RU2805231C1 - Method for assessing effectiveness of using cell-engineered construct based on polymer carrier and cell culture in experiment when replacing hyaline cartilage defects - Google Patents

Abstract

FIELD: traumatology; orthopaedics.

SUBSTANCE: invention can be used in tissue engineering to evaluate the effectiveness of using a cell-engineered construct in vivo based on a polymer carrier and cell culture in an experiment when replacing hyaline cartilage defects. The method involves performing electron microscopy, where the resulting images are analysed quantitatively and qualitatively, the degree of defect replacement is determined by the change in the area of the defect and the surface structure of the damaged area is analysed, then points are assigned. Then the points are summed up and a value from 0 to 10 is obtained, where 0 is the ineffective use of the cell-engineered construct, and 10 is the most effective use of the cell-engineered construct.

EFFECT: improved efficiency of assessing the effectiveness of using a cell-engineered construct based on a polymer carrier and cell culture in an experiment when replacing hyaline cartilage defects.

1 cl, 15 dwg, 3 ex

Description

The invention relates to the field of medicine, in particular to traumatology and orthopedics, and can be used in tissue engineering in the development of cell-engineered structures (hereinafter referred to as CECs) during in vivo experiments to replace defects in the hyaline layer of the articular surface.

Various promising techniques are currently being actively developed to replace the surface of damaged hyaline cartilage. They are tested both in vitro and in vivo on experimental animals. When using laboratory animals, first of all, a standardized injury is modeled, and then, using the developed technique, the defect is replaced, and at the final stage of research, the effectiveness of the chosen technique is analyzed. This sequence is due to the need for a comprehensive assessment of the effectiveness of the developed method of replacing hyaline cartilage.

The main purpose of experiments on laboratory animals is to evaluate tissue reactions to the influence of the method under study (implantation of a cell-engineered structure, cultured cells, electromagnetic radiation, microfracture, etc.) and the process of regeneration of hyaline cartilage. For this, clinical (assessment of joint mobility), radiation (magnetic resonance imaging) and microscopic research methods (scanning electron microscopy, histology) are most often used.

In vivo experiments must be conducted strictly in accordance with the principles of humane treatment of laboratory animals.

The most common evaluation method is the classical preparation of histological slides. Blocks of excised tissue are decalcified, then passed through various reagents to obtain a paraffin block with a sample, its further dewaxing, cutting on a microtome, followed by specific staining, including the use of modern histochemical methods. Studying the area of damage using such methods makes it possible to evaluate the prospects of the developed method for replacing hyaline cartilage and to study the dynamics of perifocal tissue reactions. Additionally, complex and labor-intensive methods are used that use confocal microscopy and specific fluorescent dyes, including the cryomicroscopy method.

In addition to the above methods, there is data on the use of scanning electron microscopy to analyze the effectiveness of various experimental methods of replacing hyaline cartilage in laboratory animals.

The possibility of using scanning electron microscopy in studies on hyaline cartilage was first announced in 1979. In an article by M.N. Pavlova [1] described the applied method of scanning electron microscopy (SEM) in comparison with the technique of classical electron microscopy. In another fundamental work, Ian C. Clarke [2,3] also considered the possibility of using SEM to analyze both the surface and internal structure of the sample under study, but the latter work did not compare the SEM technique with other methods. In the work of Rolf Zehbe [4], the SEM technique was used to analyze the surface structure of a sample, and at the same time a technological technique was presented for producing a cross section for SEM analysis of the internal structure. This extensive study also compared this method with other techniques. However, in the works described above, the issue of using scanning microscopy to analyze damage in experimental animals was not investigated, the intensity of the dynamics of regenerative or degenerative processes was not analyzed, and a scaffold was not used to replace defects.

It is known to use SEM to assess the effectiveness of replacing hyaline cartilage defects with a polymer scaffold [5]. From this patent we know the idea of not only replacing a defect with a polymer, but also pre-populating the latter with a cell culture to form an autologous regenerate and replace the defect at the final stage. However, this publication does not provide a comparative description of the various methods among themselves and does not study their correlation.

A logical continuation of the development of this technology was the work of the authors Gawlitta, Debby et al. [6], including experimental studies on rats, during which they previously modeled and analyzed the damage in dynamics and used a cell-engineered design to replace defects. As in previous studies, these studies used scanning electron microscopy techniques to analyze regenerative and degenerative lesions. However, a full comparison of different methods has not been made.

In the work of H. Katagiri et al.[7] Using experimental animals, cell culture was not used to populate the scaffold, but the methods were compared with each other.

The closest to the claimed invention is the method proposed by Pengfei Zheng [8]. During the study, the manufactured cell-engineered construct (CEC) was implanted into laboratory animals. The ICRS scale was used to analyze photographs of macrosamples. The state of the polymer carrier was assessed using the SEM method. The disadvantages of this method are the lack of quantitative assessment of the results of implantation of the developed CIC using microphotographs obtained by SEM. Also, the authors did not compare the results obtained with histological or other research methods. In addition, the authors did not consider the possibility of using SEM as an independent method for analyzing regenerative damage to hyaline cartilage.

The technical problem is the need to develop a method for quantitatively assessing the effectiveness of using CIC based on a polymer carrier and cell culture in an experiment when replacing hyaline cartilage defects.

The technical result consists in increasing the efficiency of assessing the use of a cell-engineered construct for replacing hyaline cartilage defects in in vivo experiments by obtaining a quantitative assessment of the state of the implantation zone, which allows one to objectively determine the dynamics of regenerative processes, as well as compare the results of different experiments.

The technical result is achieved by the fact that when analyzing microphotographs of the area of the replaced cell-engineered defect structure obtained by the SEM method, a quantitative assessment of the visualized changes is used, according to the proposed criteria: defect area, degree of integration of the CIC, surface structure. Changes in the defect area are assessed as 0 points for an increase in the diameter of the defect, a score of 1 point corresponds to a decrease in the diameter of the defect by no more than 10%), a score of 2 points corresponds to a decrease in the diameter of the defect from 11% to 50%, a score of 3 points corresponds to an intact undamaged surface or reduction in defect diameter by more than 51%. Then the integration of the cell-engineered structure into the intact area is assessed and in the case of the absence of biological (structural) connections between the CIC and intact cartilage, 0 points are assigned, in the case of an insignificant connection of the CIC with intact cartilage, microcracks along most of the defect border (from 76% to 100% of perimeter of the defect) is assigned 1 point, in the case of multiple microcracks along the border of the defect, occupying from 26% to 75% of it, partial connection of the CIC and intact cartilage is assigned 2 points, in the case of single microcracks along the border of the defect, occupying no more than 25% of it perimeter, tight fixation of the CIC is assigned 3 points, in the absence of breaks and microcracks, a continuous transition border (intact cartilage - regenerate area), or undamaged intact cartilage is assigned 4 points, after which the surface structure of the damage area is analyzed and if the damage is irregular in shape, single areas regeneration in the area of damage or their complete absence, there are no signs of cell proliferation, then 0 points are assigned, and if the regenerate is of the correct original shape, but there is an open surface structure of the defect area or the surface of the regenerate is porous, there are signs of cell proliferation, then 1 point is assigned, if the regenerate is of the correct original shape , and the surface of the regenerate is different from the intact one, but not porous, there are signs of cell proliferation (the presence of dividing cells in the area of the regenerate), then 2 points are assigned, and if the intact smooth surface is similar to intact cartilage or intact undamaged cartilage, then 3 points are assigned, after which sum up the points and get a value from 0 to 10, where 0 is the ineffective use of the cell-engineered construct, and 10 is the most effective use of the cell-engineered construct.

More detailed information about the assessment criteria is given in table. 1.

Table 1.-Evaluation criteria Characteristic Grade Points Degree of defect replacement intact undamaged surface or reduction in defect diameter by more than 51% 3 reduction in defect diameter from 11% to 50% 2 reduction in defect diameter by no more than 10%) 1 increase in defect diameter 0 Integration into the undamaged area absence of breaks and microcracks, continuous transition border (intact cartilage - regenerate area), or undamaged intact cartilage 4 single microcracks along the border of the defect, occupying no more than 25% of its perimeter, tight fixation of the CIC 3 multiple microcracks along the border of the defect, occupying from 26% to 75% of it, partial connection of the CIC and intact cartilage 2 insignificant connection of the CIC with intact cartilage, microcracks along most of the defect border (from 76% to 100% of the defect perimeter) 1 lack of biological (structural) connections between the CIC and intact cartilage. 0 Surface structure of the damaged area intact smooth surface - similar to intact cartilage or intact undamaged cartilage 3 The regenerate has the correct original shape, the surface of the regenerate is different from the intact one, but not porous, signs of cell proliferation (the presence of dividing cells in the area of the regenerate) 2 The regenerate has the correct original shape, but there is an open surface structure of the defect area or the surface of the regenerate is porous, there are signs of cell proliferation 1 Damage of irregular shape, single areas of regeneration in the area of damage or their complete absence, no signs of cell proliferation 0 TOTAL
0-10

The proposed method provides a more effective assessment of the use of CIC when replacing hyaline cartilage defects by obtaining a quantitative assessment of the state of the implantation zone using the SEM method using the developed scale, which allows one to objectively determine the dynamics of regenerative processes, as well as compare the results of different experiments.

The inventive method is carried out as follows.

After the animals are removed from the experiment, part of the joint is removed and samples are prepared for scanning electron microscopy. As part of the preparation, the removed joint may be treated with alcohol, trimmed (if necessary, reduced in size for microscopy) and freeze-dried. Next, the area of implantation of the cell-engineered structure is analyzed using a scanning electron microscope, and microphotographs are obtained. The resulting image is analyzed and points are assigned to quantitatively assess the area of implantation of the CIC, according to the given assessment methodology indicated in Table No. 1.

Preferably, the inventive method is used to compare several cell-engineered constructs in order to determine the most effective one.

The claimed invention is illustrated by examples.

Example 1.

The knee joint of an intact mature rat at the age of 6 months was obtained. A superficial hyaline cartilage defect was not created. The cell-engineered construct was not implanted. After obtaining the sample, SEM images of the joint surface were obtained and analyzed. According to the results of the analysis, an intact undamaged surface or a reduction in the diameter of the defect by more than 51%, based on this, the picture corresponds to 3 points, as shown in Fig. 1.

Based on the results of the analysis of the SEM image, the absence of breaks and microcracks, a continuous transition border (intact cartilage - regenerate area), or undamaged intact cartilage was established, which corresponds to 4 points and is shown in Fig. 2.

Based on the analysis of SEM images, it was found that the surface structure fully corresponds to native hyaline cartilage, as shown in Fig. 3, and therefore a score of 3 was given.

Thus, the total number of points was 10, which corresponds to the high efficiency of using a cell-engineered construct based on a polymer carrier and cell culture.

Example 2.

A superficial defect of the hyaline cartilage of the knee joint was created in a 6-month-old sexually mature rat. In the flexion position under general anesthesia, a cylindrical defect with a diameter of 1.00 mm was created using low-speed milling. The cell-engineered construct was also implanted. The cell culture was provided by the Center for Collective Use “Collection of Vertebrate Cell Cultures” of the Institute of Scientific Research of the Russian Academy of Sciences. Next, the animal was removed from the experiment 90 days after surgery. After obtaining the sample, SEM images of the joint surface were obtained and analyzed.

Based on the results of the analysis of SEM images, it was found that an increase in the diameter of the defect, the defect is clearly visualized and has increased in size by more than two times. This picture corresponds to 0 points, as shown in Fig. 4.

Based on the results of analysis of SEM images, it was established that there are no biological (structural) connections between the CIC and intact cartilage, as shown in Fig. 5 and fig. 6. The picture corresponds to 0 points.

Based on the results of the analysis of SEM images, it was found that on the surface of the regeneration area there is a regenerate of the correct original shape, but there is an open surface structure of the defect area or the surface of the regenerate is porous, there are signs of cellularity. This picture corresponds to 1 point in the proposed assessment. Examples of these phenomena are shown in Fig. 7 and fig. 8.

In fig. Figure 7 shows an implanted CIC in the area of the defect and significant damage and an uneven surface are visible.

In fig. 8 shown implanted CIC in the area of the defect. Significant damage, uneven surface, and partial cell proliferation are visible.

The total number of points is -1 point, which corresponds to low efficiency.

Example 3.

A superficial defect of the hyaline cartilage of the knee joint was created in a 6-month-old sexually mature rat. In the flexion position under general anesthesia, a cylindrical defect with a diameter of 1.00 mm was created using low-speed milling. A cell-engineered construct containing a biodegradable scaffold and a culture of cytokine-modified mesenchymal stromal cells using the TGF-β3 cytokine were implanted into the defect area. The cell-engineered construct was also implanted. The cell culture was provided by the Center for Collective Use “Collection of Vertebrate Cell Cultures” of the Institute of Scientific Research of the Russian Academy of Sciences. Next, the animal was removed from the experiment 90 days after surgery. After obtaining the sample, SEM images of the joint surface were obtained and analyzed.

Based on the results of evaluating SEM images, it was found that the reduction in the diameter of the defect from 11% to 50%, which is shown in Fig. 9 and fig. 10. The picture corresponds to 3 points according to the given methodology.

In fig. Figure 9 shows a sample of the surface of hyaline cartilage of a mature rat 90 days after the created damage and implantation of the modified CIC. The regenerate area is in the center of the picture.

In fig. Figure 10 shows a sample of the surface of hyaline cartilage of a mature rat 90 days after the damage was created and implantation of the modified CIC. The regenerate area is in the lower left corner of the picture.

Based on the results of the evaluation of SEM images, it was found that multiple microcracks along the border of the defect, occupying from 26% to 75% of it, are a partial connection of the CIC and intact cartilage, which according to the above method corresponds to 2 points. The picture is shown in Fig. 11 and 12.

In fig. 11 given sample zone - intact hyaline cartilage regenerated area 90 days after the created damage and implantation of the modified CIC. An edge crack is visible along the perimeter of the KIC. Partial connection of the CIC - intact cartilage.

In fig. Figure 12 shows the area of the regenerate after implantation of the CIC on the 90th day after the injury. An edge crack is visible along the entire circumference.

Based on the results of evaluating SEM images, it was found that the regenerate had the correct original shape, the surface of the regenerate was different from the intact one, but not porous, and there were signs of cell proliferation (the presence of dividing cells in the area of the regenerate). The picture according to the above method corresponds to 3 points. The data is presented in Fig. 13,14,15.

In fig. Figure 13 shows the area of the regenerate after implantation of the CIC on the 90th day after the injury. An edge crack is visible on part of the circle.

In fig. 14 given regenerated area after CIC implantation on the 90th day after injury. An edge crack is visible on part of the circle.

In fig. Figure 15 shows the area of the regenerate after implantation of the CIC on the 90th day after the injury. The transition between the zone of intact cartilage and the regenerated area is visible.

The total number of points is 8, which indicates a fairly good effectiveness of using the CIC.

Bibliography

1. Pavlova M.N. Electron microscopy of human articular cartilage. Archive of anatomy, histology and embryology. 1979, 77(7): 65-71

2. Clarke IC Articular cartilage: a review and scanning electron microscope study. 1. The interterritorial fibrillar architecture. J Bone Joint Surg Br. 1971. 53(4): 732-50.

3. Clarke IC Articular cartilage: a review and scanning electron microscope study. II. The territorial fibrillar architecture. J Anat. 1974 Nov;118(Pt 2):261-80.

4. Rolf Zehbe, Heinrich Riesemeier, C James Kirkpatrick, Christoph Brochhausen. Imaging of articular cartilage--data matching using X-ray tomography, SEM, FIB slicing and conventional histology. Micron. 2012 Oct;43(10):1060-7. doi: 10.1016/j.micron.2012.05.001.

5. Tissue engineered cartilage, method of making the same, therapeutic and cosmetic surgical applications using the same. United States Patent 8202551. Li, Wan-ju (Madison, WI, US). Tuan, Rocky S. (Bethesda, MD, US)

6. Engineered Devitalized Cartilaginous Tissue For Bone Regeneration. European Patent Application EP3967335. Gawlitta Debby (NL) Longoni Alessia (NL) Utomo Lizette (NL) Rosenberg Antoine Johan Wilhelm Peter (NL)

7. H Katagiri, LF Mendes, FP Luyten. Definition of a Critical Size Osteochondral Knee Defect and its Negative Effect on the Surrounding Articular Cartilage in the Rat. Osteoarthritis Cartilage. 2017 . Sep;25(9):1531-1540. doi: 10.1016/j.joca.2017.05.006

8. Pengfei Zheng, Xinyue Hu, Yue Lou, Kai Tang. A Rabbit Model of Osteochondral Regeneration Using Three-Dimensional Printed Polycaprolactone-Hydroxyapatite Scaffolds Coated with Umbilical Cord Blood Mesenchymal Stem Cells and Chondrocytes. Med Sci Monit. 2019 Oct 1;25:7361-7369. doi: 10.12659/MSM.915441.

Claims (1)

A method for assessing the effectiveness of using a cell-engineered construct based on a polymer carrier and cell culture in an experiment when replacing hyaline cartilage defects, during which the area of implantation of the cell-engineered construct is studied using scanning electron microscopy, characterized in that, according to the results of electron microscopy on the resulting images, quantitatively and qualitatively analyze the images and assign points, while determining the degree of defect replacement by changing the area of the defect and scoring 0 points when the diameter of the defect increases, a score of 1 point corresponds to a decrease in the diameter of the defect by no more than 10%, a score of 2 points corresponds to a decrease in diameter defect from 11 to 50%, a score of 3 corresponds to an intact undamaged surface or a reduction in the diameter of the defect by more than 51%, then the integration of the cell-engineered structure into the undamaged area is analyzed and, in the absence of structural connections between the cell-engineered structure and intact cartilage, assigned 0 points, in the case of microcracks from 76 to 100% of the perimeter of the defect boundary, 1 point is assigned, in the case of microcracks along the defect boundary, occupying from 26 to 75% of its perimeter, 2 points are assigned, in the case of single microcracks along the defect boundary, occupying no more than 25% of its perimeter, and fixation of the cell-engineered structure is assigned 3 points, in the absence of breaks and microcracks, a continuous transition boundary between intact cartilage and the regenerated area, or undamaged intact cartilage, 4 points are assigned, after which the surface structure of the damaged area is analyzed, and if the damage is of irregular shape, single areas of regeneration in the area of damage or their complete absence, there are no signs of cell proliferation, then 0 points are assigned, and if the regenerate is of the correct original shape, but there is an open surface structure of the defect or the surface of the regenerate is porous, there are signs of cell proliferation, then assigned 1 point, if the regenerate is of the correct original shape, and the surface of the regenerate is not porous, there are signs of cell proliferation, the presence of dividing cells in the area of the regenerate, then 2 points are assigned, and if there is an intact smooth surface or intact undamaged cartilage, then 3 points are assigned, after which sum up the points and get a value from 0 to 10, where 0 is the ineffective use of the cell-engineered construct, and 10 is the most effective use of the cell-engineered construct.