1、Designation: F 1903 98 (Reapproved 2003)Standard Practice forTesting For Biological Responses to Particles in vitro1This standard is issued under the fixed designation F 1903; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the yea
2、r of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers the production of wear debris anddegradation products from implanted materials that may lea
3、d toa cascade of biological responses resulting in damage toadjacent and remote tissues. In order to ascertain the role ofparticles in stimulating such responses, the nature of theresponses, and the consequences of the responses, establishedprotocols are needed. This is an emerging, rapidly developi
4、ngarea and the information gained from standard protocols isnecessary to interpret responses and to determine if there iscorrelation with the in vivo responses. Since there are manypossible and established ways of determining responses, asingle standard protocol is not stated. However, well describe
5、dprotocols are needed to compare results from different inves-tigators using the same materials and to compare biologicalresponses for evaluating (ranking) different materials. Forlaboratories without established protocols, recommendationsare given and indicated with an *.1.2 Since the purpose of th
6、ese studies is to predict theresponse in humans, the use of human cells would providemuch information. However in the interest of safety in labo-ratory protocols, the use of non-human and non-primate cells isdescribed.1.3 The values stated in SI units are to be regarded as thestandard.1.4 This stand
7、ard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 AS
8、TM Standards:2F 619 Practice for Extraction of Medical PlasticsF 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices3. Summary of Practice3.1 Biological responses to particles may be evaluated usingspecimens from animals being tested according to the PracticeF 748 m
9、atrix for irritation and sensitivity, or for implantation.Blood, organs, or tissues from the animals may be used.3.2 Biological responses to particles may be evaluated usingmaterials or extracts according to Practice F 619. These mate-rials or extracts may be used for in vivo tests or for the in vit
10、rotests. Particles generated by other methods may also be used.4. Significance and Use4.1 This practice is to be used to help assess the biocom-patibility of materials used in medical devices. It is designed totest the effect of particles from the materials on macrophages.For safety reasons, the use
11、 of non human, non primate cells isrecommended in this practice. For laboratories equipped andapproved to work with human blood and tissue, the use of thesesame protocols would be advantageous for development ofunderstanding of the interaction of cells and particles.4.2 The appropriateness of the me
12、thods should be carefullyconsidered by the user since not all materials or applicationsneed be tested by this practice.4.3 Abbreviations:4.3.1 LPSlipopolysaccharide (endotoxin).4.3.2 LALLimulus Amebocyte Lysate.4.3.3 ATCCAmerican Type Culture Collection.4.3.4 FCS (FBS)Fetal Calf Serum.4.3.5 NCSNewbo
13、rn Calf Serum.4.3.6 PBSPhosphate Buffered Saline.4.3.7 HANKSA balanced salt solution.4.3.8 MMPSMatrix Metallo Proteases.4.3.9 RPMI 1640Specific Growth Medium (Roswell ParkMemorial Institute).4.3.10 HEPESA buffering salt.5. Responses from Cells Grown In Vitro5.1 ParticlesDefine the nature of the part
14、icles used:5.1.1 Source,5.1.2 Chemistry,5.1.3 Size (mean and range),5.1.4 Shape,1This practice is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Devices and is the direct responsibility of Subcommittee F04.16 onBiocompatibility Test Methods.Current edition approved Nov. 1, 2003.
15、 Published December 2003. Originallyapproved in 1998. Last previous edition approved in 1998 as F 1903 98.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standa
16、rds Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.1.5 Method of sterilization,5.1.6 If the presence of bacterial lipopolysaccharide (LPS)was determined, specify how this was done and the s
17、ensitivityof the method. (LAL testing with a sensitivity of at least 0.06EU is recommended),5.1.7 Concentration of particles used as weight or number orsurface area/106cells, and5.1.8 Surface charge (if known).5.2 CellsDefine the nature of the cells used:5.2.1 Established Cell Line (if not, go to 5.
18、2.2)The use ofestablished cell lines provides a known cell type with areproducible response. Although the correlation with the invivo system may not be known at this time, careful studies withestablished cell lines could eventually allow determination ofcorrelation between in vivo and in vitro syste
19、ms.5.2.1.1 Source of cell and identifying number or code,5.2.1.2 Nature of the cell (for example, macrophage), and5.2.1.3 Special attributes of the cell line (for example,nonphagocytic),5.2.1.4 *ATCC murine macrophages such as RAW 264.7,J774A, P388D1, or IC-21 are recommended.5.2.2 Primary Isolate (
20、if not, go to 1.2.1):5.2.2.1 Source of cell including species and location (forexample, murine, alveolar),5.2.2.2 Nature of the cell (for example, macrophage),5.2.2.3 Mechanism of isolation (for example, lavage), and5.2.2.4 Specify if stimulant used and if so which one (forexample, mineral oil).5.2.
21、2.5 *Mouse (specify strain, age, and sex used) perito-neal exudate cells are recommended with a mild stimulant suchas nutrient broth.5.3 Culture Conditions:5.3.1 Specify source and type of medium. If not a commer-cial source, list ingredients and sources of ingredients.5.3.2 Specify source and type
22、of serum, and whether it washeat inactivated. If the presence of LPS was determined,specify method and sensitivity of the method.5.3.3 Specify culture conditions (for example, 37C, hu-midified, 5 % CO2incubator).5.3.4 Specify time of termination of culture or sampling ofculture medium.5.3.5 If cell
23、counts were determined specify as to when andhow. If estimates of cell number/mL specify when and how.5.3.5.1 *Medium and serum specified by the supplier of thecells are recommended. Generally RPMI 1640 with heatinactivated 10 % newborn or fetal calf serum are recom-mended. LPS levels are generally
24、provided or available fromthe distributor. Recommended culture conditions are 37C,with 5 % CO2, in a humidified incubator. Cell counts at thetime of initial plating and at the termination of the culture arerecommended using a hemocytometer with monolayer cellsresuspended by trypsin solution (not rec
25、ommended for mac-rophages), washing with Ca and Mg free PBS or Hanks, orscraping in 1 mL of Ca and Mg free PBS or Hanks. Theaddition of trypan blue is helpful. The supernatant of themedium from macrophages exposed to particles for specifiedtime periods is assayed.5.3.6 Controls:5.3.6.1 Cells not sti
26、mulated with particles should be main-tained at the same time under the same conditions.5.3.6.2 Polystyrene particles, spherical, size range 1 to 5m, should be used as a reference control.5.3.6.3 LPS is a known stimulant and is a good positivecontrol. A concentration between 0.25 and 1 ng/mL of cult
27、uremedium is sufficient.5.3.6.4 Culture medium is a recommended diluent for theassays.5.4 Products or Response DeterminedOne or more of thefollowing:5.4.1 Cell viability can be determined by any of severalmethods:5.4.1.1 From the total cell counts and viable cell count withtrypan blue dye exclusion,
28、5.4.1.2 From flow cytometry with vital stains,5.4.1.3 From uptake of DNA precursors, and5.4.1.4 From total DNA or RNA in the culture.5.4.2 Soluble Cell Products Elaborated:5.4.2.1 Reliable reagents or kits are available to detectproducts. The products are classified into three classes: growthfactors
29、 such as TNFa, TGF beta, and PGE2, interleukins suchas IL-1, IL-1 receptor antagonist, IL-1 beta, and IL-6, andreactive oxygen species such as superoxide or nitric oxide(NO) (usually measured as nitrite produced using the Griessreagent). The release of at least two products, which are fromdifferent
30、classes, should be determined.5.4.2.2 The time in culture should be specified since theseproducts peak at different times (for example, TNFa peaks by24 h, NO peaks around 72 h, the others generally peak at 48 to72 h). The detection of NO and TNFa are strongly recom-mended and the assay procedures ar
31、e described in some detailin this recommended practice.5.4.3 Assay for Nitric Oxide:5.4.3.1 The production of NO is most conveniently mea-sured by detection of the stable endproduct nitrite. This reflectsthe action of NO on arginine. The technique is simple,inexpensive and quantitative. The reagent
32、used to quantitatenitrite is the Griess reagent. This is prepared by dissolving 100mg of Sulfanilamide and 10 mg of N-(1-naphthyl)ethylenediamine in 2.5 % phosphoric acid. Assay ofculture medium: 100 uL of the culture medium is placed in a96-well microtiter plate and then 100 uL of the Griess reagen
33、tis added. This is incubated at room temperature for 10 min andthe optical density determined at 540 nm wavelength. Astandard curve is prepared with sodium nitrite in water. It isrecommended that two-fold serial dilutions be prepared en-compassing the range of 32 to 0.125 nmoles/mL. The standardcurv
34、e is prepared using 100 uL of the dilutions of sodiumnitrite and 100 uL of the Griess reagent.5.4.4 Assay for TNFa:5.4.4.1 Kits for assay of TNFa using immunological assaysare available from supply houses including, but not limited to,R growth factors; interleukins; macroph-ages; nitric oxide; parti
35、cles; TNFaAPPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The primary purpose of this practice is to describemethodology to determine the biological response to particlesusing in vitro cellular responses.X1.2 It is well recognized that the biological responses toparticles could be different fr
36、om those to solid materials. Theinteraction of the particles with phagocytic cells, notablymacrophages, is a key to the final biological response.X1.3 The interaction of macrophages with particles hasbeen an active research area for many years. Many investiga-tors have developed procedures for doing
37、 these studies. Thisdocument is intended to delineate the information necessary sothat the results from these various studies can be interpretedand to describe methodology appropriate for those investiga-tors developing such studies.X1.4 The interaction of phagocytic cells with particles willlead to
38、 the production of various soluble mediators which mayinfluence the progression of the biological response and theimmune response. It is unknown at this time which of theseresponses are favorable and which are unfavorable to the host.Similarly it is unknown whether quantitative evaluation ofthese fa
39、ctors is predictive of the biological response. Studiessuch as the ones described here are needed to determine theimportance of these responses in biocompatibility and biocom-patibility testing of materials.X1.5 The level of LAL for device evaluation is commonly0.5 EU/m/L . LAL testing with a sensit
40、ivity of 0.06 EU/mL isrecommended in this practice for evaluating in vitro responsesto particles since levels of LPS above 0.06 EU/mL may beF 1903 98 (2003)3stimulatory to macrophages. This will permit distinguishingcellular responses to particles from cellular responses to LPS.X1.6 Polystyrene (PS)
41、 particles were chosen as a referencecontrol for this standard practice so that testing results fromdifferent laboratories of from different cell lines or assaysystems could be compared and normalized to PS. Theanticipated responses of the cells to these particles and pro-duction of various cytokine
42、s and other growth factors areunknown. They may stimulate some factors and not others.These particles are not device material applicable and serve asa neutral territory, commercially available, well characterizedparticulate material for normalization of results.X2. ADDITIONAL REFERENCESGreen, L.C.,
43、DA Wagner, J Glowoski, PL Skipper, JSWishnok, SR Tannenbaum, “Analysis of Nitrate, Nitrite, and15N Nitrate in Biological Fluids,” Anal. Biochem. 126:131,1982.Flick DA, GE Gifford, “Comparison of In Vitro Cell Cyto-toxicity Assays for Tumor Necrosis Factor,” J. ImmunolMethods 68: 167, 1984.Chapekar,
44、MS, TG Zaremba, RK Kuester, VM Hitchins,“Synergistic Industion of Tumor Necrosis Factor a by Bacte-rial Lipopolysaccharide and Lipoteichoic Acid in Combinationwith Polytetrafluoroethylene Particles in a Murine MacrophageCell Line RAW 264.7,” J. Biomed. Mater. Res. 31: 1996, pp251-256.St. John K.R.,
45、(ed), Particulate Debris from Medical Im-plants: Mechanisms of Formation and Biological Conse-quences, STP 1144, ASTM, Philadelphia, PA 1992.Wright, T.M., Goodman, S.B., (eds), Implant Wear: TheFuture of Total Joint Replacement, American Academy ofOrthopaedic Surgeons, Rosemont, IL 1996.Ruff, M.R.,
46、Gifford, G.E., “Tumor Necrosis Factor,” Lym-phokines 2: 1991, pp. 235-272.Bonfield, T.L., Anderson, J.M., “Functional Versus Quanti-tative Comparison of IL-1 Beta from Monocytes/Macrophageson Biomedical Polymers,” J. Biomed. Mater. Res. 27:, 1993,pp. 1195-1199.ASTM International takes no position re
47、specting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This
48、 standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM Inter
49、national Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-
