F04.44 - Assessment for TEMPs

SIGNIFICANCE AND USE
4.1 This guide is aimed at providing a range of  in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone.  
4.2 This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.  
4.3 The user is encouraged to use appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.  
4.4 It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.  
4.5 Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs, respectively.  
4.6 Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone. TEMPs included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this guide are segmental critical size defects which, by definition, will not fill with viable tissue without treatment. Thus, these models represent a stringent test of a material’s ability to induce or augment bone growth.  
1.2 Guidelines include a description and rationale of various animal models including rat (murine), rabbit (leporine), dog (canine), goat (caprine), and sheep (ovine). Outcome measures based on radiographic, histologic, and mechanical analyses are described briefly and referenced. The user should refer to specific test methods for additional detail.  
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in the Referenced Documents (Section 2).  
1.4 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.  
1.5 Other preclinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 A common therapy to mitigate the pathological effects of blood vessel occlusion or aneurysm-related vascular wall weakening is to reroute blood flow around the diseased vascular regions. Autologous and non-autologous grafts are often used as vascular substitutes surgically to achieve this therapeutic intervention. Vascular graft TEMPs may also be used for these purposes. They may also be used to create or revise arteriovenous shunts.  
4.2 Coronary, carotid, renal, common iliac, external iliac, superficial femoral, and popliteal arteries are examples of vascular sites commonly requiring bypass surgery.  
4.3 TEMPs may be composed of biological products (for example, cells, organs, and tissues), biomaterials (for example, substrates and scaffolds composed of polymers or collagen), biomolecules (for example, recombinant proteins, native/biological proteins, amino acids, peptides, fatty acids, sugars, and other macromolecules), and various combinations thereof (see Terminology F2312). Examples of TEMPs are listed in Classification F2211.  
4.4 TEMPs may be used with the intent of facilitating the surgical outcome by improving the biological repair and/or reconstruction, by accommodating the mechanical loads at the repair site, or by a combination of these mechanisms.  
4.5 Clinical evidence of improved surgical outcomes may include patency, reduced incidence of revision surgery, reduced rate of implant infection, and improved functionality after surgery.
SCOPE
1.1 This guide is intended as a resource for individuals and organizations involved in the development, production, delivery, and regulation of tissue engineered medical products (TEMPs) intended for use in the surgical repair, replacement, shunting, and/or bypass of blood vessels. This guide is intended for use related to the in vitro assessment of TEMP vascular grafts. In vitro cellular characterization and in vivo testing are not within scope for this standard guide.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guide is aimed at providing a range of in vivo models to aid in pre-clinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone in the spine.  
4.2 This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.  
4.3 The user is encouraged to utilize appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.  
4.4 It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.  
4.5 Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs.  
4.6 Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the pre-clinical in vivo assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone in an interbody and/or posterolateral spinal environment. TEMPs included in this guide may be composed of, but are not limited to, natural or synthetic biomaterials or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this document represent a stringent test of a material’s ability to induce and/or augment bone growth in the spinal environment.  
1.2 While clinically TEMPs may be combined with hardware for initial stabilization or other purposes, the focus of this guide is on the appropriateness of the animal model chosen and evaluation of the TEMP-induced repair and as such does not focus on issues of components or constructs.  
1.3 Guidelines include a description and rationale of various animal models for the in vivo assessment of the TEMP. The animal models utilize a range of species including rat (murine), rabbit (lapine), dog (canine), goat (caprine), pig (porcine), sheep (ovine), and non-human primate (primates). Outcome measures include in vivo assessments based on radiographic, histologic, and CT imaging as well as subsequent in vitro assessments of the repair, including histologic analyses and mechanical testing. All methods are described briefly and referenced. The user should refer to specific test methods for additional detail.  
1.4 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in Referenced Documents (Section 2).  
1.5 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.  
1.6 Other pre-clinical methods may also be appropriate, and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.  
1.9 This international standard was developed in accordance with internationally recognized prin...

SIGNIFICANCE AND USE
4.1 This guide covers animal implantation methods and analysis of the explanted DBM-containing material to determine whether a material or substance possesses osteoinductive potential, as defined by its ability to cause bone to form in vivo at a site that would otherwise not support bone formation, that is, heterotopically in a skeletal muscle implant site. For in vitro evaluation see Test Method F2131 for in vitro assessment of rhBMP 2.  
4.2 The test methods described here may be suitable for defining product specifications, cGMP lot release testing, research evaluation, regulatory submission, and so forth, but a positive outcome should not be presumed to indicate that the product will be osteoinductive in a human clinical application. At present, the only direct assays to assess new bone formation are in vivo, since the property of bone conduction or induction can only be assessed in a heterotopic or orthotopic site in a living animal. When these products are implanted in an orthotopic site, osteogenic factors already present at the implantation site may contribute to and enhance bone formation in conjunction with the osteoconductive nature of the product. Thus, orthotopic implantation of products may result in bone formation by acting on existing bone-forming cells and not by causing mesenchymal stem cells to become osteochondroprogenitor cells. In contrast, when these products are implanted in a heterotopic site, no native osteogenic factors are present to contribute to or enhance bone formation. Thus, heterotopic implantation of products will only result in new bone formation by causing mesenchymal stem cells to become osteochondroprogenitor cells. In vitro assays have been described and some believe they may correlate to the results obtained from in vivo assays. However such in vitro assays measure only some of the biochemical marker(s) associated with in vivo bone formation and are therefore only indirect assays for osteoinductive activity or the capacity t...
SCOPE
1.1 This guide covers general guidelines to evaluate the effectiveness of DBM-containing products intended to cause and/or promote bone formation when implanted or injected in vivo. This guide is applicable to products that may be composed of one or more of the following components: natural biomaterials (such as demineralized bone), and synthetic biomaterials (such as calcium sulfate, glycerol, and reverse phase polymeric compounds) that act as additives, fillers, and/or excipients (radioprotective agents, preservatives, and/or handling agents) to make the demineralized bone easier to manipulate. It should not be assumed that products evaluated favorably using this guidance will form bone when used in a clinical setting. The primary purpose of this guide is to facilitate the equitable comparison of unique bone-forming products in in vivo heterotopic models of osteoinductivity. The purpose of this guide is not to exclude other established methods.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with the use of DBM-containing bone-forming/promoting products. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices involved in the development of said products in accordance with applicable regulatory guidance documents and in implementing this guide to evaluate the bone-forming/promoting capabilities of the product.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Cell Therapy Products may be used to treat clinical conditions, for example in regenerative medicine (e.g. type I diabetes, acute myocardial infarction, pediatric congenital heart disease, chronic ischemic heart failure, cancer, Crohn’s disease, chronic wound repair, nerve and spinal cord injury, musculoskeletal repair), and may be used for immunotherapy (e.g. graft versus host disease, CAR-T therapy).  
5.2 Autologous, allogeneic, and xenogeneic cells may be used to make a product.  
5.3 A product may be cells only, cells combined with an inert carrier, cells within an extracellular matrix, or cells within a synthetic scaffold, and will include tissue engineered medical products containing cells.  
5.4 Cells may be gene-modified cells.  
5.5 Cells may be adult or embryonic stem cells.  
5.6 Cells may be minimally manipulated.
SCOPE
1.1 This guide is intended as a resource for individuals and organizations involved in the development, production, delivery, and regulation of cellular therapy products (CTPs) including genetically modified cells, tissue engineered medical products (TEMPs) and combination products where cell activity is a functional component of the final product.  
1.2 This Guide was developed to include input derived from several previously published guidance documents and standards (section 2.4). It is the intent of this Guide is to reflect the current perspectives for CTP potency assays.  
1.3 CTPs can provide therapy by localized or systemic treatment of a disease or pathology.  
1.4 The products may provide a relatively short therapy, may be transient, or may be permanent and provide long-term therapy.  
1.5 The products may be cells alone, cells combined with a carrier that is transient, or cells combined with a scaffold or other components that function in the overall therapy.  
1.6 Potency assays may be in-vitro or in-vivo assays designed to determine the potency of a specific product. In-vivo assays are likely to be particularly useful to study the mechanism of action (MOA) of the therapy, but may not be desirable for final product quality control where they may be time-consuming and expensive, and where in-vitro assays may be preferable.  
1.7 It is likely that multiple assays, and possibly both in-vitro and in-vivo assays, will be required to provide a broad measure of potency. However, in-vitro assays are likely to be preferred as release assays for products, and so studies to identify potency assays should emphasize in-vitro assays that are correlative or predictive of preclinical or clinical results.  
1.8 Potency assays should be developed during the product development cycle and therefore are likely to be more comprehensive at the end of that cycle compared to the beginning of product development and testing. It is recommended that potency assays be developed as early as possible in the product development cycle (Figs. 1 and 2).
FIG. 1 Progressive Implementation of Potency Assays
FIG. 2 Flow Chart for Stages in Product Development Showing When Potency Assays Will Be Developed and Introduced  
1.9 Potency measurements are used as part of the testing for cell and cell-based products to demonstrate that product lots meet defined specifications when released for clinical use.  
1.10 Shelf life specifications should be developed during the product development process to include potency measurements.  
1.11 This standard guide is not intended to apply to drug or gene therapy products. However, genetically modified cell therapies, for example the chimeric antigen receptor-T (CAR-T) cell therapy, which the United States FDA classifies as gene therapy, are applicable.  
1.12 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limit...

SIGNIFICANCE AND USE
4.1 Tissue-engineered cartilage is prepared by seeding stem cells or chondrocytes in a three-dimensional biodegradable scaffold under controlled growth conditions. It is expected that the cells will differentiate towards chondrogenic lineage and produce an ample amount of cartilage extracellular matrix proteins, proteoglycans, and collagen type-II. Longitudinal assessment is needed weekly for the first few weeks in vitro and monthly at a later stage in vivo to determine the growth rate of tissue-engineered cartilage. Traditional testing methods such as histological staining, mechanical testing, and qPCR are invasive, destructive, and cannot be performed in vivo after the transplantation of engineered tissue as a regenerative treatment. In the regenerative medicine of cartilage, it is important to evaluate whether the implanted tissue regenerates as an articular cartilage over time. MRI is the only available non-invasive imaging modality that is utilized for post-operative monitoring and assessment of cartilage regeneration in clinics. Therefore, it is important to evaluate tissue-engineered cartilage using MRI at the preclinical stage as well.  
4.7.1 The change in calculated relaxation rate, R2(ECM), using Eq 1 have been found to be positively correlated with tissue growth (3, 6).
SCOPE
1.1 This standard is intended as a standard test method for engineered cartilage tissue growth evaluation using MRI.  
1.2 This standard is intended for use in the development of tissue engineering regenerative medical products for cartilage damages, such as in knee, hip, or shoulder joints.  
1.3 This standard has been prepared for evaluation of engineered cartilage tissue growth at the preclinical stage and summarizes results from tissue growth evaluation of tissue-engineered cartilage in a few notable cases using water spin-spin relaxation time, T2, in vitro and in vivo in small animal models.  
1.4 This standard uses the change in mean T2 values as a function of growth time to evaluate the tissue growth of engineered cartilage.  
1.5 This standard provides a method to remove the scaffold contribution to the tissue growth evaluation.  
1.6 Information in this standard is intended to be applicable to most porous natural and synthetic polymers used as a scaffold in engineered cartilage, such as alginate, agarose, collagen, chitosan, and poly-lactic-co-glycolic acid (PLGA). However, some materials (both synthetic and natural) may require unique or varied methods of MRI evaluation that are not covered in this test method.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guide covers animal implantation methods and analysis of bone void fillers to determine whether a material or substance leads to lumbar intertransverse process spinal fusion, as defined by its ability to cause bone to form in vivo.
SCOPE
1.1 Historically, the single-level rabbit posterolateral, or intertransverse, lumbar spine fusion model was developed and reported on by Dr. Scott Boden, et. al. (Emory Spine Center for Orthopedics) and the model has been proposed as a non-clinical model which may be used to replicate clinically-relevant fusion rates for iliac crest autograft in the posterolateral spine (1, 2).2 This model is used routinely in submissions to regulatory bodies for the purpose of evaluating the potential efficacy of bone void filler materials as compared to other materials or iliac crest autograft to effect spinal posterolateral fusion. The use of this standard’s recommendations as part of a regulatory submission does not provide any guarantee of regulatory clearance and should be considered as a part of the data provided for regulatory submission.  
1.2 This guide covers general guidelines to evaluate the effectiveness of products intended to cause and/or promote bone formation in the lumbar intertransverse process spinal fusion model in vivo. This guide is applicable to products that may be composed of one or more of the following components: natural biomaterials (such as demineralized bone), and synthetic biomaterials (such as calcium sulfate, glycerol, and reverse phase polymeric compounds) that act as additives, fillers, and/or excipients (radioprotective agents, preservatives, and/or handling agents). It should not be assumed that products evaluated favorably using this guidance will form bone when used in a clinical setting. The primary purpose of this guide is to facilitate the equitable comparison of bone void fillers and/or autograft extender products in vivo. The purpose of this guide is not to exclude other established methods.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with the use of bone void fillers. It is the responsibility of the user of this standard to establish appropriate safety and health practices involved in the development of said products in accordance with applicable regulatory guidance documents and in implementing this guide to evaluate the bone-forming/promoting capabilities of the product.  
1.5 This standard does not purport to address the requirements under 21 CFR Part 58 concerning Good Laboratory Practices or international standard counterpart OECD Principles of Good Laboratory Practice (GLP). It is the responsibility of the sponsor of the study to understand the requirements for conduct of animal studies whereby the data may be used to support premarket applications, including requirements for personnel, protocol content, record retention and animal husbandry.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Injuries to the knee meniscus are one of the most common orthopaedic problems. Meniscus injures include acute tears (such as occur in sports injuries), chronic degenerative tears, extrusion/subluxation, and/or degenerative dysfunction that occurs as part of the knee aging process or as a result of multiple meniscus surgeries. Knee arthroscopy for partial excision of the knee meniscus (partial meniscectomy) is the most commonly performed orthopaedic procedure.  
5.2 Complete or near complete excision of the meniscus in a young individual is associated with an early increased risk of knee osteoarthritis due to the loss of the meniscus chondroprotective effects. Lateral meniscal injuries tend to be more severe than medial injuries. Meniscus repair, augmentation, transplantation, and/or reconstruction is recommended in individuals to restore the chondroprotective effect of the meniscus, relieve pain, and prevent degenerative knee osteoarthritis. The potential of TEMPs to enhance the outcome of the surgical meniscus repair and/or reconstruction has been recognized.  
5.3 The knee joint and temporomandibular joint (TMJ) are examples of joints with meniscal structures.  
5.4 TEMPS may be used with the intent of enhancing the surgical outcome by improving the biological repair at the site of implantation, by providing mechanical function at a defect site, or by a combination of these mechanisms.  
5.5 Improving surgical outcome may include improving function relative to the pre-operative condition, shortening the recovery time after surgery, relieving pain, enabling return to normal daily activities, encouraging tissue growth into the defect site, restoring the mechanical function of the meniscus, delaying the progression of osteoarthritis, or any combination thereof.
SCOPE
1.1 This guide is intended as a resource for individuals and organizations involved in the production, delivery, and regulation of tissue engineered medical products (TEMPs) and other tissues intended for use in the surgical repair, replacement, and/or reconstruction of the knee meniscus.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 A common therapy to mitigate the pathological effects of blood vessel occlusion or aneurysm-related vascular wall weakening is to reroute blood flow around the diseased vascular regions. Autologous and non-autologous grafts are often used as vascular substitutes surgically to achieve this therapeutic intervention. Vascular graft TEMPs may also be used for these purposes. They may also be used to create or revise arteriovenous shunts.  
4.2 Coronary, carotid, renal, common iliac, external iliac, superficial femoral, and popliteal arteries are examples of vascular sites commonly requiring bypass surgery.  
4.3 TEMPs may be composed of biological products (for example, cells, organs, and tissues), biomaterials (for example, substrates and scaffolds composed of polymers or collagen), biomolecules (for example, recombinant proteins, native/biological proteins, amino acids, peptides, fatty acids, sugars and other macromolecules) and various combinations thereof (see Terminology F2312). Examples of TEMPs are listed in Classification F2211.  
4.4 TEMPs may be used with the intent of facilitating the surgical outcome by improving the biological repair and/or reconstruction, by accommodating the mechanical loads at the repair site, or by a combination of these mechanisms.  
4.5 Clinical evidence of improved surgical outcomes may include patency, reduced incidence of revision surgery, reduced rate of implant infection, and improved functionality after surgery.
SCOPE
1.1 This guide is intended as a resource for individuals and organizations involved in the development, production, delivery, and regulation of tissue engineered medical products (TEMPs) intended for use in the surgical repair, replacement, shunting and/or bypass of blood vessels. This guide is intended for use related to the in vitro assessment of TEMP vascular grafts. In vitro cellular characterization and in vivo testing are not within scope for this standard guide.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guide is aimed at providing a range of  in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone.  
4.2 This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.  
4.3 The user is encouraged to use appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.  
4.4 It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.  
4.5 Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE)), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs, respectively.  
4.6 Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the  in vivo assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone. TEMPs included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this guide are segmental critical size defects which, by definition, will not fill with viable tissue without treatment. Thus, these models represent a stringent test of a material’s ability to induce or augment bone growth.  
1.2 Guidelines include a description and rationale of various animal models including rat (murine), rabbit (leporine), dog (canine), goat (caprine), and sheep (ovine). Outcome measures based on radiographic, histologic, and mechanical analyses are described briefly and referenced. The user should refer to specific test methods for additional detail.  
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in the Referenced Documents (Section 2).  
1.4 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.  
1.5 Other pre-clinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This guide covers animal implantation methods and analysis of the explanted DBM-containing material to determine whether a material or substance possesses osteoinductive potential, as defined by its ability to cause bone to form in vivo at a site that would otherwise not support bone formation, that is, heterotopically in a skeletal muscle implant site. For in vitro evaluation see Test Method F2131 for in vitro assessment of rhBMP 2.  
4.2 The test methods described here may be suitable for defining product specifications, cGMP lot release testing, research evaluation, regulatory submission, and so forth, but a positive outcome should not be presumed to indicate that the product will be osteoinductive in a human clinical application. At present, the only direct assays to assess new bone formation are in vivo, since the property of bone conduction or induction can only be assessed in a heterotopic or orthotopic site in a living animal. When these products are implanted in an orthotopic site, osteogenic factors already present at the implantation site may contribute to and enhance bone formation in conjunction with the osteoconductive nature of the product. Thus, orthotopic implantation of products may result in bone formation by acting on existing bone forming cells and not by causing mesenchymal stem cells to become osteochondroprogenitor cells. In contrast, when these products are implanted in a heterotopic site, no native osteogenic factors are present to contribute to or enhance bone formation. Thus, heterotopic implantation of products will only result in new bone formation by causing mesenchymal stem cells to become osteochondroprogenitor cells. In vitro assays have been described and some believe they may correlate to the results obtained from in vivo assays. However such in vitro assays measure only some of the biochemical marker(s) associated with in vivo bone formation and are therefore only indirect assays for osteoinductive activity or the capacity t...
SCOPE
1.1 This guide covers general guidelines to evaluate the effectiveness of DBM-containing products intended to cause and/or promote bone formation when implanted or injected in vivo. This guide is applicable to products that may be composed of one or more of the following components: natural biomaterials (such as demineralized bone), and synthetic biomaterials (such as calcium sulfate, glycerol, and reverse phase polymeric compounds) that act as additives, fillers, and/or excipients (radioprotective agents, preservatives, and/or handling agents) to make the demineralized bone easier to manipulate. It should not be assumed that products evaluated favorably using this guidance will form bone when used in a clinical setting. The primary purpose of this guide is to facilitate the equitable comparison of unique bone-forming products in in vivo heterotopic models of osteoinductivity. The purpose of this guide is not to exclude other established methods.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with the use of DBM-containing bone-forming/promoting products. It is the responsibility of the user of this standard to establish appropriate safety and health practices involved in the development of said products in accordance with applicable regulatory guidance documents and in implementing this guide to evaluate the bone-forming/promoting capabilities of the product.

SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone in the spine.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to utilize appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs.
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the pre-clinical in vivo assessment of tissue-engineered medical products (TEMPs) intended to repair or regenerate bone in an interbody and/or posterolateral spinal environment. TEMPs included in this guide may be composed of, but are not limited to, natural or synthetic biomaterials or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this document represent a stringent test of a material’s ability to induce and/or augment bone growth in the spinal environment.
1.2 While clinically TEMPs may be combined with hardware for initial stabilization or other purposes, the focus of this guide is on the appropriateness of the animal model chosen and evaluation of the TEMP induced repair and as such does not focus on issues of hardware.
1.3 Guidelines include a description and rationale of various animal models for the in vivo assessment of the TEMP. The animal models utilize a range of species including rat (murine), rabbit (lapine), dog (canine), goat (caprine), pig (porcine), sheep (ovine), and non-human primate (primates). Outcome measures include in vivo assessments based on radiographic, histologic, CT imaging as well as subsequent in vitro assessments of the repair, including histologic analyses and mechanical testing. All methods are described briefly and referenced. The user should refer to specific test methods for additional detail.
1.4 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in Referenced Documents (Section 2).
1.5 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.
1.6 Other pre-clinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.

SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to use appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE)), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs, respectively.  
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of tissue engineered medical products (TEMPs) intended to repair or regenerate bone. TEMPs included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this guide are segmental critical size defects which, by definition, will not fill with viable tissue without treatment. Thus, these models represent a stringent test of a material’s ability to induce or augment bone growth.
1.2 Guidelines include a description and rationale of various animal models including rat (murine), rabbit (leporine), dog (canine), goat (caprine), and sheep (ovine). Outcome measures based on radiographic, histologic, and mechanical analyses are described briefly and referenced. The user should refer to specific test methods for additional detail.
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in the Referenced Documents (Section 2).
1.4 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.
1.5 Other pre-clinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue-engineered medical products (TEMPs) intended for the clinical repair or regeneration of bone.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to use appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials, TEMPs, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and effectiveness studies to support regulatory submissions (for example, Investigational Device Exemption (IDE)), Premarket Approval (PMA), 510K, Investigational New Drug (IND), or Biologics License Application (BLA) submissions in the U.S.) should conform to appropriate guidelines of the regulatory bodies for development of medical devices, biologics, or drugs, respectively.  
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of tissue engineered medical products (TEMPs) intended to repair or regenerate bone. TEMPs included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof, and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA. The models described in this guide are segmental critical size defects which, by definition, will not fill with viable tissue without treatment. Thus, these models represent a stringent test of a material’s ability to induce or augment bone growth.
1.2 Guidelines include a description and rationale of various animal models including rat (murine), rabbit (leporine), dog (canine), goat (caprine), and sheep (ovine). Outcome measures based on radiographic, histologic, and mechanical analyses are described briefly and referenced. The user should refer to specific test methods for additional detail.
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of the product. ASTM standards for these steps are available in the Referenced Documents (Section 2).
1.4 The use of any of the methods included in this guide may not produce a result that is consistent with clinical performance in one or more specific applications.
1.5 Other pre-clinical methods may also be appropriate and this guide is not meant to exclude such methods. The material must be suitable for its intended purpose. Additional biological testing in this regard would be required.
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue engineered medical products intended for the clinical repair or regeneration of articular cartilage.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to utilize appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials or devices, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and Effectiveness studies to support IDE (Investigational Device Exemption), PMA (Premarket Approval), or 510K submissions should conform to appropriate FDA guidelines for development of medical devices.
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of implantable devices intended to repair or regenerate articular cartilage. Devices included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA.
1.2 Guidelines include a description and rationale of various animal models utilizing a range of species such as rabbit (lupine), dog (canine), pig (porcine), goat (caprine), sheep (ovine), and horse (equine). Outcome measures based on histologic, biochemical, and mechanical analyses are briefly described and referenced. The user should refer to specific test methods for additional detail.
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of product. ASTM standards for these steps are available in Reference Documents.
1.4 The values stated in SI units are to be regarded as the standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

SIGNIFICANCE AND USE
Injuries to tendons or ligaments are frequently treated by surgery to repair the damaged tissues and facilitate the healing process. The potential of TEMPs to enhance the outcomes (including function, pain, anatomy) of the surgical repair has been recognized.
Examples of tissues that when injured may be appropriate for repair using TEMPs: rotator cuff with a partial or full tear; Achilles tendon; Achilles tendon after harvesting for anterior cruciate ligament repair; patella tendon; patella tendon after harvesting for anterior cruciate ligament repair; quadriceps tendon; posterior cruciate ligament; medial collateral ligaments; lateral collateral ligaments; flexor tendons.  
TEMPs may be used with the intent to improve the surgical outcome of tendon or ligament repair by (a) assuming some of the mechanical load experienced at the repair site to stabilize the surgical repair, (b) improving the natural biological healing process, or (c) a combination of these mechanisms.  
TEMPs should improve clinical outcome. This may be accomplished by reducing or eliminating pain, returning function, shortening the recovery time following surgery, facilitating early mobility, improving return of strength, improving mobility, or other clinically relevant parameters.  
The mechanism used by TEMPs to improve surgical repair should be understood and this conclusion should be supported by experimental results and should be supportive of the primary function of the TEMP.  
TEMPs with the primary function of mechanical reinforcement may also have a secondary, biological function.  
When the product is used to improve the body’s natural biological repair process of tendons or ligaments, the product should allow cell attachment, migration, infiltration, extracellular matrix deposition and organization, formation of tendon or ligament repair tissue, integration with adjacent tendon, ligament or bone, tendon-bone attachment, or more than one of these actions.  
When the TEMP is used ...
SCOPE
1.1 This guide is intended as a resource for individuals and organizations involved in the development, production, and delivery of tissue engineered medical products (TEMPs) intended to provide a mechanical (functional) reinforcement of the surgical repair of tendons and ligaments.
1.2 Surgical repair can include procedures that repair tendon to tendon, tendon to bone, tendon to muscle, ligament to ligament, and ligament to bone. In the context of this guide, a tendon is a fibrous cord or band that connects a muscle to a bone or other structure and consists of both dense collagenous fibers and rows of elongated tendon cells. In contrast, a ligament is a band or sheet of fibrous tissue connecting two or more bones, or cartilagenous structures.
1.3 Examples of TEMPs for use in reinforcement of tendon or ligament repairs include extracellular matrices (including allograft tissue, xenograft tissue, and tissue engineered extracellular matrix), polymeric matrices, membranes, or combinations of two or more of these, with or without cells and/or molecular mediators, where the function is to reinforce the surgical repair of tendon to tendon, tendon to bone, tendon to muscle, ligament to ligament, or ligament to bone.
1.4 The products may be rapidly degrading, slowly degrading, or non-degrading.
1.5 The guide is not intended to apply to TEMPs that have a primary function to induce a biological repair through cell or molecular action, although biologic activity may be a feature of the TEMPs. Examples of products or product concepts that are not included are (a) growth factors or cytokines applied to a biologic or synthetic scaffold, and (b) platelet-enriched plasma applied to or within a biologic or polymeric scaffold, where the primary function of the product is biologic.
1.6 The guide is not intended to apply to TEMPs that have a primary function to induce a chemical repair. An example of a product or product concept t...

SIGNIFICANCE AND USE
This guide is aimed at providing a range of in vivo models to aid in preclinical research and development of tissue engineered medical products intended for the clinical repair or regeneration of articular cartilage.
This guide includes a description of the animal models, surgical considerations, and tissue processing as well as the qualitative and quantitative analysis of tissue specimens.
The user is encouraged to utilize appropriate ASTM and other guidelines to conduct cytotoxicity and biocompatibility tests on materials or devices, or both, prior to assessment of the in vivo models described herein.
It is recommended that safety testing be in accordance with the provisions of the FDA Good Laboratory Practices Regulations 21 CFR 58.
Safety and Effectiveness studies to support IDE (Investigational Device Exemption), PMA (Premarket Approval), or 510K submissions should conform to appropriate FDA guidelines for development of medical devices.
Animal model outcomes are not necessarily predictive of human results and should, therefore, be interpreted cautiously with respect to potential applicability to human conditions.
SCOPE
1.1 This guide covers general guidelines for the in vivo assessment of implantable devices intended to repair or regenerate articular cartilage. Devices included in this guide may be composed of natural or synthetic biomaterials (biocompatible and biodegradable) or composites thereof and may contain cells or biologically active agents such as growth factors, synthetic peptides, plasmids, or cDNA.
1.2 Guidelines include a description and rationale of various animal models utilizing a range of species such as rabbit (lupine), dog (canine), pig (porcine), goat (caprine), sheep (ovine), and horse (equine). Outcome measures based on histologic, biochemical, and mechanical analyses are briefly described and referenced. The user should refer to specific test methods for additional detail.
1.3 This guide is not intended to include the testing of raw materials, preparation of biomaterials, sterilization, or packaging of product. ASTM standards for these steps are available in Reference Documents.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.
WITHDRAWN RATIONALE
This guide covered general guidelines for the in vivo assessment of implantable devices intended to repair or regenerate articular cartilage.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this guide was withdrawn in January 2019 in accordance with section 10.6.3 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.