Porous biological ceramic hard tissue slice as well as preparation method and application thereof

Abstract

The invention provides a porous biological ceramic hard tissue slice as well as a preparation method and application thereof. The preparation method of the porous biological ceramic hard tissue slice comprises the following steps: preparing a hydroxyl phospholime bone restoration specimen; dehydrating and transparentizing the hydroxyapatite bone restoration specimen; carrying out permeation and embedding on the hydroxyl phospholime bone restoration specimen; carrying out block trimming and slicing on the hydroxyl phospholime bone restoration specimen; mounting the hydroxyl phospholime bone restoration specimen; through the five steps, the porous biological ceramic hard tissue slice is obtained. According to the method, the defect that a tissue-material structure is easy to damage when a biological ceramic material is treated by a traditional hard tissue slicing technology is overcome; the improved hard tissue slicing technology is suitable for research of biological ceramic materials in bone tissue engineering application.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering technology, specifically relating to a porous bioceramic hard tissue section, its preparation method, and its application. Background Technology

[0002] There are two traditional methods for preparing bone tissue sections: paraffin-embedded sections and hard tissue-embedded sections. Soft paraffin-embedded sections involve decalcifying, dehydrating, and clearing the bone tissue, then embedding it in paraffin and sectioning it. Hard tissue-embedded sections involve fixing the bone tissue sample, clearing and infiltrating it with resin, embedding it in resin, sectioning it, and then staining it for observation. Paraffin sections are widely used in tissue sectioning. However, paraffin sections also have many drawbacks: the processing often leads to a decrease in enzyme activity in the tissue sample, making many sensitive indicators difficult to detect, or it can severely affect the observation results due to changes in the bone tissue structure. Especially in the field of tissue bioceramics, decalcification can easily destroy the original morphological characteristics and structural relationships between tissue cells and biomaterials, making it difficult to fully characterize the true state of cell growth. Due to the rapid development of tissue bioceramic materials, more and more indicators need to be observed through hard tissue-embedded sections. This allows for a comprehensive observation of the interaction between biomaterials and tissues in the animal body, as well as changes in osteogenic and angiogenic processes within the material, thus better deducing the osteogenic induction effect of osteoid prostheses on animals.

[0003] Traditional hard tissue sectioning techniques for bone prostheses made from hydroxyapatite (HA) scaffold materials have many limitations. Most methods use homemade chrome alum gelatin slides for mounting, but because the gluing is done manually, the adhesion quality of the slides is unpredictable, thus affecting the effectiveness of preventing slide detachment. Traditional hard tissue sectioning methods have a high detachment rate, and chrome alum gelatin slides exhibit significant dye adsorption during staining, often resulting in excessive non-specific dye adsorption that affects the observation of stained specimens. In recent years, commercially available cationic slides have been widely used in paraffin sectioning, and their effectiveness in preventing detachment is widely recognized; however, their application in hard tissue sectioning is rarely reported. Summary of the Invention

[0004] To address the challenges of tissue section detachment and dye adsorption, the first objective of this invention is to provide a method for preparing porous bioceramic hard tissue sections. The second objective is to provide porous bioceramic hard tissue sections prepared by this method. The third objective is to provide the application of these porous bioceramic hard tissue sections as scaffolds for supporting and inducing bone tissue growth in bone tissue engineering.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] On one hand, the present invention provides a method for preparing porous bioceramic hard tissue sections, which includes the following steps:

[0007] Step 1: Prepare a hydroxyphosphorus lime-based bone repair specimen;

[0008] Step two: Dehydrate and clear the hydroxyapatite-based bone repair specimen;

[0009] Step 3: Infiltrate and embed the hydroxyapatite-based bone repair specimen;

[0010] Step four: Trim and section the hydroxyapatite-based bone repair specimen;

[0011] Step 5: Mount the hydroxyapatite-based bone repair specimen;

[0012] After the above 5 steps, porous bioceramic hard tissue sections are obtained.

[0013] In the above preparation method, preferably, step one includes the following specific steps:

[0014] Chinese rural dogs aged 18–24 months and weighing 16–18 kg were selected and anesthetized by intraperitoneal injection of sodium pentobarbital.

[0015] Next, a hydroxyapatite particle stacking scaffold was implanted into the abdominal cavity of the dog to construct tissue-engineered bone. Three months after the construction, the animal was sacrificed and the osteoprosthetic specimen was removed.

[0016] The specimen was first rinsed with physiological saline to remove impurities, and then fixed in paraformaldehyde solution to prepare a hydroxyphosphorus lime-based bone repair specimen.

[0017] In the above preparation method, preferably, the injection dose of sodium pentobarbital is 10 mg / kg.

[0018] In the above preparation method, preferably, one week before the animal is euthanized, the step of injecting 40 mg / kg of calcein into the muscle is also included.

[0019] In the above preparation method, preferably, the mass concentration of the paraformaldehyde solution is 4%.

[0020] In the above preparation method, preferably, step two includes the following specific steps:

[0021] After fixing the hydroxyphosphorus lime-based bone repair specimens with paraformaldehyde for one week, the samples were rinsed with running water for 24 hours and then dehydrated with 70%, 80%, 85%, 90%, 95%, 100%, and 100% ethanol for 24 hours each, respectively; followed by clearing with xylene for 24 hours.

[0022] In the above preparation method, preferably, the volume of ethanol and xylene used is more than 20 times the volume of the sample.

[0023] In the above preparation method, preferably, step three includes the following specific steps:

[0024] After the specimens were cleared with xylene, they were soaked in permeation solutions I, II, and III for 24 hours each. Then, the specimens were carefully placed into embedding bottles in a certain direction and the embedding solution was poured in.

[0025] During embedding, the samples were numbered, placed in flat-bottomed glass bottles, embedded with embedding solution, and then transferred to a vacuum drying oven for 4 hours. Subsequently, they were placed in a water bath at 36.5℃ for 24–48 hours for polymerization, and finally placed in a constant temperature oven at 37℃ for 12–24 hours for polymerization.

[0026] In the above preparation method, preferably, the preparation of the embedding solution includes:

[0027] First, place benzoyl peroxide in a 40℃ oven to dry overnight to remove moisture, then prepare the solution. Stir the prepared solution thoroughly for 4-6 hours, filter it with filter paper, seal it completely, and store it at 4℃. After 4-6 days, it can be used for embedding.

[0028] The ratios of the permeate and embedding fluid are shown in Table 1 below.

[0029] Table 1:

[0030]

[0031] In the above preparation method, preferably, step four includes the following specific steps:

[0032] After placing the embedded tissue specimen in a 4°C refrigerator for one week, remove the embedded specimen.

[0033] Fix the specimen on the inner edge cutting machine, select an initial cutting surface, and trim the block until the cut surface is flat;

[0034] The trimmed specimen was fixed on a hard tissue microtome and sectioned. The section thickness was 4–7 μm, and the sectioning speed was controlled at 15 μm / s.

[0035] In the above preparation method, preferably, step five includes the following specific steps:

[0036] Place the slices into a 60℃ water bath mounting pan and wait until they are fully extended before removing them with a cationic anti-detachment glass slide.

[0037] Cover the slide with plastic wrap and press with your fingers to remove air bubbles between the slide and the slide. Cover each slide with a piece of smooth paper and clamp them together.

[0038] After baking in a 60℃ oven for 7 days, the slides were removed to obtain modified porous bioceramic hard tissue sections.

[0039] On the other hand, the present invention also provides a porous bioceramic hard tissue section, which is prepared by the above-described preparation method.

[0040] Furthermore, the present invention also provides the application of the above-mentioned porous bioceramic hard tissue sections as scaffolds for supporting and inducing bone tissue growth in bone tissue engineering.

[0041] The beneficial effects of this invention are:

[0042] Compared with traditional hard tissue sectioning techniques, the porous bioceramic hard tissue sectioning technique of the present invention has the following advantages:

[0043] (1) In this invention, the embedded sample is placed at 4°C for 7 days. Traditional methods use embedding solution to fix tissues, and when the section thickness is less than 10 μm, the material particles are easily broken; while this invention can better fix the sample after placing it at 4°C for 7 days, and can also ensure the integrity of the material and tissue when the section thickness reaches 4 μm.

[0044] (2) The bleaching temperature of the slides in this invention is set to 60°C and the time is 15 minutes. The traditional bleaching temperature of hard tissue sections is about 55°C and the bleaching time is 5 minutes; while the present invention increases the bleaching temperature and extends the bleaching time, which allows the specimen to spread better.

[0045] (3) This invention uses wet mounting technology. Traditional mounting technology is prone to generating air bubbles during the baking process; however, this invention bakes the film directly without baking, thus avoiding the generation of air bubbles.

[0046] (4) The baking temperature of the specimens in this invention is set at 60°C and the baking time is 7 days. When the temperature is below 60°C, the plastic wrap is easy to fall off the specimens, and when the temperature is above 60°C, the plastic wrap is easy to adhere to the specimens.

[0047] (5) This invention uses cationic slides. Traditional chrome alum gelatin slides have a high detachment rate and are prone to adsorbing dye during staining, affecting the observation effect; this invention uses cationic slides, because the surface of the cationic slides has a persistent cationic charge, tissues or cells can be better adsorbed on the glass slide.

[0048] In summary, the porous bioceramic hard tissue sections prepared by the method of this invention allow for clearer and more complete observation of the structural relationship and interaction between the tissue and the material under an inverted microscope; significantly improves the quality of the slides; overcomes the drawback of traditional hard tissue sectioning techniques that easily damage the tissue-material structure when processing bioceramic materials; and the improved hard tissue sectioning technique of this invention is suitable for research on the application of bioceramic materials in bone tissue engineering.

[0049] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 HE staining images of tissue sections from the specimen three months after implantation into the abdominal cavity (a) HE staining image before the modification of hard tissue sectioning technique; b) HE staining image after modification.

[0052] Figure 2 The images show toluidine blue staining of tissue sections from the specimen three months after implantation into the abdominal cavity (a) is the toluidine blue staining image before the modification of the hard tissue sectioning technique; b) is the toluidine blue staining image after the modification.

[0053] Figure 3 Masson staining images of tissue sections from the specimen three months after implantation into the abdominal cavity (a) Masson staining image before the modification of hard tissue sectioning technique; b) Masson staining image after modification.

[0054] Figure 4 Fluorescence images of tissue sections from the specimen one month after implantation into the abdominal cavity (a) is the fluorescence image before the modification of the hard tissue sectioning technique; b) is the fluorescence image after the modification. Detailed Implementation

[0055] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any special limitations.

[0056] Example 1:

[0057] 1. Preparation process of hydroxyapatite-based bone repair specimens

[0058] This experiment used Chinese rural dogs as experimental animals, regardless of sex, aged 18–24 months, and weighing 16–18 kg. Animals were anesthetized by intraperitoneal injection of sodium pentobarbital 10 mg / kg. A hydroxyapatite particle-stapled scaffold was implanted into the peritoneal cavity of the dogs for tissue-engineered bone construction. Three months after construction, the animals were sacrificed, and the osteogenic prosthesis was removed. One week before sacrifice, calcein 40 mg / kg was injected intramuscularly. The removed specimens were first rinsed with physiological saline to remove impurities, and then fixed in 4% paraformaldehyde solution to prepare the hydroxyapatite osteogenic prosthesis specimen.

[0059] 2. Preparation and Improvement Process of Porous Bioceramic Hard Tissue Sections

[0060] (1) After fixing the HA type bone repair specimen with 4% paraformaldehyde for 1 week, rinse the sample with running water for 24 hours, and then dehydrate it with 70%, 80%, 85%, 90%, 95%, 100%, and 100% ethanol for 24 hours each, and then clear it with xylene for 24 hours. The volume of each liquid is more than 20 times the volume of the sample.

[0061] (2) Permeation and embedding: After the specimens were cleared with xylene, they were soaked in permeation solutions I, II and III for 24 hours respectively. Then, the specimens were placed in embedding bottles in a certain direction and the embedding solution was carefully poured in.

[0062] Preparation of embedding solution: First, place benzoyl peroxide in a 40℃ oven to dry overnight to remove moisture, then prepare the solution. Stir the prepared solution thoroughly for 4-6 hours, filter it with filter paper, seal it completely, and store it at 4℃. Let it stand for 4-6 days before using it for embedding.

[0063] During embedding, the samples were numbered, placed in flat-bottomed glass bottles, and embedding solution was added. The bottles were then transferred to a vacuum drying oven and dried under vacuum for 4 hours. Subsequently, they were placed in a water bath at 36.5℃ for 24–48 hours to polymerize (the choice of temperature has a significant impact on the polymerization rate of the embedding solution). Finally, they were placed in a constant temperature oven at 37℃ for 12–24 hours to polymerize.

[0064] (3) Trimming and sectioning: After the fully aggregated tissue specimens are placed in a refrigerator at 4°C for about a week, the embedded specimens are removed. The specimens are fixed on an inner edge cutter, a preliminary cutting surface is selected, and the specimens are trimmed until the cut surface is flat. The trimmed specimens are fixed on a hard tissue microtome for sectioning, with a section thickness of 4-7 μm and a sectioning speed of 15 μm / s.

[0065] (4) Mounting: Place the slides in a 60℃ water bath mounting pot. After they are fully extended, use a cationic anti-detachment glass slide to lift them out. Cover the slides with a plastic wrap and press with your fingers to remove air bubbles between the slides and the glass slide. Cover each slide with a smooth thin paper, overlap them and clamp them tightly. Place them in a 60℃ oven to bake for 7 days and then take them out to obtain the improved porous bioceramic hard tissue slides.

[0066] Example 2: Comparative Experiment on Histological Examination of Modified Porous Bioceramic Hard Tissue Sections

[0067] The modified porous bioceramic sections prepared in Example 1 were characterized by HE staining, toluidine blue staining, Masson staining, and fluorescence staining. The tissue or cell ingrowth of the HA particle-accumulated scaffold in vivo after three months of hybridization was observed using different staining methods. The staining effects of the modified sections and the unmodified sections are shown in the light microscope images. Figures 1 to 4 As shown.

[0068] Figure 1 This is a comparison of HE staining of HA-type bone repair specimens before and after modification under a light microscope. Figure 1 As clearly observed in Figure a, HE staining without hard tissue section modification suffers from the drawback of dye adsorption and fails to clearly observe the interaction between tissue and material; the localization of blood vessels and cells is blurred. After modification, as shown in Figure a... Figure 1 Figure b shows a clear material-tissue interface, intact vascular structure, and a distinct calcium deposition zone (red area) at the junction of the material surface and tissue. Clear morphology of cells and collagen is visible in the connective tissue ingrained into the scaffold pores.

[0069] Figure 2 This is a comparison image of toluidine blue staining on HA-type bone repair specimens before and after modification, under a light microscope. Figure 2 Figure a shows poor color saturation and blurred structure in the specimen, making it impossible to accurately locate the cell nucleus. Figure 2 Figure b clearly shows the newly formed collagen within the scaffold (stained sky blue); the osteoclasts tightly attached to the material surface exhibit a typical fried egg shape (stained dark blue), and the cell nuclei are clearly visible.

[0070] Figure 3 This is a comparison image of modified Masson's trichrome staining of HA-type bone repair specimens before and after modification under a light microscope. Figure 3 As shown in Figure a, due to severe local detachment and wrinkling of the specimen, the growth state of the tissue and the interaction between the tissue and the material cannot be clearly distinguished and observed. Figure 3 Figure b accurately shows the newly formed bone tissue in the material pores (collagen is stained blue, and the calcification zone at the edge is stained red, indicating that the newly formed bone tissue is not yet mature). Osteoblasts are neatly arranged on the surface of the new bone, and osteoclast adhesion and collagen formation are visible on the material surface. The cell nuclei are stained purple, and the nucleoli are clearly visible; collagen fibers are lightly stained blue.

[0071] Figure 4 Fluorescence images under a microscope of HA-type bone repair specimens before and after modification of hard tissue sections. Figure 4Figure a shows that the overall structure of the specimen could not be well observed due to severe HA particle detachment from the scaffold. Figure 4 Figure b in the image is the improved fluorescence image. It can be seen from the image that the overall structure of the specimen is basically intact, the material-tissue interface is clear, and the fluorescence expression at the calcein deposition site is clear and bright.

[0072] In summary, the improved specimen section staining technique allows for a clearer and more complete observation of the structural relationships and interactions between tissues and materials under an inverted microscope. It significantly improves slide quality and overcomes the drawback of traditional hard tissue sectioning techniques that easily damage the tissue-material structure when processing bioceramic materials. The improved hard tissue sectioning technique is well-suited for research on the application of bioceramic materials in bone tissue engineering.

[0073] Specific embodiments have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A method for preparing porous bioceramic hard tissue sections, comprising the following steps: Step 1: Prepare a hydroxyphosphorus lime-based bone repair specimen; Step two: Dehydrate and clear the hydroxyapatite-based bone repair specimen; Step 3: Infiltrate and embed the hydroxyapatite-based bone repair specimen; Step four: Trim and section the hydroxyapatite-based bone repair specimen; Step 5: Mount the hydroxyapatite-based bone repair specimen; After the above 5 steps, porous bioceramic hard tissue sections are obtained.

2. The preparation method according to claim 1, characterized in that, The specific steps of step one include: Chinese rural dogs aged 18–24 months and weighing 16–18 kg were selected and anesthetized by intraperitoneal injection of sodium pentobarbital. Next, a hydroxyapatite particle stacking scaffold was implanted into the abdominal cavity of the dog to construct tissue-engineered bone. Three months after the construction, the animal was sacrificed and the osteoprosthetic specimen was removed. The specimen was first rinsed with physiological saline to remove impurities, and then fixed in paraformaldehyde solution to prepare a hydroxyphosphorus lime-based bone repair specimen.

3. The preparation method according to claim 2, characterized in that: The injection dose of the sodium pentobarbital is 10 mg / kg; Preferably, one week before the animal is euthanized, the procedure further includes injecting 40 mg / kg of calcein into the muscle. Preferably, the mass concentration of the paraformaldehyde solution is 4%.

4. The preparation method according to claim 1 or 3, characterized in that, The specific steps of step two include: After fixing the hydroxyphosphorus lime-based bone repair specimens with paraformaldehyde for one week, the samples were rinsed with running water for 24 hours and then dehydrated with 70%, 80%, 85%, 90%, 95%, 100%, and 100% ethanol for 24 hours each, respectively; followed by clearing with xylene for 24 hours. Preferably, the volume of the ethanol and the xylene used is more than 20 times the volume of the sample.

5. The preparation method according to claim 1 or 4, characterized in that, The specific steps of step three include: After the specimens were cleared with xylene, they were soaked in permeation solutions I, II, and III for 24 hours each. Then, the specimens were carefully placed into embedding bottles in a certain direction and the embedding solution was poured in. During embedding, the samples were numbered, placed in flat-bottomed glass bottles, embedded with embedding solution, and then transferred to a vacuum drying oven for 4 hours. Subsequently, they were placed in a water bath at 36.5℃ for 24–48 hours for polymerization, and finally placed in a constant temperature oven at 37℃ for 12–24 hours for polymerization.

6. The preparation method according to claim 5, characterized in that, The preparation of the embedding solution includes: First, place benzoyl peroxide in a 40℃ oven to dry overnight to remove moisture, then prepare the solution. Stir the prepared solution thoroughly for 4-6 hours, filter it with filter paper, seal it completely, and store it at 4℃. After 4-6 days, it can be used for embedding.

7. The preparation method according to claim 1 or 5, characterized in that, The specific steps of step four include: After placing the embedded tissue specimen in a 4°C refrigerator for one week, remove the embedded specimen. Fix the specimen on the inner edge cutting machine, select an initial cutting surface, and trim the block until the cut surface is flat; The trimmed specimen was fixed on a hard tissue microtome and sectioned. The section thickness was 4–7 μm, and the sectioning speed was controlled at 15 μm / s.

8. The preparation method according to claim 1 or 7, characterized in that, The specific steps of step five include: Place the slices into a 60℃ water bath mounting pan and wait until they are fully extended before removing them with a cationic anti-detachment glass slide. Cover the slide with plastic wrap and press with your fingers to remove air bubbles between the slide and the slide. Cover each slide with a piece of smooth paper and clamp them together. After baking in a 60℃ oven for 7 days, the slides were removed to obtain modified porous bioceramic hard tissue sections.

9. A porous bioceramic hard tissue section, which is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the porous bioceramic hard tissue section of claim 9 as a scaffold for supporting and inducing bone tissue growth in bone tissue engineering.

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