1、Designation: F 2721 09Standard Guide forPre-clinical in vivo Evaluation in Critical Size SegmentalBone Defects1This standard is issued under the fixed designation F 2721; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers general guidelines for the in vivoassessment of tissue engineered medical products (TEMPs)intended t
3、o repair or regenerate bone. TEMPs included in thisguide may be composed of natural or synthetic biomaterials(biocompatible and biodegradable) or composites thereof, andmay contain cells or biologically active agents such as growthfactors, synthetic peptides, plasmids, or cDNA. The modelsdescribed i
4、n this guide are segmental critical size defectswhich, by definition, will not fill with viable tissue withouttreatment. Thus, these models represent a stringent test of amaterials ability to induce or augment bone growth.1.2 Guidelines include a description and rationale of variousanimal models inc
5、luding rat (murine), rabbit (leporine), dog(canine), goat (caprine), and sheep (ovine). Outcome measuresbased on radiographic, histologic, and mechanical analyses aredescribed briefly and referenced. The user should refer tospecific test methods for additional detail.1.3 This guide is not intended t
6、o include the testing of rawmaterials, preparation of biomaterials, sterilization, or packag-ing of the product. ASTM standards for these steps areavailable in the Referenced Documents (Section 2).1.4 The use of any of the methods included in this guidemay not produce a result that is consistent wit
7、h clinicalperformance in one or more specific applications.1.5 Other pre-clinical methods may also be appropriate andthis guide is not meant to exclude such methods. The materialmust be suitable for its intended purpose. Additional biologicaltesting in this regard would be required.1.6 The values st
8、ated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.7 This standard 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 a
9、nd health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F 561 Practice for Retrieval and Analysis of Medical De-vices, and Associated Tissues and FluidsF 565 Practice for Care and Handling of Orthopedic Im-plants and Inst
10、rumentsF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF 981 Practice for Assessment of Compatibility of Bioma-terials for Surgical Implants with Respect to Effect ofMaterials on Muscle and BoneF 1983 Practice for Assessment of Compatibility ofAbsorbable/Resorbable Bioma
11、terials for Implant Applica-tionsF 2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical ProductsF 2451 Guide for in vivo Assessment of Implantable De-vices Intended to Repair or Regenerate Articular Cartilage2.2 Other Documents:ISO 10993 Biological
12、Evaluation of Medical DevicesPart5: Tests for in vitro Cytotoxicity321 CFR Part 58 Good Laboratory Practice for NonclinicalLaboratory Studies421 CFR 610.12 General Biological Products StandardsSterility43. Terminology3.1 Definitions:3.1.1 bone regenerationthe formation of bone that hashistologic, bi
13、ochemical, and mechanical properties similar tothat of native bone.3.1.2 bone repairthe process of healing injured bonethrough cell proliferation and synthesis of new extracellularmatrix.1This guide is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is t
14、he direct responsibility of SubcommitteeF04.44 on Assessment for TEMPs.Current edition approved June 1, 2009. Published June 2009. Originallyapproved in 2008. Last previous version approved in 2008 as F 2721 08.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cust
15、omer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.4Available from U.S. Go
16、vernment Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.3 compact boneclassification of ossified
17、bony connec-tive tissue characterized by the presence of osteon-containinglamellar bone. Lamellar bone is highly organized in concentricsheets.3.1.4 cortical boneone of the two main types of osseoustissue. Cortical bone is dense and forms the surface of bones.3.1.5 critical size defecta bone defect,
18、 either naturallyoccurring or artificially created, which will not heal withoutintervention. In the clinical setting, this term applies toexceeding a healing period of approximately 6 months (inotherwise healthy adults).3.1.6 diaphysealpertaining to the mid-section of longbones.3.1.7 endochondral os
19、sificationone of the two main typesof bone formation, where a cartilaginous matrix forms first andis subsequently replaced by osseous tissue.3.1.7.1 DiscussionEndochondral ossification is respon-sible for much of the bone growth in vertebrate skeletons,especially in long bones.3.1.7.2 DiscussionThe
20、other main mechanism for boneformation is intramembraneous ossification, where osseoustissue is formed directly, without cartilaginous precursor;occurs mainly in the formation of flat bones (skull).3.1.8 growth platethe anatomic location within the epi-physeal region of long bones corresponding to t
21、he site ofgrowth of bone through endochondral ossification.3.1.8.1 DiscussionThe growth plate in skeletally matureanimals is fused.3.1.9 long bonebone that is longer than it is wide, andgrows primarily by elongation of the diaphysis. The long bonesinclude the femurs, tibias, and fibulas of the legs,
22、 the humeri,radii, and ulnas of the arms, the metacarpals and metatarsals ofthe hands and feet, and the phalanges of the fingers and toes.3.1.10 marrowsoft, gelatinous tissue that fills the cavitiesof the bones. It is either red or yellow, depending upon thepreponderance of hematopoietic (red) or fa
23、tty (yellow) tissue.3.1.10.1 DiscussionRed marrow is also called myeloidtissue.3.1.11 matrixa term applied to either the exogenousimplanted scaffold or the endogenous extracelluar substance(otherwise known as extracellular matrix) derived from thehost.3.1.12 metaphysealpertaining to the dense end-se
24、ction oflong bones.3.1.13 remodelinga life long process where old bone isremoved from the skeleton (bone resorption) and new bone isadded (bone formation).3.1.14 residence timethe time at which an implantedmaterial (synthetic or natural) can no longer be detected in thehost tissue.3.1.15 skeletal ma
25、turitythe age at which the epiphysealplates are fused.3.1.15.1 DiscussionIn rodents, skeletally mature animalsare characterized by defined gonads.3.1.16 trabecular boneossified bony connective tissuecharacterized by spicules surrounded by marrow space.3.1.17 weight-bearing versus non-weight bearing
26、modelsweight bearing is the amount of weight a patient or experimen-tal animal puts on the leg on which surgery has been per-formed, generally described as a percentage of the bodyweight.3.1.17.1 DiscussionNon weight bearing means the legmust not touch the floor (i.e., supports 0 % of the body weigh
27、t).3.1.17.2 DiscussionFull weight bearing means the leg cancarry 100 % of the body weight on a step.4. Significance and Use4.1 This guide is aimed at providing a range of in vivomodels to aid in preclinical research and development oftissue-engineered medical products (TEMPs) intended for theclinica
28、l repair or regeneration of bone.4.2 This guide includes a description of the animal models,surgical considerations, and tissue processing as well as thequalitative and quantitative analysis of tissue specimens.4.3 The user is encouraged to use appropriate ASTM andother guidelines to conduct cytotox
29、icity and biocompatibilitytests on materials, TEMPs, or both, prior to assessment of thein vivo models described herein.4.4 It is recommended that safety testing be in accordancewith the provisions of the FDA Good Laboratory PracticesRegulations 21 CFR 58.4.5 Safety and effectiveness studies to supp
30、ort regulatorysubmissions (for example, Investigational Device Exemption(IDE), Premarket Approval (PMA), 510K, InvestigationalNew Drug (IND), or Biologics License Application (BLA)submissions in the U.S.) should conform to appropriate guide-lines of the regulatory bodies for development of medicalde
31、vices, biologics, or drugs, respectively.4.6 Animal model outcomes are not necessarily predictiveof human results and should, therefore, be interpreted cau-tiously with respect to potential applicability to human condi-tions.5. Animal ModelsNOTE 1This section provides a description of the options to
32、 considerin determining the appropriate animal model and bone defect size andlocation.NOTE 2Research using these models needs to be conducted inaccordance with governmental regulations and guidelines appropriate tothe locale for the care and use of laboratory animals. Study protocolsshould be develo
33、ped after consultation with the institutional attendingveterinarian, and need appropriate review and approval by the institutionalanimal care and use committee prior to study initiation.5.1 Defect Size:5.1.1 A high proportion of fracture injuries in humans occurin long bones. Accordingly, defects cr
34、eated in long bones arecommonly used for assessing bone repair/regeneration inanimal models.5.1.2 In principle, critical-size defects may be achieved inboth metaphyseal and diaphyseal locations. For the purpose ofthis guide, only defects created in the diaphyseal section oflong bones will be describ
35、ed.5.1.3 Significant variability exists between animal specieswith respect to the size and weight of the animal, anatomy, andgait thereby influencing kinetics, range of motion, and me-chanical forces on defects. These factors influence boneF2721092architecture and structure. These factors play a sig
36、nificant rolein the response to injury or disease of bone. The user shouldconsider carefully the animal model that is appropriate for thestage of investigation of an implanted TEMP.5.1.4 Mechanical load has been shown to affect bone repair.Amongst the mechanobiological factors, intermittent hydro-st
37、atic pressure and load-bearing stresses play an important rolein modulating bone development and maintenance, as well asbone degeneration The impact of mechanical load extent orduration on the implanted TEMP, and surrounding native bone,varies depending on the anatomic site. The defect site chosento
38、 evaluate implants should, therefore, factor the impact ofmechanical load on the performance of the implant.5.1.5 It is recommended that an appropriate species andanatomic site be chosen, that have dimensions sufficiently largeto adequately investigate and optimize the formulation, design,dimensions
39、, and associated instrumentation envisaged for hu-man use, especially in late stages of development.5.1.6 Larger animals may be more appropriate for studyingrepair in defects and locations that more closely approximatethose in humans.5.1.7 Larger defect dimensions generally require a methodof fixati
40、on to secure the implant and thereby reduce implantdislocation. The method of implant immobilization can nega-tively impact both the surrounding host tissue and repair tissue.Accordingly, the difference in the design of the test TEMP inmodels which generally do not require fixation should befactored
41、 into the interpretation of results with respect topredictability of outcomes in larger animal models and humansrequiring fixation.5.1.8 For each species, a critical size defect is defined as theminimum defect dimension that the animal is incapable ofrepairing without intervention. The dimensions of
42、 criticaldefects generally differ for each species and should be consid-ered carefully when designing the implant dimensions andmethod of fixation. As an empirical rule, the length of thedefect (created by ostectomy) should at least be equal to 1.5times the diameter of the selected bone (1, 2).5Some
43、 authorsrecommend at least 2 times the diameter of the selected bone(3).5.1.9 Whether or not the periosteum from the resectedsegment of bone is still present can influence healing within thebone defect. The periosteum is typically removed in moststudies of segmental critical-size defects. Whether or
44、 not theperiosteum has been removed should be stated when reportingresults.5.1.10 Each study should include an empty-defect controlgroup to confirm that the model is a critical-size defect. If/oncethe model is very well characterized, the use of historical datainstead of actual control animals shoul
45、d be considered, in orderto save on animal numbers, unless this would compromise theobjectives of the study. For example, in pivotal preclinicalproof-of-concept studies, concurrent controls are likely to beappropriate.5.1.11 The use of unilateral defect models is generallyrecommended. This is especi
46、ally true for weight-bearing loca-tions in animals that use all four limbs for weight bearing(especially goats, sheep, and horses).5.2 Handling:5.2.1 Exposure of implants to extreme and highly variablemechanical forces as a result of jumping and running can leadto increased variability in outcome me
47、asures.5.2.2 Potential differences in outcome when using weight-bearing versus non-weight bearing models should be carefullyconsidered.5.3 Chromosomal Sex:5.3.1 Due to the impact of circulating steroids on cartilageand bone metabolism and regeneration, the choice of chromo-somal sex should be consid
48、ered. Animals in lactation shouldnot be used. For some purposes, the use of aged or ovariecto-mized females (especially rats) may be indicated to simulateosteoporotic conditions.5.3.2 It is recommended that the chromosomal sex be thesame within the cohort, and that needs to be reported. Theinvestiga
49、tor should be aware that variances can occur betweensexes and that appropriate statistical power needs to beinstituted.5.4 Age:5.4.1 Bone undergoes dynamic changes in metabolism andremodeling during growth. Due to the impact of these physi-ologic processes on tissue repair, skeletally mature animalsshould be used. The cohorts should have fused epiphysealgrowth plates. Skeletal maturity varies between species andcan be determined radiographically if necessary.5.4.2 Older animals have a greater propensity for osteopeniaand have a decreased capacity to re
