CN111973807A - Bionic artificial temporomandibular joint disc and preparation method thereof - Google Patents
Bionic artificial temporomandibular joint disc and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/30—Joints
- A61F2/30988—Other joints not covered by any of the groups A61F2/32 - A61F2/4425
- A61F2/3099—Other joints not covered by any of the groups A61F2/32 - A61F2/4425 for temporo-mandibular [TM, TMJ] joints
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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Abstract
The invention provides a bionic artificial temporomandibular joint disc and a preparation method thereof, wherein the preparation method comprises the following steps: collecting intervertebral discs of oxtail, cleaning, performing freeze thawing treatment, extracting glycosaminoglycan and protein, crushing into flocculent, adding sodium dodecyl sulfate and DNase, treating at room temperature, and cleaning to obtain flocculent intervertebral disc samples; freeze-drying the disc flocculent sample, adding digestive juice, blending, adjusting the pH value to be neutral, and adding glycosaminoglycan and protein to obtain biological ink; and (3) blending the high polymer material and the biological ink at room temperature, and performing 3D printing to obtain the bionic artificial temporomandibular joint disc. The invention also comprises the bionic artificial temporomandibular joint disc prepared by the method. According to the invention, the biological ink is prepared from the acellular intervertebral disc, the polymer material is used as an auxiliary material, and the temporomandibular joint disc is reconstructed by using a 3D printing method, so that the problems that the physiological load of the temporomandibular joint cannot be matched, the reconstruction is difficult, the cost is high and the like in the prior art are effectively solved.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a bionic artificial temporomandibular joint disc and a preparation method thereof.
Background
The Temporomandibular joint disc (TMJ disc) is a fibrocartilage-like tissue located between the mandibular condyle and the temporal glenoid fossa, is a major component and important basis for the functioning of the Temporomandibular joint, and is mainly used to lubricate the articular surface, distribute loads, cushion concussion, and reduce the incoordination between the condyle and the bony structure of the glenoid fossa. The clinical damage or loss of the temporomandibular joint disc is one of the main causes of temporomandibular joint diseases, and further causes the dysfunction of the whole oral-jaw system, thus seriously affecting the life quality of patients. The data shows that there are as many as millions of patients worldwide each year who need to reconstruct the articular disc, with medical costs as high as hundreds of millions of dollars.
The temporomandibular joint disc is an elliptical fibrocartilaginous disc with the inner diameter and the outer diameter larger than the anteroposterior diameter, and the anatomical structure and the tissue characteristics of the temporomandibular joint disc are both used for realizing the biomechanical function of the temporomandibular joint disc. At present, biomechanical studies on articular discs have shown that: temporomandibular joint discs are also the best fibrocartilaginous organ of the human body to undergo disease such as perforation, rupture, and the like, as they are subjected to the forces of multiple components of friction, compression, tension and shear during the functional movements of the joint. Due to the lack of blood vessels and cells, the metabolism is slow, and the temporomandibular joint disc has extremely poor self-repairing ability once the temporomandibular joint disc is defected.
At present, the clinical commonly used temporomandibular joint disc repair substitute materials mainly comprise acellular allogeneic tissue patches and the transfer repair of self adjacent tissue valves, but the biomechanical properties of anatomical structures and histological components, especially substitute interpolators, are far away from temporomandibular joint discs and cannot be matched with the physiological load of temporomandibular joints. Meanwhile, the temporomandibular joint disc is special in morphology, various in components and complex in structure, and a bionic substitute with a good effect is difficult to obtain by using artificial materials, so that the temporomandibular joint disc is more difficult to reconstruct. Therefore, how to search or construct a new temporomandibular joint disc replacement material has become an important issue to be solved urgently in the field of temporomandibular joint disease treatment.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a bionic artificial temporomandibular joint disc and a preparation method thereof, wherein a biological ink is prepared from acellular intervertebral discs, a high polymer material is used as an auxiliary material, and a 3D printing method is used for reconstructing the temporomandibular joint disc, so that the problems that the physiological load of temporomandibular joints cannot be matched, the reconstruction is difficult, the cost is high and the like in the prior art are effectively solved.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: the preparation method of the bionic artificial temporomandibular joint disc comprises the following steps:
(1) collecting the intervertebral discs of the oxtail under the aseptic condition, cleaning, performing freeze thawing treatment for 4-6 cycles at the temperature of-20-37 ℃, extracting glycosaminoglycan and protein by adopting an ultrapure water/guanidine hydrochloride chemical reagent, then crushing the residual tissues into flocculent, adding 4-6 wt% of sodium dodecyl sulfate and 4-6 KU/ml of DNase, treating for 2-4 hours at room temperature, and cleaning without enzyme water to obtain an intervertebral disc flocculent sample;
(2) freeze-drying the disc flocculent sample obtained in the step (1), adding digestive juice, blending for 60-84 h at room temperature under the condition of 25-35 r/min, adjusting the pH value to be neutral, and adding the glycosaminoglycan and the protein obtained in the step (1) to obtain biological ink;
(3) and (3) blending the high polymer material with good biocompatibility and the biological ink obtained in the step (2) according to the mass ratio of 1: 1-3 at room temperature, and then carrying out 3D printing to obtain the bionic artificial temporomandibular joint disc.
Further, in the step (1), the intervertebral disc of 8-month-old oxtail is harvested under aseptic conditions and washed with physiological saline to remove blood stain.
Further, in step (1), 5 wt% of sodium lauryl sulfate and 5KU/ml of DNase were added and treated at room temperature for 3 hours.
Further, in the step (2), the digestive juice is prepared by the following method: dissolving pepsin in 0.4-0.6 vt% acetic acid solution to obtain digestive juice; the dry weight mass ratio of the pepsin to the intervertebral disc flocculent sample is 1: 9-11.
Further, the concentration of the acetic acid solution is 0.5 vt%, and the dry weight mass ratio of the pepsin to the disc flocculent sample is 1: 10.
Further, in the step (2), the pH value is adjusted to be neutral by using sodium hydroxide.
Further, in the step (3), the polymer material with good biocompatibility is norbornene modified hyaluronic acid or GelMA (methacrylated hydrogel).
Further, in the step (3), the polymer material and the bio-ink are blended at room temperature according to the mass ratio of 1: 1.
The bionic artificial temporomandibular joint disc prepared by the preparation method of the bionic artificial temporomandibular joint disc.
In summary, the invention has the following advantages:
1. the method is convenient and quick, has lower preparation cost, strong reliability, large clinical transformation value and convenient popularization and use, and effectively solves the problems that the physiological load of the temporomandibular joint cannot be matched, the reconstruction is difficult, the cost is higher and the like in the prior art. The intervertebral disc and the temporomandibular joint disc belong to fibrocartilage, and have rich sources and low cost, thus being an excellent choice for realizing the reconstruction of temporomandibular joint disc organs and solving the problems.
2. The temporomandibular joint disc has special morphology, various components and complex structure, and a bionic substitute with similar components and good substitution effect is difficult to obtain by utilizing artificial materials; however, tissues such as temporomandibular joint disc, intervertebral disc and meniscus belong to fibrocartilage, the basic components are the same, and the histological and functional characteristics are similar. Because the intervertebral disc supplies materials abundantly, several tens of each animal, the source is abundant; it is a good idea to prepare the articular disc with similar components by utilizing the intervertebral disc. However, the state of the intervertebral disc is different from that of the joint disc, so that the intervertebral disc needs to be made into biological ink for 3D printing and is in a bionic state.
3. During preparation, firstly, collecting the intervertebral disc of the oxtail and removing bloodstain, then carrying out decellularization treatment, extracting glycosaminoglycan and related protein for later use in the process, then crushing the remaining intervertebral disc tissue into flocculent, wherein the flocculent is more favorable for forming biological ink, removing cell components in the flocculent by using sodium dodecyl sulfate and DNA enzyme, and washing the flocculent intervertebral disc sample by enzyme-free water; freeze-drying the flocculent sample to remove water, adding digestive juice formed by pepsin and acetic acid solution, blending at room temperature and adjusting to be neutral, and finally adding the glycosaminoglycan and related protein extracted previously to obtain the biological ink; the bio-ink and the high polymer material (used for enhancing the mechanical property of the bio-ink) are blended at room temperature, and then the bionic printing is carried out by a 3D printing method, so that the bionic artificial temporomandibular joint disc can be obtained. The method takes the intervertebral discs of the oxtail as raw materials, is supplemented with high polymer materials, and reconstructs the temporomandibular joint disc by using a 3D printing method, the preparation method is simple and easy to operate, the bionic artificial temporomandibular joint disc can be quickly prepared, the current use requirements can be met, the intervertebral discs are wide in source and low in manufacturing cost, the cost can be effectively reduced, and the method is convenient to popularize and use.
Drawings
FIG. 1 is a flow chart of a disc decellularization process;
FIG. 2 is a flow chart of a process for preparing bio-ink for a decellularized intervertebral disc;
FIG. 3 shows the DNA content of each group of samples after completion of decellularization;
FIG. 4 is the collagen content of each group of samples after completion of decellularization;
fig. 5 is a dynamic elastic modulus mechanical property test result of the bio-ink.
In fig. 5, the larger squares and triangles are bio-ink test results, and the smaller squares and triangles are bio-ink test results after polymer reinforcement (i.e., bio-ink obtained by the present application).
Detailed Description
The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
A bionic artificial temporomandibular joint disc, the preparation method of which comprises the following steps:
(1) collecting intervertebral discs of 8-month-old oxtails under aseptic conditions, cleaning with penicillin normal saline to remove bloodstains, performing freeze thawing treatment at the temperature of-20-37 ℃ for 5 cycles, extracting glycosaminoglycan and protein by adopting ultrapure water/guanidine hydrochloride chemical reagents, then crushing the rest tissues into floccules, adding 4 wt% of Sodium Dodecyl Sulfate (SDS) and 4KU/ml of DNase, performing treatment at room temperature for 2 hours, and cleaning without enzyme water to obtain intervertebral disc flocculent samples;
(2) freeze-drying the disc flocculent sample obtained in the step (1), adding digestive juice, blending for 60 hours at room temperature at 25r/min, adjusting the pH value to be neutral by using sodium hydroxide, and adding the glycosaminoglycan and the protein obtained in the step (1) to obtain biological ink; the digestive juice is prepared by the following method: dissolving pepsin in 0.4 vt% acetic acid solution to obtain digestive juice; the dry weight ratio of the pepsin to the discogenic flocculent sample is 1: 9;
(3) blending the norbornene modified hyaluronic acid high polymer material and the biological ink obtained in the step (2) at room temperature according to the mass ratio of 1:1, and then performing 3D printing to obtain the bionic artificial temporomandibular joint disc.
Among them, the above-mentioned reagents used were SDS (Sigma, Cat # L4390-100G, Batch #096K0041), DNase (Sigma,150KU, D5025-150KU), Triton-100(Sigma,250ml, T8787-250), pepsin (Sigma, P7125-100G), glacial acetic acid (Sigma,45726-1L-F) and NaOH (Fluka,71692-1 KG).
Example 2
A bionic artificial temporomandibular joint disc, the preparation method of which comprises the following steps:
(1) collecting intervertebral discs of 8-month-old oxtails under aseptic conditions, cleaning with penicillin normal saline to remove bloodstains, performing freeze thawing treatment for 5 cycles at a temperature range of-20-37 ℃, extracting glycosaminoglycan and protein by adopting ultrapure water/guanidine hydrochloride chemical reagents, crushing the rest tissues into flocculent, adding 5 wt% of sodium dodecyl sulfate and 5KU/ml of DNase, performing treatment for 5 hours at room temperature, and cleaning without enzyme water to obtain intervertebral disc flocculent samples;
(2) freeze-drying the disc flocculent sample obtained in the step (1), adding digestive juice, blending for 72 hours at room temperature and 30r/min, adjusting the pH value to be neutral by using sodium hydroxide, and adding the glycosaminoglycan and the protein obtained in the step (1) to obtain biological ink; the digestive juice is prepared by the following method: dissolving pepsin in 0.5 vt% acetic acid solution to obtain digestive juice; the dry weight ratio of the pepsin to the discogenic flocculent sample is 1: 10;
(3) blending the norbornene modified hyaluronic acid high polymer material and the biological ink obtained in the step (2) at room temperature according to the mass ratio of 1:1, and then performing 3D printing to obtain the bionic artificial temporomandibular joint disc.
Example 3
A bionic artificial temporomandibular joint disc, the preparation method of which comprises the following steps:
(1) collecting intervertebral discs of 8-month-old oxtails under aseptic conditions, cleaning with penicillin normal saline to remove bloodstains, performing freeze thawing treatment for 5 cycles at a temperature range of-20-37 ℃, extracting glycosaminoglycan and protein by adopting ultrapure water/guanidine hydrochloride chemical reagents, crushing the rest tissues into flocculent, adding 6 wt% of sodium dodecyl sulfate and 6KU/ml of DNase, performing treatment for 4 hours at room temperature, and cleaning without enzyme water to obtain intervertebral disc flocculent samples;
(2) freeze-drying the disc flocculent sample obtained in the step (1), adding digestive juice, blending for 84h at room temperature at 35r/min, adjusting the pH value to be neutral by using sodium hydroxide, and adding the glycosaminoglycan and the protein obtained in the step (1) to obtain biological ink; the digestive juice is prepared by the following method: dissolving pepsin in 0.6 vt% acetic acid solution to obtain digestive juice; the dry weight ratio of the pepsin to the discogenic flocculent sample is 1: 11;
(3) and (3) blending GelMA (methacrylic acid hydrogel) high polymer material and the biological ink obtained in the step (2) according to the mass ratio of 1:1 at room temperature, and then performing 3D printing to obtain the bionic artificial temporomandibular joint disc.
Examples of the experiments
The procedure was the same as in example 2 except that the cell removal treatment was carried out using 5% SDS alone (sample after extraction), 5% SDS in combination with 5KU/ml DNAse (DNAse pretreatment), and 5% SDS in combination with 5KU/ml DNAse in combination with Triton-100(DNAse pretreatment + 0.1% Triton 100), respectively. After the treatment is finished, the cell removal effect, the content of residual component collagen and the mechanical property test result of the dynamic elastic modulus of the biological ink are evaluated by quantitative measurement and histology, and the result is shown in the figures 3-5.
As shown in FIG. 3, the DNA content in the tissue can be reduced to below the international cell-free standard (the dry weight content is less than or equal to 50ng/mg) by using three processing modes of simple 5% SDS (sample after extraction), 5% SDS combined with 5KU/ml DNAse (DNAse pretreatment), and 5% SDS combined with 5KU/ml DNAse combined with Triton-100(DNAse pretreatment + 0.1% Triton 100), so as to meet the clinical requirements. And 5% SDS combined with 5KU/ml DNAse pretreatment was most effective.
As can be seen from FIG. 4, the collagen content in the decellularized tissue was close to 900ug/mg in dry weight after the three treatment modes of simple 5% SDS (sample after extraction), 5% SDS combined with 5KU/ml DNAse (DNAse pretreatment), and 5% SDS combined with 5KU/ml DNAse combined with Triton-100(DNAse pretreatment + 0.1% Triton 100), indicating that the main component in the decellularized tissue is collagen, and the subsequent bio-ink preparation can be performed.
As can be seen from fig. 5, the dynamic elastic modulus of the bio-ink with the polymer material added is greater than that of the bio-ink without the polymer material added; therefore, the mechanical property of the bio-ink can be obviously improved by adding the high polymer material into the bio-ink, and the bionic artificial temporomandibular joint disc with better performance is prepared.
While the present invention has been described in detail with reference to the illustrated embodiments, it should not be construed as limited to the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (9)
1. A preparation method of a bionic artificial temporomandibular joint disc is characterized by comprising the following steps:
(1) collecting the intervertebral discs of the oxtail under the aseptic condition, cleaning, performing freeze thawing treatment for 4-6 cycles at the temperature of-20-37 ℃, extracting glycosaminoglycan and protein by adopting an ultrapure water/guanidine hydrochloride chemical reagent, then crushing the residual tissues into flocculent, adding 4-6 wt% of sodium dodecyl sulfate and 4-6 KU/ml of DNase, treating for 2-4 hours at room temperature, and cleaning without enzyme water to obtain an intervertebral disc flocculent sample;
(2) freeze-drying the disc flocculent sample obtained in the step (1), adding digestive juice, blending for 60-84 h at room temperature under the condition of 25-35 r/min, adjusting the pH value to be neutral, and adding the glycosaminoglycan and the protein obtained in the step (1) to obtain biological ink;
(3) and (3) blending the high polymer material with good biocompatibility and the biological ink obtained in the step (2) according to the mass ratio of 1: 1-3 at room temperature, and then carrying out 3D printing to obtain the bionic artificial temporomandibular joint disc.
2. The method for preparing a biomimetic artificial temporomandibular joint disc as described in claim 1, wherein in the step (1), the intervertebral disc of 8-month old oxtail is harvested under aseptic conditions and washed with physiological saline to remove blood stain.
3. The method for preparing a biomimetic artificial temporomandibular joint disc according to claim 1, wherein in step (1), 5 wt% sodium lauryl sulfate and 5KU/ml dnase are added and treated at room temperature for 3 hours.
4. The method for preparing a bionic artificial temporomandibular joint disc as claimed in claim 1, wherein in step (2), the digestive juice is prepared by: dissolving pepsin in 0.4-0.6 vt% acetic acid solution to obtain digestive juice; the dry weight ratio of the pepsin to the disc flocculent sample is 1: 9-11.
5. The method for preparing a bionic artificial temporomandibular joint disc as claimed in claim 4, wherein the concentration of the acetic acid solution is 0.5 vt% and the dry weight mass ratio of the pepsin to the discogenic sample is 1: 10.
6. The method for preparing a biomimetic artificial temporomandibular joint disc as claimed in claim 1, wherein in step (2), the pH is adjusted to neutral with sodium hydroxide.
7. The method for preparing a bionic artificial temporomandibular joint disc as claimed in claim 1, wherein in step (3), the high molecular material with good biocompatibility is norbornene modified hyaluronic acid or GelMA.
8. The method for preparing a bionic artificial temporomandibular joint disc as claimed in claim 1, wherein in step (3), the polymeric material and the bio-ink are blended in a mass ratio of 1:1 at room temperature.
9. The bionic artificial temporomandibular joint disc prepared by the preparation method of the bionic artificial temporomandibular joint disc according to any one of claims 1 to 8.
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