RU2716577C1 - Method of producing tissue-specific matrix for tissue engineering of cartilage - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to medicine, namely biotechnology. Method involves chopping articular porcine cartilage with fragments of size of not more than 0.5 cm, milling and separating particles with size from 100 mcm to 250 mcm. Then decellularisation is performed: at least 3 cycles of cooling at temperature of -196 °C for one hour with subsequent thawing at 35–40 °C for one hour, incubation in three shifts of phosphate buffer (138 mM NaCl, 2.67 mM KCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4 (FBS), pH = 7.4), volume 20–500 ml, containing 0.1 % sodium dodecyl sulphate and increasing concentration of Triton X100 (1, 2 and 3 %, respectively), incubation for 12–48 hours at temperature of 37 °C in 1–10 ml of solution containing 30–50 U/ml of DNase Type I in a buffer solution prepared from 10 mM Tris-HCl, 2.5 mM MgCl₂, 0.5 mM CaCl₂ , distilled water up to 1 l with pH 7.6. Further, cartilage particles are washed by rinsing with 100–500 ml bidistilled water, exposure for 20–500 ml of bidistilled water during 24 hours at room temperature and periodical stirring at rate of 10–500 rpm for one hour three times throughout the day, exposure for at least 48 hours at room temperature in bidistilled water containing ampicillin in amount of 10–20 mcg/ml and amphotericin at rate of 1.5–2.0 mcg/ml, rinsing in 20–500 ml bidistilled water. Thereafter, the cartilage particles are dried and sterilized by γ irradiation in dose of 1.5 Mrad to obtain samples of the desired matrix.

EFFECT: invention improves completeness of cell removal due to micronisation and proposed original physical effect (freezing/thawing), chemical (mixture of ionic and non-ionic surfactants) and biological (DNase) factors used in optimal combination, easier cellcellularisation of the matrix by cells by increasing the area for settling while maintaining volume and simplifying observation thereof by increasing the area of the matrix adhered surfaces by preliminary micronisation thereof.

3 cl, 7 dwg, 1 tbl, 7 ex

Description

The invention relates to medicine, namely to the field of biotechnology and regenerative medicine, and can be used to repair defects in human cartilage both separately and as part of long-functioning specialized biomedical products in combination with gel-like matrices, both without cells and in the form of cells - and tissue engineering equivalents.

The proposed method can be used in specialized departments involved in reconstructive surgery of damaged cartilage tissue and / or stimulation of in-situ processes of cartilage tissue regeneration, as well as in specialized laboratories in the study of cell and tissue-engineering structures (KIK and TIK) of cartilage.

Currently, regenerative medicine and tissue engineering technologies offer implantation of autologous chondrocytes or mesenchymal stromal cells (MSCs) cultured on three-dimensional biocompatible matrices to restore the structure and functions of irreversibly damaged cartilage tissues. Particular attention is paid to the structure and composition of the matrices, since, in addition to the corresponding mechanical properties that enable surgical use, they must ensure cell adhesion and proliferation, be biocompatible, bioresorbable, eventually being replaced by their own functional tissue.

Matrices for KIK and TIK cartilage are produced from non-specific polymeric materials of synthetic or natural origin in the form of hydrogels, sponges or fibrous nets. Due to the significant compliance with the above requirements, among the matrices, preference is given to polymers of natural origin (biopolymers) and their derivatives: alginates, collagen, gelatin, chitosan, hyaluronic acid, polyesters of bacterial origin [Rai V, Dilisio MF, Dietz NE, Agrawal DK. Recent strategies in cartilage repair: A systemic review of the scaffold development and tissue engineering. J Biomed Mater Res A. 2017; 105 (8): 2343-2354.]. However, these matrices do not have a tissue-specific framework and therefore need decoration (additional coating) or surface modification by introducing site structures for cell adhesion.

Known methods for the manufacture of the native matrix of various organs by decellularization: liver, kidneys, blood vessels, pancreas. Decellularization is a tissue treatment procedure that ensures cell destruction while preserving the extracellular matrix (VKM), whose proteins, unlike cells, carry a small amount of antigens that cause the transplant rejection reaction due to molecular evolutionary conservatism [Bernard MP, Chu ML, Myers JC , Ramirez F, Eikenberry EF, Prockop DJ. Nucleotide sequences of complementary deoxyribonucleic acids for the pro. alpha. 1 chain of human type I procollagen. Statistical evaluation of structures that are conserved during evolution. Biochemistry. 1983; 22 (22): 5213-5223.], While providing the most biochemically and functionally adequate microenvironment for cultured cells. The framework proteins of native matrices integratively contain the remnants of tissue structures (extracellular glycoproteins, structural proteins of intercellular contacts and cell attachment factors), which allow optimizing the conditions for prolonged vital activity of attached cells.

To implement the decellularization of tissues, whole organs or their fragments, various processing methods are used. The most widespread use for this purpose is surface-active substances (surfactants), in particular ionic sodium dodecyl sulfate and non-ionic - Triton X-100 [Porzionato A, Stocco E, Barbon S, Grandi F, Macchi V, De Caro R. Tissue- Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives. Int J Mol Sci. 2018 Dec 18; 19 (12). pii: E4117]. Note that an important problem when using surfactants is the difficulty of their removal.

It should be noted that some types of tissues, due to specific structural features, for example, increased density in the case of cartilage, cannot be effectively decellularized by treatment with surfactants or their mixtures. To remove cells from this kind of tissue, a comprehensive approach is required, consisting in combining surfactant treatment with other physical (ultrasound, temperature, ionic strength), chemical (alternative decellularizing agents) or biological (enzymes) effects [Huang Z, Godkin O, Schulze-Tanzil G. The Challenge in Using Mesenchymal Stromal Cells for Recellularization of Decellularized Cartilage. Stem Cell Rev. 2017 Feb; 13 (1): 50-67].

Stok K.S., Hildner F., van Osch G.J. Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction. Biomed. Mater. 2015; 10: 015010.]As an analogue of the proposed method, we have chosen a known method for the manufacture of a decellularized matrix [Utomo L., Pleumeekers M.M., Nimeskern L.,

Stok KS, Hildner F., van Osch GJ Preparation and characterization of a decellularized cartilage scaffold for ear cartilage reconstruction. Biomed. Mater. 2015; 10: 015010.]

The essence of the analogue method is as follows.

To obtain tissue-specific decellularized cartilage matrix, a calf or human ear was used. The human ear cartilage was used in its entirety, and fragments representing discs with a diameter of 8 mm were carved from the calf's ear.

Decellularization was carried out in four stages:

1. Two cycles of “freeze-thaw” (1 cycle includes 24 hours of incubation at -20 ° C and 24 hours at 45 ° C) in a hypotonic TRIS buffer, pH = 8.0.

2. Exposure for 24 hours in a decellularizing composition, including sodium dodecyl sulfate, ethylenediaminetetraacetic acid and aprotinin.

3. Additional treatment for 24 hours at 37 ° C with a solution of elastase in TRIS buffer (pH = 8.6) with the addition of aprotinin.

4. Treatment for 3 hours at 37 ° C with a solution of nucleases (50 u / ml DNase and 2.5 u / ml RNase) in TRIS buffer pH 7.5.

5. Decontamination in a 0.1% solution of peracetic acid in phosphate buffer.

6. The final three-time washing in phosphate buffer: 2 times for 30 minutes, and then 24 hours at a temperature of 45 ° C.

A feature of the known method is the additional treatment with an elastase solution used in low concentrations (0.03 units / ml), as a result of which it is possible to achieve a sufficiently complete decellularization of the cartilage tissue. The result is a decellularized matrix consisting of VKM proteins, which, when recellularized, allows tissue-specific cells to attach.

The disadvantages of this method include:

риск нарушения структуры матрикса за счет снижения количества эластина в ВКМ, связанного с обработкой эластазой;

the risk of disruption of the matrix structure due to a decrease in the amount of elastin in the ECM associated with elastase treatment

длительная отмывка от детергентов при повышенной температуре (45°С, 24 часа) может привести к денатурации белков ВКМ;

prolonged washing of detergents at elevated temperatures (45 ° C, 24 hours) can lead to denaturation of VKM proteins;

невозможность инъекционного введения, в связи с размерами фрагментов, превышающими диаметр игл, рекомендуемых при лечении поражений суставного хряща (510 мкм);

the impossibility of injecting, due to fragment sizes exceeding the diameter of the needles recommended in the treatment of articular cartilage lesions (510 microns);

стерилизация и хранение децеллюляризованного матрикса хряща в растворе надуксусной кислоты, что не является стандартизированным способом стерилизации и способно приводить к его деструкции и снижению биосовместимых свойств.

sterilization and storage of the decellularized cartilage matrix in a solution of peracetic acid, which is not a standardized method of sterilization and can lead to its destruction and reduction of biocompatible properties.

As a prototype, we have chosen a method for producing tissue-specific decellularized cartilage matrix [Chan LK, Leung VY, Tarn V, Lu WW, Sze KY, Cheung KM. Decellularized bovine intervertebral disc as a natural scaffold for xenogenic cell studies. Acta Biomater. 2013; 9 (2): 5262-5272], the essence of which is as follows.

To obtain tissue-specific decellularized cartilage matrix, bovine intervertebral discs were used. Using a surgical drill, fragments of 2 mm thick discs are obtained. Next, the fragments are subjected to 6 cycles of “freezing-thawing” (1 cycle includes 1 hour of incubation at 37 ° C and 1 hour of incubation in liquid nitrogen at a temperature of -196 ° C). By exposure in a special decellularizing composition, including 0.1% sodium dodecyl sulfate and ethylenediaminetetraacetic acid, followed by washing from the detergent in phosphate buffer during the day, a decellularized matrix consisting of VKM proteins is obtained, which allows tissue cells to attach to the cellulose during recellularization.

The disadvantages of the prototype include:

сохранение большого остаточного количества ядерного материала (21-24% от исходного);

preservation of a large residual amount of nuclear material (21-24% of the original);

низкая эффективность децеллюляризующих растворов и отмывки от детергентов вследствие большого диффузионного расстояния и короткого периода инкубации;

low efficiency of decellularizing solutions and washing from detergents due to the large diffusion distance and short incubation period;

невозможность инъекционного введения, в связи с размерами фрагментов, превышающими диаметр игл, рекомендуемых при лечении поражений суставного хряща (510 мкм);

the impossibility of injecting, due to fragment sizes exceeding the diameter of the needles recommended in the treatment of articular cartilage lesions (510 microns);

отсутствие стандартизованного стерилизации децеллюляризованного матрикса хряща;

lack of standardized sterilization of the decellularized cartilage matrix;

низкая площадь эффективной поверхности для адгезии и пролиферации клеток относительно объема вследствие крупного размера частиц и отсутствия пористости.

low effective surface area for cell adhesion and proliferation relative to volume due to large particle size and lack of porosity.

The technical problem lies in the development of an affordable and cheap way to obtain a cartilage matrix, preserving the native composition and tissue specificity, which can be used for implantation by injection, either separately or as part of KIK and TIK.

The technical result consists in:

- increasing the completeness of cell removal due to micronization and the proposed original complex effect of physical (freezing / thawing), chemical (a mixture of ionic and nonionic surfactants) and biological (DNase) factors applied in the optimal combination;

- facilitating the cellar matrix recellularization by increasing the area for occupation while maintaining the volume and simplifying the observation of it by increasing the area of the adhesive surfaces of the matrix by preliminary micronization;

- ensuring the preservation of tissue specificity due to the selection of an effective combination of influences and their modes that ensure effective decellularization while maintaining the archeectonics and composition of the matrix close to natural;

- eliminating the possibility of toxic effects of the matrix on the body due to standardization and the duration of the cost-effective technology of washing from surfactants using bidistilled water;

- providing a tissue-specific matrix separately and in combination with a gel-like matrix for the formation of TIK cartilage when cultured in a differentiating culture medium with both tissue-specific cells (chondrocytes) and MSCs;

- the use of a standardized sterilization regimen for tissue-specific cartilage matrix through the use of radiation at a dose of 1.5 Mrad, which does not affect cytotoxicity and destroys all types of microorganisms in both vegetative and spore forms;

- providing the possibility of injection, including in the joint cavity;

- elimination of the disadvantages of the prototype regarding the preservation of a large residual amount of nuclear material, low efficiency of decellularizing solutions and washing from detergents due to the large diffusion distance and short incubation period.

The invention consists in the following.

To obtain a tissue-specific matrix for tissue engineering of cartilage, pig pig cartilage is ground and its decellularization is carried out. In this case, first, pig articular cartilage is cut into fragments no larger than 0.5 cm in size, and then the resulting fragments are crushed and particles from 100 μm to 250 μm in size are isolated. Then perform the decellularization of the obtained particles, for this purpose

first at least 3 cycles, each of which includes cooling at a temperature of -196 ° C for one hour, followed by thawing at 35 ° -40 ° C for one hour;

then sequential incubation in solutions of surface-active substances (surfactants) with increasing concentration with stirring at a speed of 10-500 rpm, using as surfactant solutions:

a) 20-500 ml of a solution of phosphate buffer obtained from the composition of 138 mm NaCl, 2.67 mm KCl, 1.47 mm KH ₂ PO ₄ , 8.1 mm Na ₂ HPO ₄ , distilled water up to 1 l with a pH of 7.4 (PBS) containing 1% Triton X 100 and 0.1% sodium dodecyl sulfate sodium (DDS);

b) 20-500 ml of PBS solution containing 2% Triton X 100 and 0.1% DDS;

c) 20-500 ml of PBS solution containing 3% Triton X 100 and 0.1% DDS;

then incubation for 12-48 hours at 37 ° C in 1-10 ml of a solution containing 30-50 U / ml of type I DNase in a buffer solution obtained from 10 mM Tris-HCl, 2.5 mM MgCl ₂ , 0.5 mmol CaCl ₂ , distilled water up to 1 l with a pH of 7.6.

Next, the cartilage particles are washed by sequentially performing the following steps:

rinsing with 100-500 ml bidistilled water,

exposure during the day in 20-500 ml of double-distilled water at room temperature and periodic stirring at a speed of 10-500 rpm for one hour three times during the day,

exposure for at least 48 hours at room temperature in bidistilled water containing ampicillin at a rate of 10-20 μg / ml and amphotericin at a rate of 1.5-2.0 μg / ml,

rinsing in 20-500 ml of double-distilled water.

After that, the washed particles of decellularized cartilage are dried and sterilized with γ-radiation at a dose of 1.5 Mrad to obtain samples of the desired matrix.

Before sterilization, cartilage particles can be stored at a temperature of -30 ° C to -80 ° C.

Sterile cartilage matrix samples are stored at 4-8 ° C.

The method is as follows.

1. Joint pig cartilage is isolated mechanically, cut into fragments no larger than 0.5 cm in size, frozen at -80 ° C and stored at this temperature until grinding begins.

2. Get finely dispersed particles of pig cartilage (MDCHC) by grinding cartilage fragments using a cryomill (25 Hz for 2 minutes at a temperature of -196 ° C), which allows grinding under continuous cooling with liquid nitrogen.

3. Fractions of particles of the required size in the range of 100-250 μm are distinguished by sieving grinding through a set of sieves with the appropriate pore size.

4. The decellularization of the sample (1-1.5 g) MDCHC carried out in three stages:

a. by cooling the MFCC in a Dewar vessel at -196 ° C for one hour, followed by thawing at 35 ° C-40 ° C for one hour (the freeze / thaw cycle should be repeated at least three times).

b. treatment of MDC in three shifts of phosphate buffer (138 mM NaCl, 2.67 mM KCl, 1.47 mM KH ₂ PO ₄ , 8.1 mM Na ₂ HPO ₄ , pH 7.4 (PBS), pH = 7.4), containing 20-500 ml 0.1% sodium dodecyl sulfate and an increasing concentration of Triton X100 (1, 2 and 3%, respectively) at room temperature and periodic stirring on a magnetic stirrer (3 times a day, 1 hour, 10-500 rpm);

in. after rinsing with 400 ml of double-distilled water, MFCS were incubated for 12-48 hours at 37 ° C in 1-10 ml of buffer solution (10 mM Tris-HCl, 2.5 mM MgCl ₂ , 0.5 mM CaCl ₂ ; pH = 7.6), containing 30-50 U / ml of type I DNase (Qiagen, Germany).

5. Washing samples (1-1.5 g) MDCHC from detergents is carried out in four stages:

a. rinsing with 100-500 ml bidistilled water;

b. washing in 20-500 ml of double-distilled water at room temperature and periodically stirring on a magnetic stirrer for a day (3 times, 1 hour, 10-500 rpm) for 24 hours.

in. final washing at room temperature in bidistilled water (at least 48 hours) containing an antibiotic (ampicillin, 10-20 μg / ml) and antimycotic (amphotericin, 1.5-2.0 μg / ml).

d. The decellularized MFCS was rinsed in 20-500 ml bidistilled water (to remove antibiotic and antimycotics), excess moisture was removed using paper filters and nylon membranes with a pore size of 10 μm.

6. The decellularized MDCTs were transferred to 2 ml cryovials and stored until sterilization at a temperature of -30 ° C to -80 ° C.

7. The samples were sterilized by gamma radiation at a dose of 1.5 Mrad.

8. The sterile decellularized matrix was stored at a temperature of 4-8 ° C.

To prove the possibility of achieving the declared purpose - the suitability of a decellularized tissue-specific cell for planting on it and creating TIC cartilage, both when used separately and in combination with gel-like matrices, and to achieve the indicated technical result, we present the following data.

Example 1

Obtaining a decellularized matrix from pig cartilage according to the proposed method:

1. Joint pig cartilage is isolated mechanically, cut into fragments no larger than 0.5 cm in size, frozen at -80 ° C and stored at this temperature until grinding begins.

2. Get finely dispersed particles of pig cartilage (MDCHC) by grinding cartilage fragments using a cryomill (25 Hz for 2 minutes at a temperature of -196 ° C), which allows grinding under continuous cooling with liquid nitrogen.

3. Fractions of particles of the required size in the range of 100-250 μm are distinguished by sieving grinding through a set of sieves with the appropriate pore size.

4. The decellularization of the sample (1-1.5 g) MDCHC carried out in three stages:

a. by cooling MDCT in a Dewar vessel at -196 ° C for one hour, followed by thawing at 40 ° C for one hour. The freeze / thaw cycle must be repeated at least three times.

b. by treatment of MDC in three shifts of PBS, pH = 7.4, 500 ml, containing 0.1% sodium dodecyl sulfate and an increasing concentration of Triton X100 (1, 2 and 3%, respectively) at room temperature and periodically stirring on a magnetic stirrer (3 times day, 1 hour, 500 rpm);

in. after rinsing with 400 ml of double-distilled water, MFCS were incubated for 48 hours at 37 ° C in 1 ml of buffer solution (10 mM Tris-HCl, 2.5 mM MgCl ₂ , 0.5 mM CaCl ₂ ; pH = 7.6) containing 50 U / ml of type I DNase (Qiagen, Germany).

5. Washing samples (1-1.5 g) MDCHC from detergents is carried out in four stages:

a. rinsing with 500 ml of double-distilled water;

b. washing in 500 ml of double-distilled water at room temperature and periodically stirring on a magnetic stirrer for a day (3 times, 1 hour, 500 rpm) for 24 hours.

in. final washing at room temperature in bidistilled water (at least 48 hours) containing an antibiotic (ampicillin, 10 μg / ml) and antimycotic (amphotericin, 1.5 μg / ml).

d. Decellularized MFCS were rinsed in 500 ml bidistilled water (to remove antibiotic and antimycotics), excess moisture was removed using paper filters and nylon membranes with a pore size of 10 μm.

b. The decellularized MDCTs were transferred to 2 ml cryovials and stored until sterilization at -80 ° C.

7. The samples were sterilized by gamma radiation at a dose of 1.5 Mrad.

8. The sterile decellular matrix was stored at 4 ° C.

Cell removal from pig cartilage microparticles was confirmed using histological staining with hematoxylin and eosin.

In FIG. 1 presents a histological picture of tissue-specific decellularized cartilage matrix obtained using the proposed method. SW X100. Hematoxylin and eosin staining. 1 - stored extracellular matrix; 2 - empty gaps without cells.

As seen in FIG. 1, the preserved cells in the sample are not detected, only empty gaps are visualized.

The results obtained indicate the absence of whole cells, of which DNA is an integral component, in the decellularized matrix.

Example 2

Obtaining a decellularized matrix from pig cartilage according to the proposed method:

1. Joint pig cartilage is isolated mechanically, cut into fragments no larger than 0.5 cm in size, frozen at -80 ° C and stored at this temperature until grinding begins.

2. Receive MFCA with grinding fragments of cartilage using a cryomill (25 Hz for 2 minutes at a temperature of -196 ° C), allowing grinding under continuous cooling with liquid nitrogen.

3. Fractions of particles of the required size in the range of 100-250 μm are distinguished by sieving grinding through a set of sieves with the appropriate pore size.

4. The decellularization of the sample (1-1.5 g) MDCHC carried out in three stages:

a. by cooling the MDCTs in a Dewar vessel at -196 ° C for one hour, followed by thawing at 35 ° C for one hour. The freeze / thaw cycle must be repeated at least three times.

b. treatment MDC in three shifts of phosphate buffer (138 mm NaCl, 2.67 mm KCl, 1.47 mm KH ₂ PO ₄ , 8.1 mm Na ₂ HPO ₄ , pH 7.4 (PBS), pH = 7.4), a volume of 20 ml containing 0, 1% sodium dodecyl sulfate and an increasing concentration of Triton X100 (1, 2 and 3%, respectively) at room temperature and periodic stirring on a magnetic stirrer (3 times a day, 1 hour, 10 rpm);

in. after rinsing with 400 ml of double-distilled water, MFCS were incubated for 12 hours at 37 ° C in 10 ml of buffer solution (10 mm Tris-HCl, 2.5 mm MgCl ₂ , 0.5 mmol CaCl ₂ ; pH = 7.6) containing 30 U / ml of type I DNase (Qiagen, Germany).

5. Washing samples (1-1.5 g) MDCHC from detergents is carried out in four stages:

a. rinsing with 100 ml bidistilled water;

6. washing in 20 ml of double-distilled water at room temperature and periodically stirring on a magnetic stirrer for a day (3 times, 1 hour, 10 rpm) for 24 hours.

in. final washing at room temperature in bidistilled water (at least 48 hours) containing an antibiotic (ampicillin, 20 μg / ml) and antimycotic (amphotericin, 2.0 μg / ml).

g. Decellularized MFCS were rinsed in 20 ml bidistilled water (to remove antibiotic and antimycotics), excess moisture was removed using paper filters and nylon membranes with a pore size of 10 μm

b. The decellularized MDCTs were transferred to 2 ml cryovials and stored until sterilization at -30 ° C.

7. The samples were sterilized by gamma radiation at a dose of 1.5 Mrad.

8. The sterile decellularized matrix was stored at a temperature of 8 ° C.

To prove the possibility of injecting tissue-specific decellularized cartilage matrix obtained by the proposed method (see examples 1 and 2), the particle size distribution was evaluated using a SALD-7101 laser diffraction analyzer (Shimadzu, Japan). Glycerin was used as a liquid dispersion medium.

The particle size range of the tissue-specific decellularized cartilage matrix in suspension was 96-220 μm, while particles with a size of 161 ± 11 μm prevailed, indicating the possibility of the least invasive injection of the matrix.

Example 3

To prove the high degree of cell removal from the matrix obtained by the proposed method (see Example 1), the degree of decellularization of MFCS was evaluated using a modified method [Larynx decellularization: combining freeze-drying and sonication as an effective method / S.H. Hung, C.H. Su, F.P. Lee, H. Tseng // J. Voice. - 2013. - V. 27. - P. 289-294]. For the quantitative determination of decellularized and non-decellularized particles in samples subjected to decellularization in different modes, staining was performed in a 24-well plate with a solution of DNA-binding fluorescent dye DAPI at a concentration of 1 μg / ml (6 mg of cartilage per 1 well).

In each sample, using a Nikon Ti fluorescence microscope, the number of particles was determined:

не децеллюляризированных, богатых клетками;

not decellularized, rich in cells;

не децеллюляризированных, содержащих отдельные клетки;

not decellularized, containing single cells;

децеллюляризованные.

decellularized.

At the bottom of the hole, 15 fields of view were randomly selected with an increase in X40. In each field, the number of particles of each type was determined. The results for all fields were summarized, and the number of particles of each type in the sample was calculated as a percentage of the total number of particles. As a positive control, native cartilage particles were used after cryo-grinding; cartilage particles after cryoprice that underwent decellularization using a surfactant similarly to the prototype; cartilage particles after cryoprice, which have undergone decellularization, including a combination of 3 cycles of “freezing / thawing” and processing of surfactants, similarly to the prototype.

The amount of DNA in the tissue-specific decellularized cartilage matrix was also determined using PicoGreen fluorescent dye. For DNA isolation, the DNeasy Blood & Tissue Kit (QIAGEN, Germany) was used according to the instructions.

Table 1 shows the effect of the decellularization regime on the amount of DNA in the particles of decellularized cartilage.

As can be seen from the table, the proposed method for the decellularization of pig articular cartilage ensures the complete absence of particles with DNA in the samples.

Analysis of the DNA content showed a decrease in the amount of nuclear material in the tissue-specific decellularized cartilage matrix to 9.11 ± 1.13 ng / mg tissue (2.5% of the amount of DNA in the original tissue). The obtained results testified to the effectiveness of the performed decellularization, after which the matrix was significantly cleared of nuclear material (according to the accepted criteria, the quantitative DNA content should be less than 50 ng / mg in dry tissue [Crapo P.M., Gilbert T.W., Badylak SF An overview of tissue and whole organ decellularization processes // Biomaterials. - 2011. - Vol. 32. - No. 12. - P. 3233-3243.]).

The decrease in DNA confirms the high degree of decellularization of the matrix obtained by the proposed method, in contrast to the control samples.

Example 4

To prove the provision of tissue-specific matrix obtained by the proposed method (see example 1), the formation of TIC cartilage, we present the results of the study of adhesion, proliferation and differentiation of various types of cells on this matrix.

Tissue-specific cartilage cells, human chondroblasts, and mesenchymal stromal cells of human adipose tissue (MSC MTC), capable of differentiating into cartilage tissue cells, were placed on the matrix. The cell culture of the MSC of the VT was obtained from a subcutaneous fatty tissue fragment from a healthy donor according to the standard method [Surguchenko VA, Ponomareva AS, Kirsanova LA, Skaleckij NN, Sevastianov VI The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue -derived mesenchymal stromal cells (in vitro study) // J. Biomed. Mat. Soc. Part A 2015; 103 (2): 463-470]. Chondroblasts from fragments of cartilage of human costal arches obtained during surgical interventions with the informational consent of the patient were used. The material was washed in a Hanks solution with the addition of a culture antibiotic-antimycotic. The tissue was ground with scissors to obtain a homogeneous mass consisting of fragments with a volume of ~ 1 mm ³ , after which they were incubated in a 0.1% type I collagenase solution at 37 ° C for 12 hours. Cells of passage 3 were used in the experiment.

200 μl of suspension with a working concentration of 1 × 10 ⁶ cells in 1 ml of chondrogenic differentiating medium (DMEM HG culture medium supplemented with GlutaMAX ™ containing 10% ITS +, 1% sodium pyruvate, 0.25%) was placed in each conical well of a 96-well culture plate. ascorbate-2-phosphate, 0.0001% dexamethasone, 0.002% TGF-β1 and 1% culture antibiotic-antimycotic) and 20 mg of the decellularized cartilage matrix. Then the tablet was centrifuged at 500 g for 5 minutes. Next, the tablet was placed in a CO ₂ incubator under standard conditions (5% CO ₂ , 37 ° C). The culture medium in the wells was replaced daily. The study of the results of attachment, proliferation and differentiation of cells was carried out on the 14th day of cultivation. A biopolymer microheterogeneous collagen-containing matrix (BMKG) (Biomir Service JSC, Krasnoznamensk) of the Long brand was used as a control.

In FIG. Figure 2 presents TECs, including chondroblasts and a decellularized matrix of pig articular cartilage. SW X100. Alcian blue staining for glycosaminoglycans, where 3 is a tissue-specific decellularized matrix, 4 are cells, 5 is produced by VKM cells.

In FIG. Figure 3 presents TECs, including MSCs of VT and the decellularized matrix of pig articular cartilage. SW X100, staining with glycaminoglycans with Alcian blue, where 3 is a tissue-specific decellularized matrix, 4 are cells, 5 is produced by VKM cells.

In FIG. 4 presents TIC, including chondroblasts and BMKG brand "long" (control matrix). SW X100. Alcian blue staining for glycosaminoglycans, where 6 - BMKG, 4 - cells, 5 - produced by VKM cells.

In FIG. Figure 5 presents the TEC, including MSC ZhTch and BMKG brand "long" (control matrix). SW X200 .. Staining with alcian blue for glycosaminoglycans (6 - BMKG, 4 - cells, 5 - produced by VKM cells).

The analysis made it possible to establish that all cell types actively adhere and proliferate on the surface of matrix particles. Moreover, the positive staining of all samples with Alcian blue for glycosaminoglycans indicated the production of cells that entered differentiation in the chondrogenic direction, specific for the cartilage tissue of VKM. However, the cell mass on the surface of the decellularized matrix was significantly larger than on the non-tissue-specific control.

Thus, the expressed adhesive and proliferative properties of the decellularized matrix with respect to tissue-specific cells and cells with the ability to differentiate in the chondrogenic direction prove the preservation of the tissue-specific properties of the decellularized matrix and are most suitable for use as a matrix for creating TEC of cartilaginous tissue.

Example 5

The study of the decellularized matrix of pigs for cytotoxicity was carried out in accordance with GOST R ISO 10993-1-2011) [Interstate standard GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effects of medical devices. Part 4. The study of products in contact with blood "; GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effects of medical devices. Part 5. Cytotoxicity test: in vitro methods ”].

In the experiment, the decellularized matrix of pig cartilage obtained by the proposed method (see example 1), MSC MLC cells obtained by the standard method [Surguchenko V.A., Ponomareva A.S., Kirsanova L.A., Skaleckij N.N., Sevastianov V.I. The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (in vitro study) // J. Biomed. Mat. Soc. Part A 2015; 103 (2): 463-470] and a full growth culture medium (DMEM / F12 culture medium supplemented with GlutaMAX ™; fetal calf serum, sterile, tested for cytotoxicity and absence of mycoplasma - 10%; complex antimicrobial additive Gibco® Antibiotic-Antimycotic-1 %; HEPES1 mM; fibroblast growth factor (FGF-basic) 1 μg / L). In the experiment, cells of the third passage were used.

All manipulations were performed under aseptic conditions. ZhKh MSCs were seeded in 24-well flat-bottomed culture plates and incubated at 37 ° C under standard conditions: a humid atmosphere containing (5 ± 1)% CO ₂ until a monolayer with a degree of confluency of 80-85% was formed. The investigated matrix samples were placed on the surface of the formed monolayer of cells.

In the experiment, we studied both the cytotoxicity of directly MFCS by direct contact and extracts from the matrix. To prepare extracts, 1 pellet of a sample weighing 0.03 g was placed for 24 hours in 0.5 ml of culture medium under standard conditions. After that, the indicated volume of the extract was added to the complete growth medium in the well of the plate with a monolayer of cells.

The primary recording of results in the variant with ZhK MSC cells, according to the standards, was carried out after 24 hours. To identify a possible prolonged cytotoxic effect, cell growth was monitored up to 3 days.

A negative control was the culture medium for ZhKh MSC cells containing serum; a positive control was a standard solution of zinc in nitric acid Zn 1-2 wt. % HNO ₃ , 1: 200 dilution with 0.9% NaCl solution for injection).

Assessment of the cytotoxic effect of samples of finely dispersed matrix from decellularized pig cartilage, both by the method of extracts and by direct contact, showed the absence of cytotoxicity. The data of phase contrast microscopy are confirmed by the growth curves of MSC ZhTch.

The exclusion of the possibility of toxic effects of the matrix on the body due to the standardization of technology for washing from surfactants is confirmed by cytotoxicity studies.

Example 6

To prove the provision of a tissue-specific matrix obtained by the proposed method (see example 1), together with the gel-like matrix of the formation of TIC of cartilage, we present the results of a study of the adhesion, proliferation and differentiation of human chondroblasts and MSC of ZhTch.

The cell culture of the MSC of ZhTch was obtained from a subcutaneous fatty tissue fragment from a healthy donor according to the standard procedure [Surguchenko V.A., Ponomareva A.S., Kirsanova L.A., Skaleckij N.N., Sevastianov V.I. The cell-engineered construct of cartilage on the basis of biopolymer hydrogel matrix and human adipose tissue-derived mesenchymal stromal cells (in vitro study) // J. Biomed. Mat. Soc. Part A 2015; 103 (2): 463-470]. Chondroblasts from fragments of cartilage of human costal arches obtained during surgical interventions with the informational consent of the patient were used. The material was washed in a Hanks solution with the addition of a culture antibiotic-antimycotic. The tissue was ground with scissors to obtain a homogeneous mass consisting of fragments with a volume of ⋅ ~ 1 mm3, after which they were incubated in a 0.1% type I collagenase solution at 37 ° C for 12 hours. Cells of passage 3 were used in the experiment.

200 μl of suspension with a working concentration of 1 × 10 ⁶ cells in 1 ml of chondrogenic differentiating medium (DMEM HG culture medium supplemented with GlutaMAX ™ containing 10% ITS +, 1% sodium pyruvate, 0.25%) was placed in each conical well of a 96-well culture plate. ascorbate-2-phosphate, 0.0001% dexamethasone, 0.002% TGF-β1 and 1% culture antibiotic-antimycotics) and 20 mg of the decellularized cartilage matrix mixed with 60 mg of BMKG brand Light (JSC Biomir Service, Krasnoznamensk) . Then the tablet was centrifuged at 500 g for 5 minutes. Next, the tablet was placed in a CO ₂ incubator under standard conditions (5% CO ₂ , 37 ° C). The culture medium in the wells was replaced daily. The study of the results of attachment, proliferation and differentiation of cells was carried out on the 14th day of cultivation.

In FIG. Figure 6 shows TECs, including chondroblasts and a decellularized matrix of pig articular cartilage mixed with light BMKG. SW X100. Alcian blue staining for glycosaminoglycans, where 3 is a tissue-specific decellularized matrix, 4 are cells, 5 is produced by VKM cells.

In FIG. 7 shows TECs, including MSC ZhTch and the decellularized matrix of pig articular cartilage, mixed with BMKG brand "light". SW X100. Alcian blue staining for glycosaminoglycans, where 3 is a tissue-specific decellularized matrix, 4 are cells, 5 is produced by VKM cells.

After 14 days of cultivation in a chondrogenic medium, active proliferation of chondroblasts and MSCs of VT was observed, while resorption of tissue-specific decellularized cartilage matrix was visualized. Note that the increase in the number of cells was accompanied by the production of tissue-specific VKM containing glycosaminoglycans in the cells.

The study confirms the suitability of tissue-specific decellularized matrix when used in combination with a gel-like matrix for the formation of TEC of human cartilage tissue.

Similar results were obtained when conducting experiments in accordance with examples 3-6 using the cartilage matrix obtained in accordance with example 2.

Example 7

To prove the biocompatibility of decellularized fine particles of pig cartilage, a method for determining hemolysis was used.

In the experiment, rabbit blood mixed in a 9: 1 ratio with 3.8% sodium citrate solution as an anticoagulant was used.

Samples of MDCTs were transferred to bottles and 0.9% sodium chloride solution was added in the ratio of sample weight (g): extraction liquid (ml) = 1:30.

To prepare a control sample, 3 ml of 0.9% sodium chloride solution was poured into a test tube. To prepare a sample with 100% hemolysis, 3 ml of distilled water was poured into a test tube.

All test tubes with solutions were incubated at a temperature of 37 ° C for 120 min. Then, rabbit citrate blood was added to each tube at a rate of 200 μl per 10 ml of extract, stirred and re-incubated in an incubator at 37 ° C for 1 hour. After incubation, the tubes were placed in a centrifuge to precipitate blood at an acceleration of 2000 rpm for 20 minutes. Optical density of the supernatant at a wavelength of 545 nm was measured on a Stat Fax 1904+ biochemical analyzer. The quantitative criterion of the method was the relative value of hemolysis (α _r ) in%, determined by the formula:

where: E _op - the optical density of the experimental sample;

E _to - the optical density of the control sample;

E ₁₀₀ is the optical density of the sample with 100% hemolysis.

A sample is recognized as hemocompatible by a hemolysis test if α _g ≤ 2%.

The results of testing the biocompatible properties of MDCHc, decellularized according to the proposed method, showed that hemolysis induced by contact with a foreign surface is practically absent (α _g ≤ 0.1%), therefore, the decellularized MDCCHs comply with the requirements of GOST R ISO 10993-1-2011 to biocompatible materials.

Claims (15)

1. A method of producing tissue-specific matrix for tissue engineering of cartilage, including grinding pig cartilage, its decellularization, characterized in that the pig’s articular cartilage is first cut into fragments no larger than 0.5 cm in size, and then the fragments obtained are crushed and particles from 100 microns to 250 microns; after which they perform the decellularization of the obtained particles, while first at least 3 cycles, each of which includes cooling at a temperature of -196 ° C for one hour, followed by thawing at 35-40 ° C for one hour; then sequential incubation in solutions of surface-active substances (surfactants) with increasing concentration with stirring at a speed of 10-500 rpm, using as surfactant solutions: a) 20-500 ml of a solution of phosphate buffer obtained from the composition of 138 mm NaCl, 2.67 mm KCl, 1.47 mm KH ₂ PO ₄ , 8.1 mm Na ₂ HPO ₄ , distilled water up to 1 l with a pH of 7.4 (PBS) containing 1% Triton X 100 and 0.1% sodium dodecyl sulfate sodium (DDS); b) 20-500 ml of PBS solution containing 2% Triton X 100 and 0.1% DDS; c) 20-500 ml of PBS solution containing 3% Triton X 100 and 0.1% DDS; then incubation for 12-48 hours at 37 ° C in 1-10 ml of a solution containing 30-50 U / ml of type I DNase in a buffer solution obtained from 10 mM Tris-HCl, 2.5 mM MgCl ₂ , 0.5 mmol CaCl ₂ , distilled water up to 1 l with a pH of 7.6; then wash cartilage particles by sequentially performing the following steps: rinsing with 100-500 ml bidistilled water, exposure during the day in 20-500 ml of double-distilled water at room temperature and periodic stirring at a speed of 10-500 rpm for one hour three times during the day, exposure for at least 48 hours at room temperature in bidistilled water containing ampicillin at a rate of 10-20 μg / ml and amphotericin at a rate of 1.5-2.0 μg / ml, rinsing in 20-500 ml of double-distilled water; after which the washed particles of decellularized cartilage are dried and sterilized with γ-radiation at a dose of 1.5 Mrad to obtain samples of the desired matrix. 2. The method according to p. 1, characterized in that until sterilization the cartilage particles are stored at a temperature of from -30 ° C to -80 ° C. 3. The method according to p. 1, characterized in that sterile cartilage matrix samples are stored at a temperature of 4-8 ° C.

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