1、Designation: F2721 09 (Reapproved 2014)Standard Guide forPre-clinical in vivo Evaluation in Critical Size SegmentalBone Defects1This standard is issued under the fixed designation F2721; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisi
2、on, the year of 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 (
3、TEMPs)intended to 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 m
4、odelsdescribed in 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 variousa
5、nimal models including 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 i
6、s not intended to 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 i
7、s consistent with 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
8、.6 The values stated 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
9、-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F561 Practice for Retrieval and Analysis of MedicalDevices, and Associated Tissues and FluidsF565 Practice for Care and Handling of Orthopedic Implan
10、tsand InstrumentsF895 Test Method forAgar Diffusion Cell Culture Screeningfor CytotoxicityF981 Practice for Assessment of Compatibility of Biomate-rials for Surgical Implants with Respect to Effect ofMaterials on Muscle and BoneF1983 Practice for Assessment of Selected Tissue Effects ofAbsorbable Bi
11、omaterials for Implant ApplicationsF2150 Guide for Characterization and Testing of Biomate-rial Scaffolds Used in Tissue-Engineered Medical Prod-ucts2.2 Other Documents:21 CFR Part 58 Good Laboratory Practice for NonclinicalLaboratory Studies321 CFR 610.12 General Biological Products StandardsSteril
12、ity33. Terminology3.1 Definitions:3.1.1 bone regenerationthe formation of bone that hashistologic, biochemical, 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
13、is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.44 on Assessment for TEMPs.Current edition approved Nov. 1, 2014. Published March 2015. Originallyapproved in 2008. Last previous version approved in 2009
14、as F2721 09. DOI:10.1520/F2721-09R14.2For 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 standards Document Summary page onthe ASTM website.3Available from U.S. Gov
15、ernment Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.3 compact boneclassification of ossified bo
16、ny 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, e
17、ither 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 ossi
18、ficationone 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 ot
19、her 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 theepiphyseal region of long bones corresponding to the s
20、ite 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, the
21、 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 fatty
22、(yellow) tissue.3.1.10.1 DiscussionRed marrow is also called myeloidtissue.3.1.11 matrixeither the exogenous implanted scaffold orthe endogenous extracelluar substance (otherwise known asextracellular matrix) derived from the host.3.1.12 metaphysealpertaining to the dense end-section oflong bones.3.
23、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 maturitythe age at whic
24、h 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 modelsweight bearing
25、is the amount of weight a patient or experimen-tal animal puts on the leg on which surgery has beenperformed, 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 weight).3.1.17.2 DiscussionF
26、ull 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 theclinical repair or regeneratio
27、n 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 cytotoxicity and biocompatibil
28、itytests 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 support regulatorysubmissio
29、ns (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 medicaldevices, biologics, or dr
30、ugs, 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 considerin determining
31、 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 developed after consultation
32、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 created in long bones are
33、commonly 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 described.5.1.3 Significant va
34、riability exists between animal specieswith respect to the size and weight of the animal, anatomy, andF2721 09 (2014)2gait thereby influencing kinetics, range of motion, and me-chanical forces on defects. These factors influence bonearchitecture and structure. These factors play a significant rolein
35、 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-static pressure a
36、nd 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 evaluate impla
37、nts 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, and associate
38、d 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 fixation to secure th
39、e 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 into the inter
40、pretation 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 criticaldefect
41、s 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).4Some authorsrecomme
42、nd 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 not theperiost
43、eum 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 should be considered
44、, 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 especially true for w
45、eight-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 measures.5.2.2 Po
46、tential 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 considered. Animals i
47、n 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. Theinvestigator should be a
48、ware 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.
49、 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 repair bone defects. If specificconditions are considered important for the intended TEMPassessment, then an appropriate model should be used.5.4.3 The mesenchymal stem cell pool, growth factorresponsiveness, and metabolic activity of cells generally de-creases with age. Thus, reparative processes that are dependento
