A method for detecting and analyzing new bone tissue in non-section specimens

Abstract

The present invention discloses a method for detecting and analyzing new bone tissue of non-section specimens, and belongs to the field of bone tissue engineering technology. The method uses a high-resolution in vivo two-photon laser scanning microscopy system to perform fluorescence scanning detection on type I collagen and fluorescently labeled mineralized structures in the specimen's new bone tissue to obtain a bone matrix image; a three-dimensional scanning method including three mutually perpendicular axes, namely, an x-axis, a y-axis, and a z-axis, is adopted, and the high-resolution in vivo two-photon laser scanning microscopy system is used to perform a full-layer scan of the new bone tissue sample, and the new bone tissue is quantitatively analyzed through the fluorescent image obtained by scanning. The method of the present invention solves the problems of waste of bone tissue specimens, incomplete display of new bone tissue, uncertain labeling positioning, and inaccurate quantification caused by the use of hard tissue sections for new bone detection. The method is simple to operate, and the quantitative data is reliable, and it can fully reflect the situation of new bone.

Description

Technical Field

[0001] The invention belongs to the technical field of bone tissue engineering, and in particular relates to a method for detecting and analyzing new bone tissue of non-slice specimens. Background Art

[0002] The evaluation of new bone tissue is a focus of bone repair research. Bone tissue is composed of osteocytes and surrounding bone matrix, which contains type I collagen and surrounding mineralized structures. Collagen has the characteristic of spontaneous fluorescence, and the surrounding mineralized structures can also be fluorescently labeled by complexing fluorescent substances with calcium ions. At present, the main method for detecting new bone is to label the animals with fluorescent substances before death, then kill them, take specimens, and make hard tissue sections for observation under a fluorescence microscope or laser confocal microscope (Tissue engineering Part B: Reviews, 2010, 16, 209-217.), and then evaluate the rate of new bone formation based on the quantification of fluorescence or the distance between different fluorescence (Frontiers in Bioengineering and Biotechnology, 2020, 8.), (RSC Advances. 2020, 10, 25652-25661.).

[0003] The current method for evaluating new bone tissue requires the specimen to be dipped into plastic to make a hard tissue slice, which is cumbersome to operate, and the specimen cannot be subjected to other biological tests, resulting in a waste of bone tissue specimens and resources; and the existing method only focuses on the mineralized structure in the bone matrix of the new bone, and cannot fully display the type I collagen in the bone matrix, let alone combine the two for imaging; at the same time, the position of the mineralized structure of the marked new bone is uncertain, so the slices at a certain level may not have excited fluorescence, which is easy to miss or misjudge; in addition, although there are some quantitative analysis methods for the new bone marked by the slices, the data obtained can only represent the situation at one level, and cannot represent the overall picture of the new bone, and cannot accurately quantify the new bone. The prior art provides a method for analyzing new bone tissue, which is to slice the bone tissue sample, then stain it, and then observe the bone morphology under a two-photon confocal microscope, but the operation is cumbersome and still requires slicing. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for detecting and analyzing new bone tissue in non-sectioned specimens, in order to solve the problems of waste of bone tissue specimens caused by the need to slice hard tissue for new bone detection, incomplete display of new bone tissue, uncertain marking positioning and inaccurate quantification.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a method for detecting and analyzing new bone tissue of a non-section specimen, comprising the following steps:

[0007] 1) Combining collagen second harmonic imaging and the wavelength of excitation light of the fluorescent substance that marks the mineralized structure, a high-resolution in vivo two-photon laser scanning microscopy system was used to perform fluorescence scanning detection of type I collagen and mineralized structures in the new bone tissue of non-section specimens to obtain the fluorescence excitation conditions of collagen and mineralized structures;

[0008] The fluorescent substances used to mark the mineralized structures are calcein and alizarin red. The absorption wavelength of mineralized tissues marked by calcein is 503-555nm, and the absorption wavelength of minerals marked by alizarin red is 578-634nm.

[0009] 2) Under the condition of fluorescence excitation of collagen and mineralized structure, a three-dimensional scanning method including three mutually perpendicular axes including x-axis, y-axis and z-axis is adopted, and a high-resolution in vivo two-photon laser scanning microscopy imaging system is used to perform a full-layer scan of the non-sectioned new bone tissue sample to obtain the fluorescence images of collagen and mineralized structure of N sub-layers;

[0010] 3) reconstructing a three-dimensional image of the bone matrix in the bone tissue sample using the fluorescent images of the collagen and mineralized structures of the N sublayers;

[0011] 4) According to the three-dimensional image of collagen and mineralized structure reconstructed in step 3), the morphological structure of bone matrix in the non-slice specimen new bone tissue sample is obtained, thereby completing the qualitative and quantitative analysis of the non-slice specimen new bone tissue sample.

[0012] Preferably, in step 1), before the fluorescence scanning detection, the skull defect specimen is surface treated and trimmed to ensure the imaging effect, and then placed on a glass slide to obtain a non-sectioned specimen of new bone tissue for observation and imaging;

[0013] Among them, surface treatment and trimming are to remove all surface muscles, periosteum and other soft tissues, and trim the bone tissue around the specimen to ensure that the new bone is on the same level.

[0014] Preferably, the processed specimen is fixed on a glass slide with biological glue or adhesive tape, a drop of PBS is dropped on the defect area, a cover glass of appropriate size is placed above the defect, and then placed on a stage for observation and imaging.

[0015] Preferably, in step 1), the fluorescence excitation conditions of the collagen and mineralized structure are: the excitation light wavelength is 940 nm.

[0016] Preferably, in step 1), the second harmonic imaging absorption wavelength of collagen is 465-475 nm.

[0017] Preferably, in step 2), the full-layer scan refers to: scanning the plane fixed by the x-axis and y-axis directions of the circular defect in N sublayers in the z-axis direction, where N is an integer greater than 5. In the process of scanning each sublayer, the collagen and mineralized structure of each sublayer in the new bone tissue sample are stimulated to produce fluorescence, thereby obtaining fluorescent images of the collagen and mineralized structure of the N sublayers.

[0018] Preferably, in step 3), the fluorescence image reconstruction process of the collagen and mineralized structures of the N sub-layers is as follows: the scanned collagen or mineralized structure images of the N sub-layers are used to obtain the collagen or mineralized structure fluorescence three-dimensional images respectively using software, and then the fluorescence of the collagen or mineralized tissue is quantified, and the obtained value is the quantitative parameter of the collagen or mineralized tissue in the non-sectioned specimen.

[0019] Preferably, the software is Leica Application Suite X software or Image J software.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention discloses a method for detecting and analyzing new bone tissue of non-slice specimens. A high-resolution in vivo two-photon laser scanning microscopic imaging system is used to perform fluorescence scanning detection on type I collagen and mineralized structure in the new bone tissue of the non-slice specimens. Compared with the image obtained by scanning a hard tissue slice fluorescence microscope, the method can not only display the mineralized tissue labeled with fluorescence, but also can fully display collagen, so that the imaging of the new bone tissue is more comprehensive. A three-dimensional scanning method including three mutually perpendicular axes, namely, an x-axis, a y-axis and a z-axis, is adopted. The high-resolution in vivo two-photon laser scanning microscopic imaging system is used to perform full-layer scanning on the new bone tissue sample of the non-slice specimen. Compared with the hard tissue slice fluorescence microscope scanning method, the method can realize full-range scanning of the x-axis, the y-axis and the z-axis, can accurately locate the new bone tissue labeled with fluorescence, and can more accurately quantify the generation amount and generation speed of the new bone tissue. In addition, compared with the hard tissue slice method, the invention adopts non-slice specimens, is simple to operate, and the specimens can be used for other biological tests, and will not cause waste of bone tissue specimens.

[0022] Furthermore, the fluorescence excitation wavelength of collagen and mineralized structures is 940 nm. Under this excitation wavelength, clear emission wave images of collagen and mineralized tissues can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1It is a fixed diagram of the specimen used in the high-resolution in vivo two-photon laser scanning microscopic imaging system of the present invention;

[0024] Figure 2 The present invention uses a high-resolution in vivo two-photon laser scanning microscopy system to perform two-dimensional imaging of a specimen in a certain XY plane, including (a) collagen image, (b) mineralized structure labeled with calcein, (c) mineralized structure labeled with alizarin red, and (d) a merged image of the three;

[0025] Figure 3 The present invention uses Leica Application Suite X software to reconstruct the three-dimensional image obtained by imaging sequence images of a high-resolution in vivo two-photon laser scanning microscopy imaging system; wherein, (a) collagen, (b) mineralized structure labeled with calcein, (c) mineralized structure labeled with alizarin red;

[0026] Figure 4 It is a schematic diagram of the imaging operation and imaging effect of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0028] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0029] The skull defect model of rats and mice is the most commonly used animal model for evaluating bone regeneration, so the non-sliced ​​specimens of new bone tissue involved in the present invention are all from the skull defect model of rats or mice. The thickness of the skull domain of the studied mice and the skull modeling area of ​​the rats are both less than 1000μm, which meets the second harmonic imaging depth and imaging conditions, and there is no interference from other tissue structures around them.

[0030] The present invention provides a method for detecting and analyzing new bone tissue of a non-section specimen, comprising the following steps:

[0031] Step a: Surface treatment and trimming of rat or mouse skull defect specimens, removal of soft tissues such as surface muscles and periosteum, and trimming of bone tissue around the specimens to ensure that the new bone sites are at the same level as much as possible to ensure imaging effects, and then placing the specimens on a glass slide for observation and imaging; the specific treatment method is to fix the treated specimens on a glass slide with biological glue or adhesive tape, drip 1 drop of PBS on the defect area, and then place a cover glass of appropriate size on top of the defect, and then place the specimen on a stage for observation. For the fixation diagram of the specimen, see Figure 1 ;

[0032] Step b, combining collagen second harmonic imaging and the excitation light wavelength of the labeled fluorescent substance, using a high-resolution in vivo two-photon laser scanning microscopy system to perform a fluorescence scanning experiment on type I collagen and mineralized structure in the new bone tissue of the non-section specimen, specifically: the excitation light is uniformly gradually reduced from 1000nm, and then the collagen second harmonic signal and the emission wave of the mineralized tissue are collected, the second harmonic signal is received in channel 1, using a 465-475nm filter; the mineralized tissue labeled with calcein receives the emission wave in channel 2, using a 503-555nm filter; the mineralized tissue labeled with alizarin red receives the emission wave in channel 3, using a 578-634nm filter. Preliminary experiments determined that the fluorescence labeled with calcein and alizarin red can be excited by 940nm excitation light, so the excitation wavelength used in the experiment is 940nm, the collagen second harmonic imaging absorption wavelength is 465-475nm, the calcein-labeled mineralized tissue absorption wavelength is 503-555nm, and the alizarin red-labeled mineral absorption wavelength is 578-634nm.

[0033] Figure 2 The present invention uses a high-resolution in vivo two-photon laser scanning microscopy system to perform two-dimensional imaging of the specimen in a certain XY plane. Figure 2 As shown in a and 2b, the high-resolution in vivo two-photon laser scanning microscopy system can clearly display the two-dimensional images of the mineralized structure labeled with alizarin red and calcein. Figure 2 c It can be seen that the high-resolution in vivo two-photon laser scanning microscopy system clearly displays the two-dimensional image of collagen generated by the second harmonic generation, and can also display the orientation of collagen; Figure 2 d clearly shows the mineralized structure labeled by Alizarin Red and Calcein, as well as the merge image of the collagen tissue using the second harmonic generation.

[0034] Step c, under the fluorescence excitation condition of the collagen and mineralized structure, a three-dimensional scanning method including three mutually perpendicular axes including an x-axis, a y-axis and a z-axis is adopted, and the high-resolution in vivo two-photon laser scanning microscopy imaging system is used to perform a full-layer scan of the new bone tissue sample of the non-section specimen, wherein the full-layer scan refers to: scanning the N sublayers of the circular defect plane (i.e., the plane fixed by X and Y) in the z-axis direction, where N is an integer greater than 5, and in the process of scanning each sublayer, the collagen and mineralized structure of each sublayer in the new bone tissue sample are stimulated to generate fluorescence, thereby obtaining a fluorescence image of the collagen and mineralized structure of the N sublayers;

[0035] Step d, using the fluorescence images of the collagen and mineralized structures of the N sub-layers to reconstruct the three-dimensional image of the bone matrix in the non-section specimen new bone tissue sample; specifically: the scanned collagen or mineralized structure images of the N sub-layers are used with Leica Application Suite X software or imported into Image J software to obtain the collagen or mineralized structure fluorescence three-dimensional images respectively.

[0036] Figure 3 The present invention uses Leica Application Suite X software to reconstruct the three-dimensional image from the imaging sequence of the high-resolution in vivo two-photon laser scanning microscopy imaging system. Figure 3 a and 3b show three-dimensional images of mineralized tissue labeled with alizarin red and calcein, respectively. Figure 3 c shows a three-dimensional image of collagen generated by second harmonic generation. Figure 3 It can be seen that the method provided by the present invention can be used to reconstruct the collagen and mineralization structure in the new bone tissue sample to form a three-dimensional structural image, which is not only conducive to the observation of the overall morphology of the bone, but also provides an accurate and fast method for the quantification of collagen and mineralization structure in the new bone tissue.

[0037] Step e: According to the three-dimensional images of collagen and mineralized structures reconstructed in step d, the morphological structure of the bone matrix in the non-slice specimen new bone tissue sample is obtained, and the collagen or mineralized structure images of the N sub-layers obtained by scanning are imported into the Image J software to obtain the three-dimensional fluorescence images of the collagen or mineralized structure, and then the fluorescence amount of the collagen or mineralized tissue is quantified using the Image J software, and the obtained value is the quantitative parameter of the collagen or mineralized tissue in the non-slice specimen. Thus, the qualitative and quantitative analysis of the non-slice specimen new bone tissue sample is completed.

[0038] Figure 4This is a schematic diagram of the imaging operation and imaging effect of the present invention. It can be concluded from the figure that the use of a high-resolution in vivo two-photon laser scanning microscopy system to directly perform specimen detection can directly and quickly image the collagen and mineralization structure in the new bone tissue, thereby providing more new bone tissue information, which is an efficient and convenient analysis method.

[0039] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting and analyzing new bone tissue in non-section specimens, characterized in that: The following steps are involved: 1) Combining collagen second harmonic imaging and the wavelength of excitation light of the fluorescent substance that marks the mineralized structure, a high-resolution in vivo two-photon laser scanning microscopy system was used to perform fluorescence scanning detection of type I collagen and mineralized structures in the new bone tissue of non-section specimens to obtain the fluorescence excitation conditions of collagen and mineralized structures; Among them, the fluorescent substances marked by the mineralized structure are calcein and alizarin red. The absorption wavelength of the mineralized tissue marked by calcein is 503-555nm, and the absorption wavelength of the mineralized material marked by alizarin red is 578-634nm. In step 1), the collagen second harmonic imaging absorption wavelength is 465-475 nm; The fluorescence excitation conditions of the collagen and mineralized structure are: the excitation light wavelength is 940nm; 2) Under the condition of fluorescence excitation of collagen and mineralized structure, a three-dimensional scanning method including three mutually perpendicular axes including x-axis, y-axis and z-axis is adopted, and a high-resolution in vivo two-photon laser scanning microscopy imaging system is used to perform a full-layer scan of the non-sectioned new bone tissue sample to obtain the fluorescence images of collagen and mineralized structure of N sub-layers; 3) reconstructing a three-dimensional image of the bone matrix in the bone tissue sample using the fluorescent images of the collagen and mineralized structures of the N sublayers; In step 3), the fluorescence image reconstruction process of the collagen and mineralized structures of the N sub-layers is as follows: the collagen or mineralized structure images of the N sub-layers obtained by scanning are used with software to obtain the three-dimensional fluorescence images of the collagen or mineralized structure, and then the fluorescence of the collagen or mineralized tissue is quantified, and the obtained value is the quantitative parameter of the collagen or mineralized tissue in the non-sectioned specimen; 4) According to the three-dimensional image of collagen and mineralized structure reconstructed in step 3), the morphological structure of bone matrix in the non-slice specimen new bone tissue sample is obtained, thereby completing the qualitative and quantitative analysis of the non-slice specimen new bone tissue sample.

2. A method for detecting and analyzing new bone tissue in non-section specimens according to claim 1, characterized in that: In step 1), before the fluorescence scanning test, the skull defect specimen is surface treated and trimmed to ensure the imaging effect, and then placed on a glass slide to obtain the new bone tissue of the non-sliced ​​specimen for observation and imaging; wherein the surface treatment and trimming are to remove all soft tissues such as surface muscles and periosteum, and at the same time trim the bone tissue around the specimen to ensure that the new bone site is on the same horizontal plane.

3. A method for detecting and analyzing new bone tissue in non-section specimens according to claim 2, characterized in that: Fix the processed specimen on the slide with biological glue or tape, drop 1 drop of PBS on the defect area, place a coverslip of appropriate size on the defect, and then place it on the stage for observation and imaging.

4. The method for detecting and analyzing new bone tissue in non-section specimens according to claim 1, characterized in that: In step 2), the full-layer scan refers to: scanning the plane fixed by the x-axis and y-axis directions of the circular defect in N sublayers in the z-axis direction, where N is an integer greater than 5. In the process of scanning each sublayer, the collagen and mineralized structure of each sublayer in the new bone tissue sample are stimulated to produce fluorescence, thereby obtaining fluorescent images of the collagen and mineralized structure of the N sublayers.

5. The method for detecting and analyzing new bone tissue in non-section specimens according to claim 1, characterized in that: The software is Leica Application Suite X software or Image J software.

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