RU2770558C2 - Method for creating tissue-engineering structures by bioprinting with bioink for cartilage tissue regeneration under body conditions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and relates to a method for creating tissue engineering structures by bioprinting with bioink for regeneration of cartilage tissue in the body, including the preparation of bioink, which is obtained immediately before the 3D bioprinting process, and then used to create a tissue engineering structure, where the bioink includes the following components: constructions, where the bioink includes the following components: collagen, mesenchymal stem cells (MSCs) and decellularized extracellular matrix (dECM) powder, moreover, dECM was obtained from the costal cartilages of female rats that were subjected to decellularization and lyophilization, the resulting powder is sieved through a sieve and before use stored at +4°C, in the preparation of bioink, sterile type I porcine atelocollagen is used, which is mixed with MSC, with a buffer solution and dECM powder, then the scaffold is printed and filled with a warm nutrient medium, after which the incubation process is carried out in a CO₂ incubator.

EFFECT: invention ensures the creation of tissue-engineering structures that recreate the cellular structure and function of the cartilage tissue to be replaced, as well as high accuracy in the creation of a tissue-engineered structure by means of 3D bioprinting.

1 cl, 3 dwg, 1 tbl, 1 ex

Description

The invention relates to tissue engineering and 3D bioprinting and can be used to create tissue engineering structures (scaffolds).

3D bioprinting is a promising technology for manufacturing tissue structures of complex external geometry and/or internal structure, with strictly defined mechanical properties, with a personalized composition. The main component in 3D bioprinting is bioink, which is a biomaterial in the form of a hydrogel containing a certain number and types of cells. Bioink should have such biological characteristics as biocompatibility, cytocompatibility, biodegradation at a controlled rate, close to the rate of regeneration of the replaced tissue. The preparation of bioink is carried out using various natural and synthetic materials and their combinations, such as collagen, gelatin, agarose, silk fibroin, alginate, etc. However, most of these materials are not able to recreate the microenvironment typical of living tissues, internal cellular morphology and functions .

An ideal sample of such a material can be the basis of the target tissue itself – its extracellular matrix (ECM). The composition of the ECM of each tissue is unique. It is represented by a complex network of components, the main of which are fiber-forming proteins such as collagens, elastin, fibronectin, laminins, glycoproteins, proteoglycans and glycosaminoglycans [Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev. 2016, 97: 4-27. DOI: 10.1016/j.addr.2015.11.001]. ECM regulates many cellular processes, including growth, migration, differentiation, homeostasis, and morphogenesis [K.C. Clause, T.H. barker. Extracellular matrix signaling in morphogenesis and repair. Curr. Opin. Biotechnol., 2013.24: 830-833; Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev. 2016; 97:4-27. DOI: 10.1016/j.addr.2015.11.001.]. It is the main source and conductor of biochemical and biomechanical signals to ensure the organization and functioning of the tissue as a whole [Herrera J, Henke CA, Bitterman PB. Extracellular matrix as a driver of progressive fibrosis. J Clin Invest. 2018, 128(1): 45-53. DOI: 10.1172/JCI93557.], providing a physical and mechanical microenvironment of cells [Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev. 2016, 97: 4-27. DOI: 10.1016/j.addr.2015.11.001].

Recently, data have appeared on the use of hydrogels based on decellularized HMC (dVCM) in bioprinting. Bioinks based on dVCM initially have tissue-specific properties.

However, the use of dVCM in the form of a hydrogel is limited by the weak mechanical properties of the resulting tissue engineering structures and the low accuracy of the printing process itself.

There is a variant of creating bio-ink based on fibrin gel with the addition of dVKM microparticles for the reconstruction of the cornea of the eye.[H. Yin, Q. Lu, X Wang, et al. Tissue-derived microparticles reduce inflammation and fibrosis in cornea wounds. Acta Biomater. 2019, 85:192-202. doi: 10.1016/j.actbio.2018.12.027; A Chandru, P Agrawal, SK Ojha, et al. Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds Biomolecules 2021, 11(4). 10.3390/biom11040532]. In these works, the hydrogel was used without 3D bioprinting.

The disadvantage of the above methods is the inapplicability of these hydrogels for bioprinting due to the rheological properties of the hydrogels.

Closest to the claimed invention (prototype) is a method of using bioink presented in the work of M. Kimetal. [Kim MK, Jeong W, Lee SM, et al. Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties. Biofabrication 2020; 12: 025003], where dVKM was used in the form of microparticles as part of a gelatin hydrogel (hyaluronic acid 3 mg/ml + gelatin 37.5 mg/ml + fibrinogen 3 mg/ml). The resulting bioink formulations were examined for cytocompatibility under in vitro conditions. This research is primarily aimed at creating bio-inks that can correct liver defects, they cannot be applied to repair cartilage tissue defects.

The technical result is the creation of tissue engineering structures that recreate the cellular structure and function of the cartilage tissue to be replaced, as well as ensuring high accuracy in creating a tissue engineering structure using 3D bioprinting.

The technical result is solved by the fact that, in the same way as in the known method, bioink is prepared, which is obtained immediately before the 3D bioprinting process and bioprinting of a tissue engineering structure capable of recreating the cellular structure and function of the replaced tissue is carried out with an extrusion nozzle.

A feature of the proposed method is that the bioink includes the following components: collagen, mesenchymal stem cells (MSCs) and decellularized extracellular matrix (dECM) powder, and dECM was obtained from the costal cartilage of female Wistar rats, which were subjected to decellularization and lyophilization, the resulting powder sieved through a sieve with a pore diameter of 280 μm and stored at +4°C until use; when preparing bioink with a collagen concentration of 4%, sterile type I porcine atelocollagen is used - 80 mg / ml, which is mixed with MSCs - 5 × 10 ⁶ per ml, with a buffer solution - 250 µl per ml and dVKM powder - 25 mg/ml, then the scaffold is printed and poured with warm nutrient medium t37°C, after which the incubation process is carried out at 37°C in a CO ₂ incubator.

The invention is illustrated by a detailed description, examples of use and illustrations, which show:

Fig. 1 - Test print to assess the printability of bioink (schematic representation of the printed object).

Fig. 2 - Scaffold 1 week after implantation, about. ×10.

Fig. 3 - Formation of cartilage tissue 2 weeks after implantation, about. ×10.

The method is carried out as follows.

To create tissue engineering constructs, collagen hydrogel, powdered ECM and MSC culture are used. Powdered ECM is presented in the form of dry lyophilized granules, their diameter does not exceed the diameter of the bioprinter nozzle. If necessary, cell growth factors, their addition to the bioink is carried out at the mixing stage. Printing is carried out on laboratory culture dishes (for example, Petri dishes, well plates) or on glass located on top of the printing table. After the bioprinting process, the scaffold is flooded with warm nutrient medium (from 25 to 37°C), for example, RPMI-1640, DMEM, F-12. The incubation process is carried out at 37°C in a CO ₂ incubator. The medium is replaced during the incubation process once a day.

To assess the printability of bioink based on 4% collagen and dVKM powder, printing was carried out in a test mode (Fig. 1), after which it becomes possible to evaluate the line thickness (3 layers of filament) in 12 places and the area of formed niches in 8 places (indicates ongoing polymerization process). Both parameters were evaluated in dynamics: immediately after printing and after 24 hours of incubation in PBS buffer. Data on bioink and hydrogel based on collagen (without dVKM) are presented in Table 1.

Table 1

Results of test printing with bio-ink based on 4% collagen with and without dVCM granules

Collagen Collagen + dVKM X ± S _x [confidence interval] After printing After incubation After printing After incubation Niches, mm ² 1.516±0.125
[1.427÷1.605] 1.411±0.103
[1.338÷1.485] 1.286±0.126
[1.195÷1.376] 1.097±0.094
[1.030÷1.164] Lines, mm 0.886±0.017
[0.874÷0.898] 0.901±0.027
[0.882÷0.920] 0.920±0.036
[0.894÷0.946] 0.957±0.027
[0.938÷0.976]

An example of creating a tissue engineering construct in vivo to create a complete de novo tissue.

ECM was obtained from the costal cartilage of Wistar rats, females, 1 to 1.5 months old, which had been pre-treated, decellularized and lyophilized. Lyophilized cartilage was ground with a pestle in a mortar. The resulting powder was sifted through a stainless steel sieve with a pore diameter of 280 μm and stored in a sealed package at +4°C until use. For the preparation of bioink with a collagen concentration of 4%, sterile type I porcine atelocollagen - 80 mg/ml was used, which was mixed with MSC - 5 × 10 ⁶ per ml, Tris-HCl buffer solution - 250 μl per ml, dVKM powder - 25 mg/ml . After preparing the bi-ink, the scaffold was bioprinted. The scaffold was flooded with warm (37°C) DMEM nutrient medium. Further in the CO ₂ -incubator held the process of incubation at 37°C.

The scaffolds were implanted in outbred male white rats. After 1 and 2 weeks, the animals were euthanized and the material was taken for histological examination. One week after implantation, a connective tissue capsule was formed around the scaffolds, which grew into collagen, dividing it into separate fragments, the cells were unevenly distributed, and cartilage tissue formed in some areas of the scaffolds (Fig. 2). Connective tissue cells were also visualized between bundles of collagen fibers. Two weeks after implantation in animals, the scaffolds were replaced by newly formed cartilage tissue (Figure 3).

The proposed method makes it possible to create tissue engineering structures that recreate the cellular structure and function of the replaced cartilage tissue.

Claims (1)

A method for creating tissue engineering constructs by bioprinting with bioink for regeneration of cartilage tissue in the body, including the preparation of bioink, which is obtained immediately before the 3D bioprinting process, and then used to create a tissue engineering construct, characterized in that the bioink includes the following components: collagen, mesenchymal stem cells (MSCs) and powder of decellularized extracellular matrix (dECM), moreover, dECM was obtained from costal cartilages of female Wistar rats, which were subjected to decellularization and lyophilization, the resulting powder was sifted through a sieve with a pore diameter of 280 μm and stored at +4°C until use , in the preparation of bioink with a collagen concentration of 4%, sterile type I porcine atelocollagen is used - 80 mg / ml, which is mixed with MSC - 5 × 10 ⁶ per ml, with a buffer solution - 250 μl per ml and dVKM powder - 25 mg / ml, then the scaffold is printed and filled with a warm nutrient medium t 37 ° C, after which it is carried out Incubation process at 37°C in a CO ₂ incubator.

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