Biocompatibility Test Cases

biocompatibility refers to the ability of a biological material to generate complex biological, physical, and chemical reactions at specific sites within the body. It evaluates whether certain materials or drugs, when in contact with or implanted into the human body, can "compatibly" coexist without causing harm. The testing includes various aspects, such as cytotoxicity, sensitization, irritation, systemic toxicity (acute toxicity), subchronic toxicity (subacute toxicity), genetic toxicity, implantation, chronic toxicity, carcinogenicity, reproductive and developmental toxicity, and biodegradability.

The ISO 10993 standard typically includes the following core tests for biological evaluation:

- Cytotoxicity: The sample’s extract is cultured in a suspension of rapidly growing cells, and cell morphology is observed. (GB/T 16886.5-2003, iso 10993-5-1999)

- Skin Irritation: The sample extract is applied to absorbent gauze or the gel material is directly placed on the depilated sides of an animal’s back. The site is observed for reactions. (GB/T 16886.10-2005, iso 10993-10)

- Sensitization Test: The extract is intradermally injected into animal skin, followed by patching with gel material on the depilated back, inducing and stimulating the skin to observe reactions. (GB/T 16886.10-2005, ISO 10993-10)

cytotoxicity test Model

1. Cell Lines

- Mouse fibroblast cells L929, NIH3T3; human normal liver cells LO2.

2. Preparation of Extracts

- Gel material + culture medium, incubated at 37°C for 72 hours, filtered through 0.22 µm filter for use.

3. Cell Grouping (n=3)

1. Normal cells

2. Normal cells + Extract Concentration A

3. Normal cells + Extract Concentration B

4. Normal cells + Extract Concentration C

Intervention time: 24h, 48h, 72h; Cytotoxicity measured using CCK-8.

Skin Irritation Test Animal Model

1. Materials

- Animals: Rabbits.

- Other Materials: Gel, extract (gel material + culture medium, incubated at 37°C for 72 hours, filtered through 0.22 µm, and placed on gauze for use).

2. Model Setup

- Shaving: Shave the back regions of the rabbit along the spine, apply gel/extract on the left side, and solvent/excipient on the right side. Bandage and fix for 4 hours before removing the substance and washing the area with warm water.

- Observation: Score based on erythema and edema.

3. Evaluation Standards (GB/T16886 Part 10: Irritation and Skin Sensitization Tests):

1. Primary Irritation Score: The average score for each group divided by the number of observed test points.

2. Primary Irritation Index (PII): The primary irritation score divided by the number of animals in each group.

4. Reaction Types:

- 0.0–0.4: Mild response

- 0.5–1.9: Slight response

- 2.0–4.9: Moderate response

- 5.0–8.0: Severe response

Sensitization Test Animal Model

1. Materials

- Animals: Guinea pigs

- Drugs: Extract (gel material + culture medium, incubated at 37°C for 72 hours, filtered through 0.22 µm), Freund’s complete adjuvant, positive control (5% formaldehyde), negative control (physiological saline).

2. Model Setup (Intradermal and Epicutaneous Combined Method):

- Intradermal Induction: Divide guinea pigs into 3 groups (test group, negative control, and positive control). Shave the back, set 3 injection points symmetrically on each side, and inject 0.1 mL of the following solutions:

 - Test Group: (A) Freund’s complete adjuvant/physiological saline 1:1, (B) Gel extract, (C) A solution/B solution 1:1

 - Positive Control: (A) Freund’s complete adjuvant/physiological saline 1:1, (B) 5% formaldehyde, (C) A solution/B solution 1:1

 - Negative Control: (A) Freund’s complete adjuvant/physiological saline 1:1, (B) physiological saline, (C) A solution/B solution 1:1

After 7 days, proceed to the local induction phase.

3. Local Induction:

- Shave back hair, disinfect, and massage with 10% SDS to prepare the skin. Apply gauze soaked with gel extract, saline, and 5% formaldehyde solutions to their backs. Bandage, remove after 48 hours, and repeat this for 14 days before moving to the activation phase.

4. Activation Phase:

- Apply gauze soaked in gel extract, saline, and 5% formaldehyde to the abdomen of the guinea pigs. After 24 and 48 hours, observe and record skin changes.

5. Magnusson and Kligman Grading Criteria:

- If the negative control group scores below 1, and the test group scores ≥ 1, sensitization is confirmed.

- If the negative control group scores ≥ 1, and the test group shows stronger reactions than the negative group, sensitization is confirmed.

Hemolysis Testing

1. Materials

- Samples: Fresh human anticoagulated whole blood.

- Drugs: Extract (gel material + culture medium, incubated at 37°C for 72 hours, filtered through 0.22 µm), PBS, TritonX-100.

2. Procedure:

- Prepare anticoagulated blood with 3.2% sodium citrate, centrifuge at 1000 rpm for 10 minutes, mix the red blood cells with PBS, and adjust to 5% concentration.

- Mix 6 mL of extract, PBS, and TritonX-100, adding to 4 mL of red blood cell dilution. Incubate for 1 hour at 37°C, then centrifuge at 3000 rpm for 10 minutes.

- Measure absorbance at 540 nm.

3. Hemolysis Rate Calculation:

\[ \text{Hemolysis ratio (\%)} = \frac{(A_s - A_0)}{(A_p - A_0)} \times 100 \]

Where:

- \( A_s \) = Absorbance of each group

- \( A_0 \) = Absorbance of the blank control group

- \( A_p \) = Absorbance of the positive control group

Tissue Compatibility Testing

1. Materials

- Animals: SD rats

- Drugs: Gel

2. Procedure:

- Anesthetize rats and implant the gel into the muscle on the back. After 0, 7, and 14 days, euthanize the rats and fix the surrounding tissue for 48 hours, then dehydrate, embed in paraffin, and section for HE staining.

3. Histological Analysis:

- HE Staining: If no significant cell infiltration is observed, the material is considered to have good tissue compatibility.

- IHC Staining: If CD68 positive expression does not significantly differ from the control group, the material does not induce inflammation, suggesting good immunocompatibility.

Application Fields

The study of biocompatibility of gel materials spans across several fields, including medicine, pharmacy, and bioengineering:

1. Medical Biocompatibility: In the medical field, gel materials are widely used in tissue engineering, repair, and regeneration. Biocompatibility is a crucial criterion to assess whether these materials induce toxicity or immune responses upon contact with biological tissues.

2. Drug Delivery Systems: Gel materials can serve as carriers for drug delivery systems, offering controlled drug release and enhancing bioavailability. The study of their biocompatibility helps to evaluate interactions between the gel, drugs, and biological tissues.

3. Surgical Assistance Materials: In surgical applications, gel materials are used for wound closure, hemostasis, and soft tissue repair. Their biocompatibility ensures the safety and efficacy of these materials in surgical procedures.