Biological evaluation of medical devices - Part 6: Tests for local effects after implantation
1 Scope
This document specifies test methods for the assessment of the local effects after implantation of biomaterials intended for use in medical devices.
This document applies to materials that are
——solid and non-solid, such as porous materials, liquids, gels, pastes, and particulates, and
——non-absorbable, and
——degradable and/or absorbable, which may be solid or non-solid.
This document can also be applied to medical devices that are intended to be used topically in clinical indications where the surface or lining might have been breached, in order to evaluate local tissue responses.
Note 1: The test sample is implanted into a site and animal species appropriate for the evaluation of the biological safety of the material. The objective of the test methods is to characterize the history and evolution of the tissue response after implantation of a medical device/biomaterial including final integration or absorption/degradation of the material. In particular for degradable/absorbable materials, the degradation characteristics of the material and the resulting tissue response should be determined. The local effects are evaluated by a comparison of the tissue response caused by a test sample to that caused by control materials used in medical devices whose clinical acceptability and biocompatibility characteristics have been established.
These implantation tests are not intended to evaluate or determine the performance of the test sample in terms of mechanical or functional loading.
This document does not deal with systemic toxicity, carcinogenicity, teratogenicity or mutagenicity.
Note 2: The long-term implantation studies intended for evaluation of local biological effects might provide insight into some information about systemic toxicity, carcinogenicity, teratogenicity or mutagenicity. Systemic toxicity studies conducted by implantation might satisfy the requirements of this document. When conducting combined studies for evaluating local effects and systemic effects, the requirements of both standards is to be fulfilled.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute indispensable provisions of this document. For dated references, only the edition cited applies. For undated references, the latest edition (including any amendments) applies.
ISO 10993-1 Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process
Note: GB/T 16886.1-2022 Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process (ISO 10993-1: 2018, IDT).
ISO 10993-2 Biological evaluation of medical devices - Part 2: Animal welfare requirements
Note: GB/T 16886.2-2011 Biological evaluation of medical devices - Part 2: Animal welfare requirements (ISO 10993-2: 2006, IDT).
ISO 10993-4 Biological evaluation of medical devices - Part 4: Selection of tests for interactions with blood
Note: GB/T 16886.4-2022 Biological evaluation of medical devices - Part 4: Selection of tests for interactions with blood (ISO 10993-4:2017, IDT)
ISO 10993-12 Biological evaluation of medical devices - Part 12: Sample preparation and reference materials
Note: GB/T 16886.12-2017 Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2012, IDT)
ISO 10993-16 Biological evaluation of medical devices - Part 16: Toxicokinetic study design for degradation products and leachables
Note: GB/T 16886.16-2021 Biological evaluation of medical devices - Part 16: Toxicokinetic study design for degradation products and leachables (ISO 10993-16:2017, IDT)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-2, ISO 10993-12, ISO 10993-16 and the following apply.
3.1
absorb/absorption
action of a non-endogenous (foreign) material or substance, or its decomposition products passing through or being assimilated by cells and/or tissue over time
3.2
degradation
decomposition of a material
[Source: ISO 10993-9:2009, 3.1]
3.3
degradation product
any intermediate or final by-product which results from the physical, metabolic, and/or chemical decomposition of a material or substance
[Source: ISO/TR 37137:2014, 2.2, modified]
3.4
degrade
to physically, metabolically, and/or chemically decompose a material or substance
[Source: ISO/TR 37137:2014, 2.3]
3.5
biomaterial
material or substance intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body
[Source: European Society Biomaterials Conference II]
4 Common provisions for implantation test methods
4.1 General
It is important that the study be planned in sufficient detail such that all relevant information can be extracted from the use of each animal and each study (see ISO 10993-2, ISO 10993-11 and ISO 10993-16)
All animal studies shall be performed in a facility approved by a nationally recognized organization and in accordance with all appropriate regulations dealing with laboratory animal welfare to comply with the requirements of ISO 10993-2. These studies shall be performed under good laboratory practices or other recognized quality assurance systems.
The provisions of this Clause shall apply to the test methods specified in Annex A, Annex B, Annex C, and Annex D.
4.2 Preparation of samples for implantation
4.2.1 Test sample and reference or control material preparation shall be in accordance with ISO 10993-12. The implant size and shape shall be documented and justified. Test samples for various implant sites are described in Annex A, Annex B, Annex C, and Annex D. Physical characteristics (such as form, density, hardness, surface) can influence the character of the tissue response to the test material and shall be recorded and taken into account when the response is characterized. Control articles should be matched as closely as reasonably possible for physical characteristics.
4.2.2 Each implant sample shall be manufactured, processed, cleaned of contaminants, and sterilized by the method intended for the final product and this shall be confirmed in the study documentation. After final preparation and sterilization, the implant samples shall be handled aseptically and in such a way as to ensure that they are not damaged or contaminated in any way prior to or during implantation.
4.2.3 For materials used as scaffolds for tissue-engineered medical products, it may be appropriate not to use the final preparation pre-populated with cells and/or proteins, as the immune reaction of the animal to the cellular/protein components of such products and the reaction of the cells to the animal may interfere with the resulting local tissue response, making it difficult to interpret.
4.2.4 For composite materials (e.g. bone cements, dental materials), the components may be mixed before use and allowed to set before implantation. For multicomponent materials designed to be cured prior to placement, the components may be mixed before use and allowed to set before implantation. However, materials that are designed to polymerize in situ (e.g. bone cements, many dental materials) shall be introduced in a manner such that in situ polymerization occurs. The procedure used shall be documented and justified.
4.2.5 Non-solid materials (including powders) may be contained in open-ended cylindrical tubes for the purpose of testing for local effects after implantation (see ISO 10993-12 for the selection of materials for tubes). Prepare the test material according to the manufacturer’s instructions and insert the material into the tube until level with the end, taking care not to contaminate the outer surface of the tube with the test material. If contamination occurs, the sample shall not be implanted. Avoid entrapment of air in the tube and ensure that the end surfaces of the inserted material in the tube and the tube ends are smooth.
Polyethylene (PE), polypropylene (PP), or polytetrafluoroethylene (PTFE) tubes are commonly used for this purpose. PE tubes can be deformed by autoclaving.
4.2.6 Evaluation shall be performed by comparing the tissue reaction to that of a similar sample/material whose clinical acceptability and biocompatibility characteristics have been established.
Note: For further guidance, see ISO 10993-12.
4.2.7 The physical characteristics such as shape, and especially the surface condition of the control(s), shall be as similar to that of the implant test samples as is practical, with any deviations being explained and justified. When the test material is contained in a tube, the control shall be of the same material as the tube and have the same diameter as the outer diameter of the tube. The choice of the control rod or tube shall be documented and justified.
4.2.8 For implantation studies, the amount or size of the test and control sample shall be documented.
4.3 Study design
For devices comprising/composed of two or more different materials, the test articles should be of similar composition or multiple implants may be needed, e.g. if a device is made of HDPE and titanium then the test article should be made of HDPE and titanium.
5 Test methods, general aspects
5.1 Tissue and implantation site
5.1.1 The test sample shall be implanted into the tissues most relevant to the intended clinical use of the material. The justification for the choice of sample numbers, tissue and implantation sites shall be documented. Test methods for various implantation sites are given in Annex A, Annex B, Annex C, and Annex D. If other implantation sites are chosen, the general scientific principles behind the test methods described in Annex A, Annex B, Annex C, and Annex D shall still be adhered to and the justification provided.
Note: For some devices, there are vertical standards prescribing specific implant studies to evaluate local tissue responses, e.g. intraocular lens implant and dental usage tests. These studies can be used to satisfy the requirements in this document.
5.1.2 For absorbable materials, the implantation site shall be marked in a manner suitable for identification of the site at the end of the designated time periods. The use of a non-invasive permanent skin marker and/or a template marking the placement of the sample is recommended for short-term study intervals only. In most circumstances, a location marker comprised of an appropriate nonabsorbable negative control (e.g. HDPE 1mm by 2mm by 5mm, PP suture, gold band, clips) may be used to identify the location of the implant site. These location markers can be removed without inducing artefacts to the test article-tissue interface prior to histology processing.
Exceptionally, a sham surgical procedure might be used to evaluate the impact of the procedure on the tissue involved; in these cases, the specific justification shall be provided.
5.2 Animals
5.2.1 All aspects of animal care and accommodation shall be in accordance with ISO 10993-2. In general, small laboratory animals such as mice, rats, hamsters, or rabbits are preferred.
5.2.2 The use of larger animals may be justified based upon special scientific considerations of the particular biomaterial under study, or if needed to accommodate implant size, with whole device testing.
5.2.3 Select an animal species in line with the principles set out in ISO 10993-2, giving due consideration to the size of the implant test samples, the number of implants per animal, the intended duration of the test in relation to the expected lifespan of the animals, as well as potential species’ differences regarding biological response.
5.2.4 For short-term testing, animals such as rodents or rabbits are commonly used. For long-term testing, animals such as rodents, rabbits, dogs, sheep, goats, pigs, and other animals with a relatively long life expectancy are suitable.
5.2.5 Before starting an animal study with degradable materials, relevant information from in vitro degradation studies should be considered. For absorbable materials, a pilot study in rodents may be considered to determine the expected rate of degradation before embarking on studies on larger animals.
5.2.6 The samples of test and control materials shall be implanted under the same conditions in animals of the same species and of the same age, sex, and strain in corresponding anatomical sites. The number and size of implants inserted into an animal depends on the size of the species and the anatomical location. Whenever possible, the reference control and the test samples should be implanted into the same animal.
5.2.7 However, when a neuroimplantation study (see Annex D) is conducted or when the local effects after implantation are investigated as part of a systemic toxicity study by implantation, control and test samples shall not be placed in the same animal.
5.3 Test periods
5.3.1 The test period shall be determined by the likely clinical exposure time or be continued until or beyond when a steady-state with respect to the biological response has been reached. The time points selected shall be explained and justified.
5.3.2 For non-absorbable materials, the short-term responses are normally assessed from 1 week up to 4 weeks and the long-term responses in tests exceeding 12 weeks. The local biological response to implanted materials depends both on the properties of the materials and on the response to the associated trauma of surgery. The tissue configuration in the vicinity of an implant changes with the time elapsed after surgery. During the first two weeks after implantation, the reaction due to the surgical procedure itself may be difficult to distinguish from the tissue reaction evoked by the implant. In muscle and connective tissue, depending on the species, and the severity of the surgical trauma, a steady-state is seen in the cell population after 9 weeks to 12 weeks. Implantation in bone tissue may need longer observation periods before a steady-state is reached.
5.3.3 For absorbable materials, the test period shall be related to the estimated degradation time of the test product at a clinically relevant implantation site. When determining the time points for sample evaluation, an estimation of the degradation time shall be made. This can be accomplished in vitro by real-time or accelerated degradation studies or in certain circumstances by mathematical modelling. In general, study duration should extend up to or beyond the point of complete absorption. The evaluation period for absorbable materials will depend in part on the degradation rate of the materials. Study intervals should span a significant portion of the degradation time frame for the implant, and shall include, as a minimum, the following time points:
a) early time frame (where there is no or minimal degradation)——For absorbable materials, usually a study interval of between 1 week and 2 weeks post-implantation should be used to assess the early tissue response.
b) mid time frame (when degradation is taking place)——Subsequent study intervals for absorbable devices should be guided by the degradation profile of the specific absorbable material. The target interval should allow assessment of histological response when the tissue response is expected to be most pronounced (e.g. substantial structural disruption and/or fragmentation of the device is most likely to occur). Implants with longer-term degradation profiles may require multiple assessment time points, with intervals targeted in accordance with the expected pattern of degradation.
c) when a device with multiple materials with differing absorption rates is implanted, implant intervals reflecting the degradation profile of those components should be included.
d) late time frame (when the implant is essentially absorbed)——This interval is targeted to observe when minimal amounts of the absorbable component remain at the implant site.
Gross and microscopic evaluation after complete implant absorption is highly desirable. However, in the absence of complete absorption, the overall data collected should be sufficient to allow characterization of the local effects after implantation if:
——the affected tissue’s response, structure, and function have achieved an acceptable steady-state condition, and
——the absorbable material and/or its degradation products are in a state of limited visually-identifiable presence.
Foreword i
Introduction iii
1 Scope
2 Normative references
3 Terms and definitions
4 Common provisions for implantation test methods
5 Test methods, general aspects
6 Test report
Annex A (Normative) Test methods for implantation in subcutaneous tissue
Annex B (Normative) Test method for implantation in muscle
Annex C (Normative) Test method for implantation in bone
Annex D (Normative) Test methods for implantation in brain tissue
Annex E (Informative) Examples of evaluation of local biological effects after implantation
Bibliography
醫(yī)療器械生物學(xué)評價 第6部分：植入后局部反應(yīng)試驗
1 范圍
本文件規(guī)定了用于評估醫(yī)療器械所用生物材料植入后局部反應(yīng)的試驗方法。
本文件適用于下列材料：
——固形和非固形材料，如多孔材料、液體、膠狀、膏狀和顆粒材料，
——非吸收性材料，和
——可降解和/或可吸收性固形或非固形材料。
本文件也適用于評價臨床上預(yù)期用于局部損傷表面或損傷內(nèi)表面的醫(yī)療器械產(chǎn)生的局部組織反應(yīng)。
注1：將試驗樣品植入適宜種屬的動物和部位以評價材料的生物學(xué)安全性，其目的為表征醫(yī)療器械/生物材料植入后組織反應(yīng)的進(jìn)程和演變，包括材料最終的組織整合或吸收/降解，尤其對可降解/可吸收性材料，確定材料的降解特性以及所產(chǎn)生的組織反應(yīng)。通過比較試驗樣品與已確立臨床可接受性和生物相容性的醫(yī)療器械所用對照材料產(chǎn)生的組織反應(yīng)，對局部反應(yīng)進(jìn)行評價。
本文件不適用于預(yù)期評價或測定試驗樣品在機(jī)械或功能負(fù)荷方面的性能的植入試驗。
本文件不涉及全身毒性、致癌性、致畸性或致突變性。
注2：用來評價局部生物學(xué)反應(yīng)的長期植入研究可能提供全身毒性、致癌性、致畸性或致突變性方面的一些信息。通過植入進(jìn)行的全身毒性研究可能滿足本文件的要求。當(dāng)進(jìn)行聯(lián)合研究來評價局部反應(yīng)和全身反應(yīng)時，兩個文件的要求都要滿足。
2 規(guī)范性引用文件
下列文件中的內(nèi)容通過文中的規(guī)范性引用而構(gòu)成本文件必不可少的條款。其中，注日期的引用文件，僅該日期對應(yīng)的版本適用于本文件；不注日期的引用文件，其最新版本(包括所有的修改單)適用于本文件。
ISO 10993-1 醫(yī)療器械生物學(xué)評價 第1部分：風(fēng)險管理過程中的評價與試驗(Biological evaluation of medical devices—Part 1：Evaluation and testing within a risk management process)
注：GB/T 16886.1—2022 醫(yī)療器械生物學(xué)評價 第1部分：風(fēng)險管理過程中的評價與試驗(ISO 10993-1：2018，IDT)。
ISO 10993-2 醫(yī)療器械生物學(xué)評價 第2部分：動物福利要求(Biological evaluation of medical devices—Part 2：Animal welfare requirements)
注：GB/T 16886.2—2011 醫(yī)療器械生物學(xué)評價 第2部分：動物福利要求(ISO 10993-2：2006，IDT)。
ISO 10993-4 醫(yī)療器械生物學(xué)評價 第4部分：與血液相互作用試驗選擇(Biological evaluation of medical devices—Part 4：Selection of tests for interactions with blood)
注：GB/T 16886.4—2022 醫(yī)療 器械生物學(xué)評價 第4部分：與血液相互作用試驗選擇(ISO 10993-4：2017，IDT)。
ISO 10993-12 醫(yī)療器械生物學(xué)評價 第12部分：樣品制備與參照材料(Biological evaluation of medical devices—Part 12：Sample preparation and reference materials)
注：GB/T 16886.12—2017 醫(yī)療器械生物學(xué)評價 第12部分：樣品制備與參照材料(ISO 10993-12：2012，IDT)。
ISO 10993-16 醫(yī)療器械生物學(xué)評價 第16部分：降解產(chǎn)物與可瀝濾物毒代動力學(xué)研究設(shè)計(Biological evaluation of medical devices—Part 16：Toxicokinetic study design for degradation products and leachables)
注：GB/T 16886.16—2021 醫(yī)療器械生物學(xué)評價 第16部分：降解產(chǎn)物與可瀝濾物毒代動力學(xué)研究設(shè)計(ISO 10993-16：2017，IDT)。
3 術(shù)語和定義
ISO 10993-1、ISO 10993-2、ISO 10993-12和ISO 10993-16界定的以及下列術(shù)語和定義適用于本文件。
3.1
吸收/吸收性 absorb/absorption
某一非內(nèi)源性(外部的)材料或物質(zhì)，或其分解產(chǎn)物逐步通過細(xì)胞和/或組織或被細(xì)胞和/或組織同化的作用。
3.2
降解 degradation
材料的分解。
[來源：ISO 10993-9：2009，3.1]
3.3
降解產(chǎn)物 degradation product
某一材料或物質(zhì)由于物理、代謝和/或化學(xué)分解而產(chǎn)生的所有中間或最終副產(chǎn)物。
[來源：ISO/TR 37137：2014，2.2，有修改]
3.4
(使)降解 degrade
將某一材料或物質(zhì)通過物理、代謝和/或化學(xué)途徑進(jìn)行分解。
[來源：ISO/TR 37137：2014，2.3]
3.5
生物材料 biomaterial
預(yù)期與生物系統(tǒng)相互作用的材料或物質(zhì)，用于評價、治療、填充或替代任何人體組織、器官或功能。
[來源：歐洲學(xué)會生物材料會議]
4 植入試驗方法通則
4.1 總則
充分詳細(xì)地設(shè)計試驗方案非常重要，這樣能從所用的每只動物和每項研究中獲得全部相關(guān)信息(見ISO 10993-2、ISO 10993-11和ISO 10993-16)。
所有的動物研究均應(yīng)在經(jīng)國家認(rèn)可機(jī)構(gòu)認(rèn)可的實驗室內(nèi)進(jìn)行，并應(yīng)遵守與實驗動物福利有關(guān)的全部適用法規(guī)，以符合ISO 10993-2的要求，這些研究應(yīng)在良好實驗室質(zhì)量管理規(guī)范或其他經(jīng)批準(zhǔn)的質(zhì)量保證體系的控制下進(jìn)行。
附錄A、附錄B、附錄C和附錄D中規(guī)定的試驗方法均應(yīng)采用本章的規(guī)定。
4.2 植入樣品的制備
4.2.1 應(yīng)按照ISO 10993-12制備試驗樣品和參照或?qū)φ詹牧稀Ｖ踩霕悠返某叽绾托螤顟?yīng)形成文件并進(jìn)行論證。附錄A、附錄B、附錄C和附錄D中給出了各種植入部位的試驗樣品。物理特性(例如形態(tài)密度、硬度、表面)可能影響試驗材料組織反應(yīng)的性質(zhì)，因此應(yīng)予以記錄并在表征組織反應(yīng)時考慮這些因素。對照材料宜與試驗樣品具有盡可能相近的物理特性。
4.2.2 應(yīng)根據(jù)最終產(chǎn)品預(yù)期所用方法對每個植入樣品進(jìn)行加工、處理、清洗污染物和滅菌，并應(yīng)在研究文件中進(jìn)行確認(rèn)。植入樣品在最終制備和滅菌后，應(yīng)進(jìn)行無菌操作，以保證植入樣品在植入前和植入時不會以任何方式被損壞或污染。
4.2.3 對于用于組織工程醫(yī)療產(chǎn)品的支架材料，可能不宜使用預(yù)先裝有細(xì)胞和/或蛋白質(zhì)的最終產(chǎn)品，因為動物對這種產(chǎn)品細(xì)胞/蛋白質(zhì)成分產(chǎn)生的免疫反應(yīng)，以及細(xì)胞對動物產(chǎn)生的反應(yīng)可能會干擾局部組織反應(yīng)，造成結(jié)果難以解釋。
4.2.4 對于復(fù)合材料(如骨水泥、牙科材料)，在使用之前可能要混合組分，并在植入前進(jìn)行固化。設(shè)計為在放置前固化的多組分材料，可在使用前進(jìn)行組分混合并在植入前進(jìn)行固化。但設(shè)計用于原位聚合的材料(例如骨水泥、許多牙科材料) ，應(yīng)以在原位聚合的方式予以植入。應(yīng)對所使用的步驟形成文件并進(jìn)行論證。
4.2.5 非固形材料(包括粉劑)可裝在兩端開口的圓柱形管內(nèi)用于植入后局部反應(yīng)試驗(見ISO 10993-12中給出的管材選擇)。按照制造商的使用說明書制備試驗材料，將材料裝入管內(nèi)直至與端口平齊，謹(jǐn)慎操作防止試驗材料污染管的外表面；如出現(xiàn)污染不應(yīng)植入樣品。避免空氣進(jìn)入管內(nèi)，并確保裝入管內(nèi)的材料端口面和管的端口均光滑。
聚乙烯(PE)、聚丙烯(PP)或聚四氟乙烯(PTFE)管常用于本試驗。PE管經(jīng)高壓蒸汽處理可能會變形。
4.2.6 應(yīng)通過與已確立臨床可接受性和生物相容性的類似樣品/材料的組織反應(yīng)進(jìn)行比較來進(jìn)行評價。
注：詳細(xì)指南見ISO 10993-12。
4.2.7 對照品的物理特性如形狀，特別是表面狀況，如實際可行應(yīng)與植入試驗樣品相似，任何差異都應(yīng)說明并論證。如試驗材料裝入管內(nèi)，對照品應(yīng)是與管相同的材料，直徑與管的外徑相同。對照棒材或管的選擇應(yīng)形成文件并論證。
4.2.8 對于植入研究，應(yīng)對試驗和對照樣品的數(shù)量或尺寸形成文件記錄。
4.3 研究設(shè)計
對于包含兩種或多種不同材料/由兩種或多種不同材料組成的器械，試驗樣品宜具有相似組分或可能需要多個植入物，例如，如果某一器械由高密度聚乙烯(HDPE)和鈦組成，那么試驗樣品宜由HDPE和鈦組成。
5 試驗方法的基本要求
5.1 組織與植入部位
5.1.1 試驗樣品應(yīng)植入與材料預(yù)期臨床應(yīng)用最相關(guān)的組織，對樣品數(shù)量、組織和植入部位的選擇理由應(yīng)形成文件。附錄A、附錄B、附錄C和附錄D給出了各種植入部位的試驗方法。如選擇其他植入部位，仍應(yīng)遵循附錄A、附錄B、附錄C和附錄D給出試驗方法的基本科學(xué)原理，并進(jìn)行論證。
注：對于某些器械，具有給出特定植入研究的產(chǎn)品標(biāo)準(zhǔn)來評價局部組織反應(yīng)，例如，人工晶狀體植入物和牙科應(yīng)用試驗。這些研究可用于滿足本文件的要求。
5.1.2 對于可吸收性材料，應(yīng)采用一種適當(dāng)?shù)姆绞綐?biāo)記植入部位，用于在特定的時間段結(jié)束時識別該部位。推薦僅在間隔較短的研究中使用非侵入持久性的皮膚標(biāo)記和/或模板標(biāo)記樣品植入位置。在大多數(shù)情況下，可采用適宜的非吸收性陰性對照(例如，HDPE 1 mm×2 mm×5 mm，PP縫合線、金線、夾)組成的位置標(biāo)記物來標(biāo)記植入位置。這些位置標(biāo)志物可以在組織學(xué)處理之前，在不影響試驗樣品組織接觸面的情況下被移除。
特殊情況下，可采用假手術(shù)步驟來評價手術(shù)步驟對有關(guān)組織的影響；這種情況應(yīng)提供具體的論證。
5.2 動物
5.2.1 動物管理和飼養(yǎng)應(yīng)完全執(zhí)行ISO 10993-2。一般情況下首選小型實驗動物，比如小鼠、大鼠、倉鼠或兔。
5.2.2 基于研究中針對具體生物材料的特殊科學(xué)考慮，可論證使用較大型動物，或者如果需要調(diào)整植入物尺寸，以進(jìn)行完整器械試驗。
5.2.3 動物種屬的選擇符合ISO 10993-2規(guī)定的原則，適當(dāng)考慮植入試驗樣品的尺寸、每只動物植入物的數(shù)量、根據(jù)動物預(yù)期壽命確定的試驗周期，以及動物種屬可能存在的生物學(xué)反應(yīng)差異性。
5.2.4 對于短期試驗，通常使用嚙齒類動物或兔。對于長期試驗，適宜使用嚙齒動物、兔、犬、綿羊、山羊、豬及平均壽命相對較長的其他動物。
5.2.5 在開始可降解材料的動物研究之前，宜考慮體外降解研究的相關(guān)信息。對于可吸收性材料，在開展較大型動物研究之前，宜先采用嚙齒動物進(jìn)行預(yù)試驗，以測定材料的預(yù)期降解率。
5.2.6 應(yīng)在同樣條件下將試驗和對照材料樣品植入相同年齡、性別和品系的同一種屬動物的對應(yīng)解剖部位，根據(jù)動物種屬體型大小和解剖位置情況，確定植入物的數(shù)量和尺寸。在可能的情況下，參照/對照樣品和試驗樣品宜植入同一只動物。
5.2.7 然而，當(dāng)進(jìn)行神經(jīng)植入研究(見附錄D)，或植入后局部反應(yīng)是作為通過植入進(jìn)行全身毒性試驗的一部分而被研究時，則不應(yīng)將對照樣品和試驗樣品植入同一只動物。
5.3 試驗周期
5.3.1 應(yīng)根據(jù)臨床可能接觸時間，或是持續(xù)至相應(yīng)生物學(xué)反應(yīng)達(dá)到或超過某一穩(wěn)定狀態(tài)的時間，來確定試驗周期。所選擇的時間點(diǎn)應(yīng)進(jìn)行說明和論證。
5.3.2 對于非吸收性材料，短期反應(yīng)評估一般為1周至4周，長期反應(yīng)評估則一般超過12周。植入材料的局部生物學(xué)反應(yīng)與材料特性和手術(shù)創(chuàng)傷反應(yīng)有關(guān)，術(shù)后植入物周圍組織結(jié)構(gòu)的改變隨時間而變化。植入后的最初2周，可能很難將外科手術(shù)所致的反應(yīng)與植入物引起的組織反應(yīng)區(qū)分開來。在肌肉和結(jié)締組織中，植入后9周至12周時細(xì)胞群呈穩(wěn)定狀態(tài)，這取決于動物種屬和手術(shù)創(chuàng)傷的嚴(yán)重程度。在骨組織中，則可能需要較長的觀察期才能達(dá)到穩(wěn)定狀態(tài)。
5.3.3 對于可吸收性材料，試驗周期應(yīng)與試驗產(chǎn)品在臨床相關(guān)植入部位估計的降解時間相關(guān)。確定樣品評價時間點(diǎn)時，應(yīng)估算降解時間。這可以通過體外實時或加速降解試驗來實現(xiàn)，也可以在某些情況下通過數(shù)學(xué)模型方法來實現(xiàn)。一般情況下，研究周期宜涵蓋或超過材料的完全吸收終點(diǎn)。可吸收性材料的評價周期將部分依賴于材料的降解速率。研究間隔宜跨越植入物降解時間框架的重要部分，并應(yīng)至少包括下列時間點(diǎn)：
a)早期時間框架(無或微量降解)——對于可吸收性材料，通常宜使用植入后1周和2周之間的一個時間點(diǎn)來評估早期組織反應(yīng)。
b)中期時間框架(發(fā)生降解時)——宜根據(jù)特定可吸收性材料的降解特性來指導(dǎo)選擇可吸收器械的后續(xù)的研究時間間隔。目標(biāo)時間間隔宜允許評估預(yù)期最明顯的組織學(xué)反應(yīng)(例如，最有可能發(fā)生實質(zhì)性結(jié)構(gòu)紊亂和/或器械碎裂)。降解時間較長的植入物可能需要多個評估時間點(diǎn)，并根據(jù)預(yù)期的降解模式確定目標(biāo)間隔。
c)當(dāng)植入含有不同吸收速率的復(fù)合材料時，植入時間間隔宜包含反映這些組分降解特征的時間間隔。
d)末期時間框架(當(dāng)植入物基本被吸收時)——該時間間隔是為了在可吸收性組分在植入部位有微量殘余時進(jìn)行觀察。
植入物完全吸收后的大體和顯微學(xué)評價非常重要。然而，在沒有完全吸收情況下，如果滿足以下條件，則收集到的全部資料宜足以表征植入后的局部反應(yīng)：
——受影響組織的反應(yīng)、結(jié)構(gòu)和功能已經(jīng)達(dá)到了一個可接受的穩(wěn)定狀態(tài)，和
——可吸收性材料和/或其降解產(chǎn)物處于一種肉眼難以發(fā)現(xiàn)的狀態(tài)。
