WO2016065684A1 - Method for maintaining bone morphogenetic protein-2 activity under the irradiation sterilization conditions - Google Patents

Abstract

Disclosed is a method for maintaining BMP-2 activity using ray irradiation. BMP-2 solution is added into a centrifuge tube, sealed well with a seal membrane, and then the tube is put in a Thermos bottle, the bottle is filled with ice, and irradiated with ⁶⁰Co, ¹³⁷Cs and ¹⁹²Ir source ray, wherein the doses are set to be 0.01-100 kGy. The irradiation process for BMP-2 is realized. The problem of inactivation during the BMP-2 irradiation process is effectively solved, the research and development of medical equipments loaded with BMP-2 are promoted, and the application prospects are greatly improved.

Description

Method for maintaining bone morphogenetic protein-2 activity under irradiation sterilization condition Technical field

The invention relates to the activity maintenance of the bone morphogenetic protein-2 (BMP-2) in the irradiation sterilization process, the irradiation object is BMP-2, and the irradiation source of ⁶⁰ Co, ¹³⁷ Cs, ¹⁹² Ir is used, and the irradiation dose is 0.01. -100kGy.

Background technique

Bone morphogenetic protein (BMP) is widely used mainly because it induces the formation of bone and cartilage, can be used as a therapeutic agent for the treatment of fractures and periodontal defects, and can induce bone growth around implants and artificial prostheses. Traditional gene knockout experiments have shown that bone morphogenetic proteins possess a variety of biological activities, such as early embryogenesis, multi-dimensional organ formation and other functions can be achieved by regulating cell proliferation, differentiation and apoptosis. Through in vitro and in vivo experiments, a series of human bone morphogenetic proteins have been shown to stimulate bone formation, and recombinant human bone morphogenetic proteins have also been applied. To date, more than 20 BMPs have been identified and characterized, all of which are members of the transforming growth factor beta (TGF-beta) superfamily. Among them, BMP-2 has almost the same bone repairing ability as autologous bone and is considered to have the strongest osteoinductive activity.

Sterilization is required for the clinical use of BMP-2 and BMP-2 loaded medical devices. However, several conventional sterilization methods currently deactivate BMP-2. Ethylene oxide can chemically react with proteinaceous materials to inactivate BMP-2; autoclaving sterilizes BMP-2 and inactivates it.

Irradiation sterilization is an effective method for killing microorganisms on most substances by emitting radiation from radioactive materials. The temperature is not significantly increased throughout the sterilization process, and it is a good sterilization method for heat sensitive compounds.

Summary of the invention

It is an object of the present invention to effectively sterilize BMP-2 while maintaining its activity by radiation.

The technical solution of the present invention is as follows:

The invention adds the BMP-2 solution into the centrifuge tube, seals it with a sealing film, puts it into a thermos flask, fills it with ice, irradiates with ⁶⁰ Co, ¹³⁷ Cs, ¹⁹² Ir source rays, and sets the dose to be 0.01-100 kGy. Preferably, the dosage is set to 15-50 kGy; the irradiation process to BMP-2 is achieved.

The cells were then routinely cultured in DF12 complete medium to establish unirradiated BMP-2. The group and irradiated BMP-2 group were tested for ALP content in each well after 7 days of cell culture. The activity of BMP-2 was determined by the level of ALP, and the detection of BMP-2 activity was achieved.

The principle of detecting BMP-2 activity in the present invention is that ALP is an important index for cell differentiation into osteoblasts, BMP-2 activity is high, and the tendency of cells to differentiate into osteoblasts is obvious, and the ALP expression level is high, and the content of ALP can be high or low. The level of BMP-2 activity was measured.

The invention has the advantages that the problem of deactivation in the process of BMP-2 irradiation sterilization is effectively solved, and the application prospect thereof is improved.

DRAWINGS

Figure 1: Changes in ALP content in cells after ⁶⁰ Co-irradiation at 25 kGy;

Figure 2: Changes in ALP content in cells after ⁶⁰ CoG irradiation at 50 kGy;

Figure 3: Changes in ALP content in cells after ⁶⁰ Co-ray irradiation at 35 kGy.

Specific implementation method

The invention patent is further illustrated by way of example below.

Example 1:

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ⁶⁰ Co source radiation to set the dose to 0.01kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 2:

1. Prepare BMP-2 solution into the centrifuge tube, seal it with sealing film, put it into the thermos bottle, fill it with ice, irradiate with ¹³⁷ Cs source radiation, set the dose to 0.01kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, its ALP table The amount of ALP expression was similar to that of unirradiated BMP-2, and there was no significant difference, which proved that BMP-2 activity was maintained after irradiation.

Example 3:

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹⁹² Ir source radiation to set the dose to 0.01kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 4:

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ⁶⁰ Co source radiation to set the dose to 100kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 5:

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹³⁷ Cs source radiation to set the dose to 100kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 6

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹⁹² Ir source radiation to set the dose to 100kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 7

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ⁶⁰ Co source radiation to set the dose to 25kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained. The experimental results are shown in Figure 1.

Example 8

1. Prepare BMP-2 solution into the centrifuge tube, seal it with sealing film, put it into the thermos bottle, fill it with ice, irradiate with ⁶⁰ Co source radiation, set the dose to 50kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained. The experimental results are shown in Figure 2.

Example 9

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ⁶⁰ Co source radiation to set the dose to 35kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained. The experimental results are shown in Figure 3.

Example 10:

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹³⁷ Cs source radiation to set the dose to 25kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 11

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹⁹² Ir source radiation to set the dose to 50kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 11

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹⁹² Ir source radiation to set the dose to 28kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 12

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ⁶⁰ Co source radiation to set the dose to 15kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Example 13

1. Prepare the BMP-2 solution into the centrifuge tube, seal it with a sealing film, place it in a thermos flask, fill it with ice, and irradiate with ¹³⁷ Cs source radiation to set the dose to 15kGy.

2. Umbilical cord blood mesenchymal stem cells were routinely cultured in DF12 complete medium, and unirradiated BMP-2 group and irradiated BMP-2 group were established. The ALP content of each well was measured after 7 days of cell culture.

3. By analyzing the ALP content, we can see that after BMP-2 irradiation, the ALP expression level is similar to that of unirradiated BMP-2, and there is no significant difference, which proves that after irradiation BMP-2 activity is maintained.

Claims (2)

A method for maintaining BMP-2 activity by irradiation with radiation, characterized in that the radiation source is ⁶⁰ Co, ¹³⁷ Cs or ¹⁹² Ir, and the irradiation dose is 0.01-100 kGy. The method of claim 1 wherein the irradiation dose is 25-50 kGy.

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