RU2802060C1 - Method of assessing the biocompatibility of an implant with a mesh structure - Google Patents

Abstract

FIELD: biotechnology; implantology.

SUBSTANCE: invention can be used to determine the biocompatibility of the material. A method of assessing the biocompatibility of an implant with a mesh structure is proposed, including the introduction of a sterile implant into a sterile container, the introduction of a cell suspension, incubation in a CO₂ incubator, and subsequent control of cell growth on the implant, where the implant has a mesh structure.

EFFECT: use of the proposed method makes it possible to evaluate the cellular compatibility of implants with a mesh structure by counting the number of cells moving to the mesh loops.

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Description

The invention relates to medicine, namely to cellular biotechnology, immunology, implantology, and can be used to determine the biocompatibility of a material.

Titanium mesh is widely used in surgery, in particular, in cranioplasty, in maxillofacial surgery, in plastic surgery of the abdominal wall. One of the key problems associated with the implantation of the material is its survival. To increase the biocompatibility of the material, various methods of physical and chemical modification of its surface are used. The biocompatibility of the material can be tested both in vivo, by implantation in laboratory animals, and in vitro. In the study of implantation material in vitro for biocompatibility, most often, its two main parameters are evaluated: 1) the ability of the implantation material to integrate into surrounding tissues (cell compatibility), in this case, adhesion and proliferation of cells on its surface are mainly evaluated; 2) the level and nature of immunological reactions to the implant material.

When assessing the cellular compatibility of the material, it is necessary to seed the cell culture on the implant and assess the level of their adhesion and proliferation. Seeding is usually carried out by the sedimentation method: a suspension of cells is applied to the implant surface, which passively settle on the implant and adhere to its surface [Kawai, 2017; Groessner-Schreiber, 2003; Kopf, 2015], which makes it possible to estimate the proportion of adherent cells, the degree of their spreading over the surface, the rate of proliferation, and a number of other indicators. This assumes that the implant should have a flat surface.

A patent is known (RU, No. 2761266, IPC G01N 33/49, G01N 33/68, G01N 33/15, publ. 12/06/2021), describing a method for assessing the suitability for use in medicine of synthetic polymers by incubation of implant material with mononuclear cells peripheral blood and subsequent assessment of the amount of anti-inflammatory cytokines secreted by mononuclear cells.

This method is applicable to materials in the form of a grid. At the same time, it evaluates only certain parameters of the immunological reaction to the material, but does not provide any information about the ability of the material to adhere to cells, and, accordingly, to integrate into the tissues surrounding the implant (cell compatibility).

A device is also known (SU, No. 1120221, IPC G01N 19/04, publ. 10/23/1984), which evaluates the adhesive ability of animal cells by tearing them off the surface using a liquid jet.

This method uses a fundamentally different technique and determines the resistance to detachment of cells associated with the surface, but does not provide any information about the ability of cells to proliferate on this surface. In addition, the method is not suitable for mesh surfaces.

The closest technical solution is a method for assessing the biocompatibility of bone implants based on bones of allogeneic origin (Volov L.T., Trunin D.A., Ponomareva Yu.V., Popov N.V. Study of biocompatibility and cytotoxicity of personalized bone implants using cellular technologies , Bulletin of the Medical Institute "REAVIZ", No. 5, 2017, pp. 32-39).

1) Fragments of the implant material 3×3×5 mm in size, pre-sterilized, are placed in Petri dishes. The implant material has a monolithic non-mesh structure.

2) Prepare a cell suspension. In this work, a culture of chondroblasts was used. Cells were resuspended in growth medium.

3) The implant material is placed in a container and a cell suspension is applied to it. The cell suspension is applied directly to the implant.

4) Containers with implants are transferred to the CO ₂ incubator. Cells adhere and further divide directly on the surface of the implant.

5) Control of cell growth was carried out microscopically, by assessing the shape of cells and an approximate qualitative assessment of their number.

This method involves the seeding of cells on its surface by the method of sedimentation and involves the use of an implant with a flat surface, and therefore is not suitable for a mesh implant.

Existing methods for assessing the cellular compatibility of implants are not applicable to implants with a mesh structure.

The objective of the invention is to improve the methodology for assessing the biocompatibility of cells and implants with a mesh surface.

The technical result of the claimed invention lies in the fact that it allows you to evaluate the cellular compatibility of implants with a mesh structure, which allows you to determine the ability of such an implant to integrate into surrounding tissues.

The technical result is achieved by the method for assessing the biocompatibility of an implant with a mesh structure, including the introduction of a sterile implant into a sterile container, the introduction of a cell suspension, incubation in a CO ₂ incubator and subsequent control of cell growth on the implant, according to the invention, the implant has a mesh structure, it is sterilized in an autoclave , pour the growth medium, consisting of wt.%:

medium Needle, in the modification of Dulbecco (DMEM) 45% nutritional formula F-12 44.965% fetal calf serum 10% L-glutamine 0.03% gentamicin 0.005%;

followed by incubation in a CO ₂ incubator for 16-24 hours, adding cell suspension to the wells of the plates with a seeding density of 3-7 thousand cells / cm ² of the area of the wells, while the ratio of the volume of the cell suspension area to the growth medium is 2: 1, repeated incubation of plates with grids in CO ₂ incubator at T 37°C, CO ₂ -5% for 5-14 days and then removing them from grids with trypsin solution and counting the number of cells.

The mesh structure of the implants ensures their lower mass, elasticity, and the possibility of tissue ingrowth into the mesh cells.

Sterilization of the implant in an autoclave ensures the sterility of the test material and prevents bacterial/fungal growth during the test.

Preliminary filling of the sterilized implant with a growth medium consisting of masses. %: Eagle's medium, in Dulbecco's modification (DMEM) - 45%, nutrient mixture F-12 - 44.965%, fetal calf serum - 10%, L-glutamine - 0.03% and gentamicin - 0.005%, followed by incubation in CO ₂ incubator for 16-24 hours, allows you to remove air bubbles from the grids, which can lead to their ascent or uneven location at the bottom of the well.

The seeding density is 3-7 thousand cells / cm ² of the area of the wells, it is necessary to provide the cells with 3-4 cycles of proliferation until the formation of a confluent monolayer and provides a sufficiently active cell proliferation, which makes it possible for them to migrate to the loops of the mesh implant, the ratio of the volume of the cell area suspension to the growth medium 2:1, allows the distribution of the medium already present in the well over the surface to ensure uniform cell seeding.

Repeated incubation of plates with meshes in a CO ₂ incubator at T 37°C, CO ₂ -5% for 5-14 days until a confluent monolayer on the well surface is reached provides a sufficient level of cell migration from plastic to a mesh implant to assess its cell compatibility.

Removal of cells from the meshes with a trypsin solution and counting their number makes it possible to quantify the cell compatibility of the implant.

The invention is carried out as follows.

A mesh implant is taken up to 3 mm thick, 5-15 mm x 5-15 mm in size. The mesh implant material sterilized by autoclaving is introduced into the wells of culture plates and filled with a growth medium consisting of wt.%: Eagle's medium, in Dulbecco's modification (DMEM) - 45%, nutrient mixture F-12 - 44.965%, fetal calf serum - 10%, L-glutamine - 0.03% and gentamicin - 0.005% above the level of grids. The loops of the nets must be in contact with the bottom of the hole over the entire area. The grid plates are incubated in a CO ₂ incubator for 16-24 hours. A cell suspension is introduced into the wells with meshes with a seeding density of 3-7 thousand cells/cm ² of the area of the wells, with a ratio of the volume of the area of the cell suspension to the growth medium of 2:1. The suspension should be spread over the surface of the medium already present in the well to ensure uniform seeding of the cells.

The grid plates are transferred and incubated in a CO ₂ incubator (37° C., 5% CO ₂ ) for 5-14 days. During incubation, the formation of a monolayer at the bottom of the wells should be monitored daily using an inverted microscope. Incubation continues until a confluent monolayer is formed on the plastic surface, when the cells fill the entire space of the well bottom and begin to "support" each other. Cells adhering to the plastic and proliferating in this case are partially transferred to the wire base of the mesh, provided that it is suitable for cell adhesion.

At the end of the incubation, the mesh implants are removed from the wells and transferred to the wells of a new plate. The number of cells adhered to the mesh surface is counted. To do this, the grids are washed from the remnants of the medium and filled with a 0.25% trypsin solution so that the grids are completely flooded. The trypsinized meshes are incubated at 37° C. for 15 minutes, after which the remaining cells on the meshes are removed by gentle pipetting. The number of cells in trypsin is counted using a Goryaev chamber or an automatic cell counter.

Example 1

1) The level of adhesion and proliferation of the culture of dermal fibroblasts (obtained from donor 1) was assessed on titanium meshes 1 mm thick made of titanium grade VT0-1.

2) Meshes 15x15 mm in size and 1 mm thick, sterilized by autoclaving, were placed in the wells of 12-well culture plates and filled with growth medium in a volume of 1 ml above the level of the meshes.

3) Mesh plates were incubated in a CO ₂ incubator for 16 hours.

4) At the end of the incubation, a suspension of dermal fibroblasts (donor 1) was introduced into the wells with grids, respectively, in a volume of 2 ml and with a density of 15,000 cells/ml (in terms of the surface area of the well - 8000 cells/cm ² ). The total volume of medium in each well was 3 ml.

5) Plates with grids were transferred to a CO ₂ incubator (37°C, 5% CO ₂ ) and incubated for 7 days until a confluent cell monolayer formed on the plastic surface.

6) At the end of the incubation, the plates were removed from the CO ₂ incubator, the grids with adherent cells were transferred to empty plates. The grids were washed from the remnants of the medium with a phosphate buffer solution.

7) The grids in the wells were filled with 1 ml of 0.25% trypsin solution and incubated in trypsin at 37°C for 15 min. Cells were finally removed from the grids by vigorous pipetting.

8) The number of cells in the obtained cell suspensions (and, accordingly, on the surface of the grids) was determined by counting them in a cell counter MS-20 (Biorad). The cell concentration was 52.5 thousand cells/ml.

Example 2

1) The level of adhesion and proliferation of the culture of dermal fibroblasts (obtained from donor 2) was assessed on titanium meshes 1 mm thick made of titanium grade VT0-1.

2) Meshes 5x5 mm in size and 1 mm thick, sterilized by autoclaving, were placed in the wells of 24-well culture plates and filled with growth medium in a volume of 0.5 ml above the level of the meshes.

3) Mesh plates were incubated in a CO ₂ incubator for 16 hours.

4) At the end of the incubation, a suspension of dermal fibroblasts (donor 2) was introduced into the wells with grids in a volume of 1 ml and with a density of 5700 cells/ml (in terms of the surface area of the well - 3000 cells/cm ² ). The total volume of medium in each well was 1.5 ml.

5) Plates with grids were transferred to a CO ₂ incubator (37°C, 5% CO ₂ ) and incubated for 14 days until a confluent cell monolayer formed on the plastic surface.

6) At the end of the incubation, the plates were removed from the CO ₂ incubator, the grids with adherent cells were transferred to empty plates. The grids were washed from the remnants of the medium with a phosphate buffer solution.

7) The grids in the wells were filled with 0.3 ml of 0.25% trypsin solution and incubated in trypsin at 37°C for 15 min. Cells were finally removed from the grids by vigorous pipetting.

8) The number of cells in the obtained cell suspensions (and, accordingly, on the surface of the grids) was determined by counting them in a cell counter MS-20 (Biorad). The cell concentration was 83.4 thousand cells/ml.

Example 3

1) The level of adhesion and proliferation of a transplanted human osteosarcoma cell culture SaOS-2 was assessed on titanium meshes 3 mm thick made of titanium grade VT0-1.

2) Autoclaved meshes 9x9 mm in size and 3 mm thick were placed in the wells of 24-well culture plates and filled with growth medium in a volume of 0.75 ml above the level of the meshes.

3) Mesh plates were incubated in a CO ₂ incubator for 24 hours.

4) At the end of the incubation, a suspension of SaOS-2 osteoblasts, respectively, in a volume of 1.5 ml and with a density of 6300 cells/ml (in terms of the surface area of the well - 5000 cells cm ² ) was added to the wells with grids. The total volume of medium in each well was 2.25 ml.

5) Plates with grids were transferred to a CO ₂ incubator (37°C, 5% CO ₂ ) and incubated for 5 days until a confluent monolayer formed on the plastic surface.

6) At the end of the incubation, the plates were removed from the CO ₂ incubators, the grids with adherent cells were transferred to empty plates. The grids were washed from the remnants of the medium with a phosphate buffer solution.

7) The grids in the wells were filled with 0.75 ml of 0.25% trypsin solution and incubated in trypsin at 37°C for 15 min. Cells were finally removed from the grids by vigorous pipetting.

8) The number of cells in the obtained cell suspensions (and, accordingly, on the surface of the grids) was determined by counting them in a cell counter MS-20 (Biorad). The cell concentration was 22 thousand cells/ml.

The use of the proposed technique makes it possible to evaluate the cellular compatibility of implants with a mesh structure by counting the number of cells moving to the mesh loops.

Claims (3)

A method for assessing the biocompatibility of an implant with a mesh structure, including the introduction of a sterile implant into a sterile container, the introduction of a cell suspension, incubation in a CO ₂ incubator and subsequent control of cell growth on the implant, characterized in that the implant has a mesh structure, sterilizing it in an autoclave, pouring growth environment consisting wt.%: medium Needle, in the modification of Dulbecco (DMEM) 45% nutritional formula F-12 44.965% fetal calf serum 10% L-glutamine 0.03% gentamicin 0.005% followed by incubation in a CO ₂ incubator for 16-24 hours, adding cell suspension to the wells of the plates with a seeding density of 3-7 thousand cells / cm ² of the area of the wells, while the ratio of the volume of the area of the cell suspension to the growth medium is 2: 1, repeated incubation of plates with grids in a CO ₂ incubator at T 37°C, CO ₂ -5% for 5-14 days and then removing them from the grids with trypsin solution and counting the number of cells.