a key component of the Total Product Life Cycle (TPLC) approach embraced by the Center for device evaluation. Under the Tissue/Material Program Area, OST scientists have undertaken mechanistic studies to better understand the role of macrophages in the phagocytosis of particles and the ability of endotoxin to potentiate of latex allergy. The expertise and data obtained from these research projects position the Center to ask the right questions for regulatory decision making. OST molecular biologists in collaboration with ORA/WEAC initiated participation in the International Laboratory Study on Chemical Disinfectants. This includes over 20 laboratories around the world in a round-robin (Ring Test) study to validate methods for disinfecting medical devices. The OST/ORA labs are the only United States participants.

Developing new approaches for medical device toxicology and risk assessment. Medical device toxicology and risk assessment pose a unique set of challenges, and OST staff are developing new, cutting-edge approaches to address these challenges. Efforts in toxicology methods development are focused on developing more sensitive, predictive tests for screening medical device materials and to reduce the use of animals for testing purposes. OST has an extensive, productive research program to develop new molecular biomarkers of exposure and toxicity for compounds released from medical device materials. OST scientists are also adapting existing toxicology test methods to use in assessing medial device materials. For example, new approaches are being developed to assess the potential for devices that polymerize in tissue, such as bone cements, dental acrylics and tissue sealants, to produce cytotoxic effects. Furthermore, research efforts in latex glove allergies have provided the Center with an independent source of data to support regulatory decision-making. Similar to the adaptation of toxicology research methods for medical devices, the methods used to assess the risk posed by exposure to industrial or environmental compounds must be modified when assessing the risk posed by exposure to compounds released from medical devices. OST contributed extensively to the major risk assessment of the re-use of single use devices, extensive contributions through the preparation of materials, review of documents, consultations on requested concerns, development of testing strategies and presentations at the Center level, and to external CDRH regulatory authorities. In response to the challenge, OST has spearheaded a risk assessment methods development program. Much of the work being done in this program is specifically intended to address issues raised during the development of the ISO/DIS 10993-17 standard.

Large Animal Models of Vascular Disease and Therapeutic Device Interventions
Key words: cardiovascular disease, balloon angioplasty, restenosis

CDRH has established a large animal cardiovascular research program to develop and study models of cardiovascular disease and therapeutic device interventions. OST is establishing animal models of vascular disease in order to investigate the safety and effectiveness of therapeutic interventions such as balloon angioplasty or stents in coronary and carotid blood vessels. This work has demonstrated that angioplasty balloon-induced coronary stenosis is dependent on balloon sizing, is gender-differentiated, and is influenced by the hormonal milieu in a domestic pig. These animal models provide the basis for investigating the biologic response (e.g., restenosis) to long-term medical device implants such as balloons or stents. The research goals include improved understanding of both the mechanisms of action and the failure modes for these interventions.

Large Animal Models of Vascular Disease and Therapeutic Device Interventions
Key words: cardiovascular disease, balloon angioplasty, restenosis

CDRH has established a large animal cardiovascular research program to develop and study models of cardiovascular disease and therapeutic device interventions. OST is establishing animal models of vascular disease in order to investigate the safety and effectiveness of therapeutic interventions such as balloon angioplasty or stents in coronary and carotid blood vessels. This work has demonstrated that angioplasty balloon-induced coronary stenosis is dependent on balloon sizing, is gender-differentiated, and is influenced by the hormonal milieu in a domestic pig. These animal models provide the basis for investigating the biologic response (e.g., restenosis) to long-term medical device implants such as balloons or stents. The research goals include improved understanding of both the mechanisms of action and the failure modes for these interventions.

Evaluation of In Situ Polymers – Test Methods Development
Key words: in situ polymers, biocompatibility standard, bone cements, dental acrylics

Current biocompatibility standards, i.e., ASTM (F748) and ISO (TC194-10993-1), provide a framework testing medical devices that are in a final end-stage, finished form. They do not address devices that polymerize in the tissue, such as bone cements, dental acrylics, and tissue sealants. These polymers, such as dental and bone cements, are characterized by the rapid unification of two or more compounds placed directly in tissues to form a unique product. This type of reaction can generate heat, acid, free radicals, and other products with unknown modes of action, even though the final polymer may be biocompatible. To better estimate the cytotoxicity of these in situ polymers, various means of introducing the reacting material to cells in culture were developed. Coating the material on a sterile glass cover slip, then adding the cover slip to the in vitro test system immediately provided reasonable cytotoxicity data that reflected actual use conditions. For in situ polymeric devices that are more viscous, such as dental materials and bone cements, a mold was used which was placed directly into cell culture. As manufacturers submit new materials and applications for in situ polymers, the independent data and new test methods developed by OST scientists will provide CDRH with the knowledge and expertise to evaluate product safety.

Particulate Debris and Chronic Inflammatory Responses – In Vitro Methods
Key words: in vitro methods, macrophages, phagocytosis, particles, wear debris, titanium, cadmium oxide, immunomodulation, inflammation

OST scientists are using cultured macrophages to understand the immunotoxic and inflammatory potential of particulate materials. Particles from orthopedic implants have been shown to induce inflammatory responses in the host. Titanium alloy (TiAlV) particles were tested alone and with lipopolysaccharide (LPS) in an in vitro system to determine their effects on the induction of cytokines. It was found that TiAlV particles are not very inflammatory in terms of the three cytokines and nitric oxide that were measured. The in vitro data on various particulate debris were used for an ASTM test method to assess biocompatibility of device particles. The data are being used to determine if there is a correlation between in vitro and in vivo findings. Together, the in vitro and in vivo studies will be used to develop risk analyses for particulates from medical devices, to write guidance documents, and to improve ASTM and ISO/AAMI standards.

Because of the important role of macrophages in the phagocytosis of foreign particles related to wear debris from implants, OST scientists assessed the effects of particulate (enter cells via phagocytosis) and soluble (enter cells via diffusion or transport mechanisms) forms of the same chemical on several endpoints (cytokine production, stress protein synthesis, nitric oxide production, and cytotoxicity). This approach should distinguish which effects, if any, may be specifically associated with the particulate or soluble forms. Cadmium chloride (soluble) and cadmium oxide (particulate) were chosen since cadmium possesses known immunomodulatory properties. The results showed that macrophages respond similarly in vitro to a particulate and soluble form of the same material. They were published in August 2000 in In Vitro and Molecular Toxicology in a paper titled "Effects of Particulate and Soluble Cadmium Species on Biochemical and Functional Parameters in Cultured Murine Macrophages."

Particulate Debris and Chronic Inflammatory Responses – In Vivo Methods
Key words: in vivo methods, immunotoxicity, PMMA, polystyrene, polyethylene, spleen, lymphocytes

The purpose of this study is to evaluate the in vivo effects of biomaterial particles on macrophages and possible long-term impacts on immune functions of the host. The studies in mice indicated that polystyrene (PS) and polyethylene (PE) particles injected intraperitoneally are relatively inert materials, deposited in the omentum fatty tissues and caused only a transient inflammatory reaction. However, the polymethylmethacrylate (PMMA) particle injection lead to infiltration of activated macrophages into the spleen and to a marked involvement of splenic lymphocytes. The data show that the chemistry of material is a determining factor in signaling macrophage migration and chemotactic factor production. In addition, PE appeared to be a good candidate for use as a negative reference in the evaluation of biomaterial immunotoxicity.

Improvement of Natural Rubber Latex (NRL) Protein Quantitation Accuracy 
Key words: natural rubber latex, latex allergies, protein quantitation

OST was involved in developing a method for quantitation of natural rubber latex (NRL) proteins, which recently became a national standard, ASTM D6499. The research was focused on modifying the protocol to include the measurement of glove powder-bound proteins, which may contain 10-20% of total amount of the protein on the NRL product. To validate the ASTM D6499 measurements for potential sensitization, they were compared with other NRL protein methods. Depending on the source of antigen, the ASTM D6499 correlated either with total protein level or with allergen level obtained by the RAST test. The data showed that a proper selection of standard reagent is critical for the test endpoint.

Effects of Hospital Environmental Factors on NRL Allergy
Key words: latex proteins, endotoxin, glutaraldehyde, IgE antibodies

This study is designed to determine if the endotoxin and glutaraldehyde, present on the medical devices and in the hospital environment, may be contributing factors in the development of latex allergy. Studies indicated that animals exposed to endotoxin concomitant with latex proteins developed an elevated level of IgE antibodies. Exposure to endotoxin (LPS) and latex proteins resulted in both suppression and enhancement of the IgE antibody level, depending on the dose of LPS. The data indicate that hospital environment may contain factors that may contribute to the sensitization to NRL proteins.

Medical Glove Reclassification Rule
Key words: medical gloves, natural rubber latex, proteins, reclassification, proposed rule, labeling

Experimental and clinical studies demonstrate that cornstarch on surgical gloves can enhance foreign body reactions, increase infections, and act as a carrier of natural latex allergens. OST has provided leadership in defining the issues and developing a proposed rule to address these adverse health effects. The proposed rule, published in 1999, would require (1) new label caution statements that include recommended maximum limits for protein, glove powder, and powder-free residue; (2) labeling of protein levels on NRL gloves; (3) labeling powder levels on powdered NRL and synthetic gloves; and (4) expiration labeling supported by stability studies. The comments received on the proposed rule have been analyzed, responses to the issues raised have been drafted, and options to the proposed labeling statements were tested by a focus group. Based on the analysis of comments and the results of the focus testing, the text of the final regulation has been drafted and the preamble to the rule and the medical glove guidance document are being revised. After senior staff approval, the final rule will be sent to FDA/OCC.

Molecular Biomarkers for Preclinical Evaluation of Medical Device Materials
Key words: kidney, nephrotoxicity, stress proteins, heat shock proteins, preclinical test method development

OST scientists develop predictive preclinical methods for improved safety assessments, standards, and risk assessments. New molecular biomarkers of exposure and toxicity must be carefully validated with traditional assays and standards for use in preclinical safety evaluation and in risk assessment activities. The rationale for assessing biomarkers at the cellular and molecular level are that such targets are usually the first responses induced by potentially hazardous materials and chemicals. OST scientists are evaluating the "stress" protein response, sometimes called heat shock proteins, as a method that will serve as a screening assay to more readily predict potential adverse effects of device materials and other chemicals in major target systems in the body, such as the kidney, liver, and endocrine system. To be effective and useful, a biomarker should be detectable earlier than the onset of overt tissue damage. OST scientists published a paper in February 2000 in Toxicological Sciences titled "Mercury induces regional and cell-specific heat shock protein expression in rat kidney." The data demonstrated that the expression of specific proteins in response to mercury exposure correlated with the ensuing development of kidney damage. Further evaluation of these biomarkers within specific major morphological regions and cell types within the kidney demonstrated that cells that do not or are unable to express these proteins in response to nephrotoxicant exposure might be more susceptible to damage.

Molecular Biomarkers for Endocrine Disruption by Medical Device Materials
Key words: endocrine disruption, women's health, bisphenol A, hazard analysis, mode of action

Certain plasticizers found in some medical device plastics or used in device manufacture may be potential endocrine disrupters, i.e., mimic or interfere with endogenous endocrine and hormone homeostasis. OST scientists are collaborating with researchers at the George Washington University assessing the estrogenic properties of bisphenol A (BPA), a plasticizer found in medical tubing and dental sealants. Three endpoints (uterine hypertrophy, histology, and expression of specific proteins, called heat shock proteins) were used to evaluate modifications and enhancements to the traditional assay for estrogenic effects of BPA in mice. The results have shown that expression of specific heat shock proteins is an estrogenic effect independent of uterine swelling and is a more sensitive indicator of estrogenic effect than uterine swelling. The OST modifications provide additional biomarkers of effect that expand the mechanistic information derived from the assay and will reduce the number of uncertainties in assessing risk from exposure to estrogenic materials. Results of these experiments were published in Toxicological Sciences, a leading toxicology journal. The paper was designated "Highlight Paper of the Month" and was titled "Bisphenol-A induced increase in uterine weight and alterations in uterine morphology in ovariectomized mice: Role of the estrogen receptor".

DEHP Risk Assessment
Key words: DEHP, phthalate, PVC medical devices, risk assessment

In late FY 1999, a citizen’s petition was submitted to the FDA Commissioner, requesting the Agency 1) initiate a rule or issue guidance consistently requiring all PVC medical devices that may leach phthalate plasticizers include a prominent, clearly worded warning label as to the potential for DEHP or other phthalate plasticizers to leach out of the PVC and to enter the body, and 2) establish a program to expedite the development and use of substitutes for PVC medical devices that leach phthalate plasticizers. To determine if such actions are warranted, it is important to first assess the risk posed by patient exposure to DEHP. In FY 2000, OST prepared a risk assessment of DEHP released from medical devices and submitted it for review to each FDA Center. Based on the comments received, the risk assessment was revised in FY 2000. Because of the cross-cutting nature of the issues addressed in the risk assessment, the document was also submitted to the FDA Senior Science Council, where it is currently under review.

Risk Assessment of Dioxin in Tampons
Key words: dioxins, tampons, risk assessment

Over the past 2 years, OST scientists have developed a risk assessment examining the potential for harmful affects from dioxin in tampons. Congress and the press initially raised concerns because most tampons contain rayon made from cellulose fibers that might contain dioxin due to the manufacturing process of the rayon. Openly published data of the chemical analysis of tampons indicated that little or no dioxin was present, but that there were small amounts of related compounds. OST scientists evaluated the results of the chemical analysis, usage patterns and exposures, and a new risk assessment approach was undertaken to revise this document. The hazards from tampon use were calculated, and the amount of additional exposure to dioxin from tampons was found to be infinitesimal, presenting negligible additional risk for adverse effects.

Tissue Engineering

The Molecular Biology Branch of OST served as Chair of the FDA InterCenter Tissue Engineering Working Group, providing leadership in programs to develop guidance and planning options to the Center and Agency. These encompass the following: 1) technology monitoring and assessment; 2) evaluating applications in medical products; 3) 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 to facilitate scientific and regulatory assessment of new biotechnology-derived and tissue-engineered medical products (TEMPs).

These products have focused on addressing the scientific and regulatory considerations for new products and developing information for Center/Agency decision making. 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 research and development community, and participating in the development of cooperative programs with other Federal agencies, such as the MATES Working Group of the Subcommittee on Biotechnology, under the broad umbrella of the Office of Science Technology Policy.