YY/T 1680-2020 English PDF (YYT1680-2020)

This Standard provides methods and guidelines for evaluating the effectiveness of allogeneic demineralized bone products in causing or promoting bone formation when implanted (or injected) into the human body. This Standard is applicable to the evaluation of osteoinductive performance of demineralized bone and medical device products containing demineralized bone.

YY/T 1680-2020
YY
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 11.040.40
C 45
Allogeneic Grafts - In Vivo Evaluation of
Osteoinductive Potential for Materials Containing
Demineralized Bone
ISSUED ON: FEBRUARY 21, 2020
IMPLEMENTED ON: JANUARY 01, 2021
Issued by: National Medical Administration of Traditional Chinese
Medicine
Table of Contents
Foreword ... 3
Introduction ... 4
1 Scope ... 5
2 Normative References ... 5
3 Terms and Definitions ... 5
4 Animal Model ... 6
5 Test Procedures ... 8
6 Judgment of Results ... 11
7 Report Contents ... 12
Bibliography ... 13
Allogeneic Grafts - In Vivo Evaluation of
Osteoinductive Potential for Materials Containing
Demineralized Bone
1 Scope
This Standard provides methods and guidelines for evaluating the effectiveness of allogeneic demineralized bone products in causing or promoting bone formation when implanted (or injected) into the human body.
This Standard is applicable to the evaluation of osteoinductive performance of demineralized bone and medical device products containing demineralized bone. NOTE: Bone implants containing human gene recombinant BMP protein can also refer to this Standard for the evaluation of in vivo osteoinductive performance.
2 Normative References
The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) is applicable to this document.
GB/T 16886.2 Biological Evaluation of Medical Devices - Part 2: Animal Welfare Requirements
YY/T 0513.3 Allogeneic Grafts - Part 3: Demineralized Bone Grafts
3 Terms and Definitions
For the purpose of this document, the terms and definitions given in YY/T 0513.3 and the following apply.
3.1 Materials containing demineralized bone
Including but not limited to demineralized bone matrix, growth factors, differentiation factors, biologically active carriers and/or non-biologically active carriers. cytotoxic effector cells and graft-versus-host reaction. Therefore, there is no T cell response to exogenous materials.
NOTE 1: Athymic animals can still produce an innate immune response (macrophages, giant cells and granular red blood cells), therefore, have the ability to produce an immune response to non-biocompatible or microbial (culture) positive products.
NOTE 2: The choice of nude rat or nude mouse as the experimental animal model depends on the size (volume) of the implant to be evaluated.
4.2.2 Male athymic animals should be used in implantation experiments. If female animals are used, it is necessary to prove that their estrus cycle shall not negatively affect the experiment.
4.2.3 Adolescent athymic animals should be used in implantation experiments (age at the time of implantation is 6 ~ 9 weeks). Older animals may not respond effectively to osteoinductive materials.
4.3 Heterotopic implantation site
In athymic mouse/rat models, the intermuscular pocket spaces of the gluteal muscle or biceps femoris are suitable implant sites. In clinical applications, there are blood, bone tissue and osteogenic components at the bone tissue implantation site; while the blood, bone tissue and osteogenic components shall cause false positive results during heterotopic implantation.
4.4 Number of implants and sample volume/mass
4.4.1 Number of implants: In the product effectiveness test, 3 independent batches of products should be used for testing. It is recommended to use 3 animals for each batch of products; each animal has 2 implantation sites. The positive control and the negative control are implanted into 3 animals, respectively; and each animal has 2 implantation sites. So that ensure that each group shall have no less than 4 samples for evaluation. NOTE: This design can obtain 83% inclusion probability within the 95% inclusion interval. This allows a 12% difference in the detection of new bone formation. The hypothesis is as follows: newly-formed bone accounts for 40%; the standard deviation is 5%; the statistical significance level is 0.05.
4.4.2 Sample volume/mass: Generally, the mass of the implanted sample containing demineralized bone material is (15±5) mg; and the size of the implanted sample should be 3mm~5mm in length, 3mm~5mm in width, and 1mm~3mm in thickness (powder or granular materials can be added with excipients; and the used excipients shall not contain minerals and osteogenic active substances, which shall not affect the osteoinductive ability of the raw materials).
4.4.3 The test group shall have the same implant dose and shape of demineralized for subsequent implantation operations. The technician shall monitor the animal's breathing during the operation.
5.3.5 Place the anesthetized animal in the prone position on the clean test bench in the operating room and fix it. Wipe the animal back and waist area with iodine solution from the center of the operation to the edge of the operation; and finally wipe off the iodine solution with a clean sterile gauze pad.
5.3.6 The detailed procedures of the gluteus medius and gluteus maximus intermuscular implantation are as follows: Carefully lift the skin above the spine at the back and waist; and make a 0.5cm~1cm long unidirectional incision. Open the skin incision and use the blunt peeling method to create a small pocket gap in the gluteus medius and gluteus maximus tissue parallel to the direction of the muscle fibers, taking care to avoid blood vessels. If there is a small amount of bleeding, stop the bleeding in time and try to remove the blood. The sample is implanted in the gap between the gluteus medius and the gluteus maximus intermuscular. After implantation, suture the muscle layer. Use the same method to implant other test or control samples in the gap between the gluteus medius and gluteus maximus on the other side. Finally, suture the skin layer.
NOTE: When implanting the sample, pay attention to prevent the sample from contacting blood. For example, try to avoid bleeding during the operation. If accidentally bleeding, try to compress the bleeding and clean the blood residue before implanting the sample. When suturing after surgery, the inserting position of the sutures shall be as far away as possible from the sample. 5.4 Postoperative treatment of animals
5.4.1 Put the animal back into the breeding cage and raise it separately. 5.4.2 Observe and record the health of the animals once a day after the operation. The health status of the animal may be judged by direct contact with the animal or observation outside the cage. If abnormal clinical symptoms or symptoms of inflammation and infection are observed, corresponding treatment shall be carried out as soon as possible.
5.4.3 If the experimental animal died during the experiment, the animal is required to be autopsy. If the animal died after 14d (including 14d) after operation, the implantation site and surrounding tissues shall be removed and fixed until its potential use in histological research is determined. If the animal died within 14d after the operation, an autopsy was performed. The final weight of the animal shall be recorded each time. 5.5 Methods of collecting materials
5.5.1 Collect materials at the designed time point; and record the final weight, health status, and whether there are abnormal conditions of each experimental animal before collecting the materials.
5.5.2 Touch the implant area to determine the approximate implant position. Use a scalpel to make a longitudinal incision in the gluteus medius. Observe whether there is blood outflow from the operation site. If there is blood outflow, perform hemostasis treatment first.
5.5.3 Remove the fascia, sharply separate the tissue around the muscle and take out the muscle as a whole. If the implanted material migrates away from the muscle tissue, the observations are recorded in detail for subsequent analysis of the impact of sample migration on the osteoinduction process. For example, when the implant migrates near the bone tissue of the experimental animal, it shall cause false positive results. 5.5.4 Remove irrelevant tissue around the implant.
5.5.5 Record any abnormal color or shape of the muscle and implant, and/or the condition of the implant and the connected muscle tissue. To avoid tissue dehydration, place the sample in a tissue box or specimen bottle containing 8~10 times the volume of neutral buffered formalin and fix it for at least 72h. Or put the sample into the tissue box and freeze and fix the specimen quickly.
5.6 Histological evaluation of samples
5.6.1 The samples were frozen sectioned by quick freezing, or fixed by routine histological staining procedures. After fixation, perform decalcification (if necessary), dehydration, embed in paraffin or other media and trim. Before embedding, cut the sample in half along the long axis; and embed the tissue block with the section facing downward. The thickness of the slices is generally 4μm~7μm. They are spread out on a glass slide and made into slices. Each slice contains at least 3 different sections. Then choose the best new bone formation observation method to stain the slices. The dyeing methods are: hematoxylin-eosin staining (HE), toluidine blue staining, alcian blue staining, masson trichrome staining, Goldner trichrome staining and Von Kossa staining. If using Von Kossa staining, special attention shall be paid to distinguish stains and calcification of new bone.
5.6.2 At least 3 slices are prepared for each sample; and the slices with the least wrinkles, the most uniform staining, and the largest amount of implant material are used for the evaluation of osteoinductive ability.
5.6.3 Insert a 5×5 (24mm2) grid into the eyepiece of the microscope. Athymic mouse implant sample slices are observed by using a 10x objective lens, with a total magnification of 100 times; and athymic rat sample slices are observed by using a 4x objective lens, with a total magnification of 40 times. When placing the specimen on the stage, adjust the position of the sample slice so that the objective lens is above one of the four corners of the sample slice. Check each field of view until the entire sample slice is observed. A field of view is the area that can be observed within a 5×5 grid under 100/40 times magnification. If it is impossible to determine whether there is bone formation in a specific division area, use a high magnification objective lens to