阅读说明：本技术 腱膜支架及其制备方法和应用 (Aponeurosis stent and preparation method and application thereof ) 是由 范存义 崔昊旻 余雅玲 文根 陈帅 于 2020-08-11 设计创作，主要内容包括：本发明提供了一种腱膜支架及其制备方法和应用,涉及生物材料技术领域。本发明提供的腱膜支架的制备方法,包括以下步骤：将腱膜细胞破碎、然后破碎细胞核,再通过DNA酶去除脱氧核糖核酸,最后经过切片制备得到腱膜支架。本发明的制备方法简单快捷,成本低,不涉及有害化学物质。本发明的腱膜支架,生物活性好,无免疫排斥反应,柔韧性好,能有效防止细胞透过,避免肌腱粘连。(The invention provides an aponeurosis scaffold and a preparation method and application thereof, and relates to the technical field of biological materials. The preparation method of the aponeurosis scaffold provided by the invention comprises the following steps: and (3) breaking the aponeurosis cells, then breaking cell nuclei, removing deoxyribonucleic acid by using DNase, and finally slicing to obtain the aponeurosis scaffold. The preparation method is simple and quick, has low cost and does not relate to harmful chemical substances. The aponeurosis scaffold has good biological activity, no immunological rejection and good flexibility, can effectively prevent cells from permeating and avoid tendon adhesion.)

1. The preparation method of the aponeurosis scaffold is characterized by comprising the following steps: and (3) breaking the aponeurosis cells, then breaking cell nuclei, removing deoxyribonucleic acid by using DNase, and finally slicing to obtain the aponeurosis scaffold.

2. The method of preparing a aponeurosis scaffold according to claim 1, further comprising a first rinse prior to disrupting the aponeurosis cells;

preferably, the rinsing liquid of the first rinsing comprises a buffer solution containing an anticoagulant, wherein the anticoagulant is heparin, and the buffer solution is PBS buffer solution;

preferably, the time of the first rinsing is 20-40min, preferably 30 min.

3. The method of preparing a aponeurosis scaffold according to claim 1, wherein the disruption of aponeurosis cells is performed by shaking the aponeurosis into a surfactant solution;

preferably, the surfactant solution comprises NP-40 solution or Triton X-100 solution, preferably Triton X-100 solution;

preferably, the concentration of the Triton X-100 solution is 1-3% (w/v), preferably 2% (w/v);

preferably, the temperature of the shaking is 2-10 ℃, preferably 4 ℃;

preferably, the time of the oscillation is 18 to 30 hours, preferably 24 hours.

4. The method of preparing a aponeurosis scaffold according to claim 1, wherein the disrupting cell nuclei is shaking of aponeurosis in a surfactant solution;

preferably, the surfactant solution comprises an SDS solution, a sodium glycocholate solution, or a sodium taurocholate solution, preferably an SDS solution;

preferably, the concentration of the SDS solution is 0.1-0.5% (w/v), preferably 0.25% (w/v);

preferably, the temperature of the shaking is 2-10 ℃, preferably 4 ℃;

preferably, the time of the oscillation is 18 to 30 hours, preferably 24 hours.

5. The method of preparing a aponeurosis scaffold according to claim 1, wherein the removing deoxyribonucleic acid by dnase is digesting aponeurosis in a dnase solution;

preferably, the concentration of the DNase solution is 0.5-2g/L, preferably 1 g/L;

preferably, the temperature of the digestion is 36-38 ℃, preferably 37 ℃;

preferably, the time of digestion is 0.5-1.5h, preferably 1 h.

6. Method for preparing a aponeurosis scaffold according to claim 1, characterized in that said sectioning is performed to a thickness of 150 and 250 μm, preferably 200 μm.

7. The method of preparing a aponeurosis scaffold according to claim 1, wherein the disrupting aponeurosis cells and disrupting cell nuclei further comprises a second rinse;

preferably, a third rinsing is further included between the disruption of the cell nuclei and the removal of the deoxyribonucleic acid by DNase;

preferably, the removing of deoxyribonucleic acid by DNase further comprises a fourth rinsing between the sections;

preferably, a fifth rinse is also included after the slicing.

8. The method for preparing the aponeurosis scaffold according to claim 7, wherein the rinsing solutions of the second rinsing, the third rinsing, the fourth rinsing and the fifth rinsing respectively independently comprise a buffer solution, preferably a PBS buffer solution;

preferably, the time of the second rinsing, the third rinsing and the fourth rinsing is 20-40min, preferably 30 min;

preferably, the time of the fifth rinsing is 5-15min, preferably 10 min.

9. A aponeurosal scaffold produced by the method of making an aponeurosal scaffold according to any one of claims 1 to 8.

10. Use of a aponeurosis scaffold according to claim 9 in the manufacture of a product for the prevention of tendon adhesions.

Technical Field

The invention relates to the technical field of biological materials, in particular to an aponeurosis stent and a preparation method and application thereof.

Background

With the development of social mechanization and the popularization of sports competition, the incidence of tendon injury increases year by year, causing huge economic and social burdens. According to statistics, the tendon diseases account for 19.2% of skeletal muscle system diseases, tendon adhesion occurs after 30-40% of flexor tendon injuries, and operations caused by the tendon adhesion are about 900 ten thousand cases per year. Tendon adhesions are manifested by loss of the sliding function of the tendon and restricted motor function of the adjacent joints, which seriously affect the quality of life and the working ability of the patient. In clinic, tendon adhesion is usually treated mainly by a loosening operation and is treated with biological materials or medicines, so the treatment effect is not good.

Tendon adhesion is a pathological phenomenon occurring during tendon healing. Tendon healing comprises endogenous healing and exogenous healing, wherein the endogenous healing is a tendon self-repair process completed by proliferation of tendon cells in a tendon; and the exogenous healing is that synovial membrane and granulation tissues around the tendon grow into the injured area of the tendon to form granulation tissues, and the repair of the tendon is completed through the growth of the granulation tissues. Exogenous healing is often the major factor leading to tendon adhesion. Therefore, prevention of exogenous healing, and completion of self-repair of damaged tendons by endogenous healing as much as possible, are key to avoiding adhesions.

For this reason, intensive research has been conducted on methods and theories for preventing tendon adhesion, such as improvement of tendon suture technology, early functional exercise after surgery, oral administration of drugs, and placement of physical barriers between tendons and surrounding tissues to block exogenous healing, and various therapeutic products have been developed, but these products still have serious drawbacks while achieving certain therapeutic effects:

(1) the absorbable polylactic acid anti-adhesion barrier film comprises: is made of medical synthetic polymer and is in a film shape, and is placed between tendon and peritendinous tissue to form a physical barrier for preventing and treating tendon adhesion. But it can only be used as a mechanical barrier, lacks biological activity, and has the disadvantages of low strength, poor flexibility, immunological rejection and slow degradation speed, and often forms a fiber package block locally.

(2) Hyaluronic acid gel: the polysaccharide has viscoelasticity, can maintain lubrication, and can be applied around tendon in operation to play a role in three-dimensional separation and prevent postoperative adhesion. However, the liquid easily flowing substance has the characteristic of flowing to the lower part, and in clinical practical application, the liquid easily flowing substance flows out of a body along with the change of the body position of an affected limb along with the drainage strip in an incision, so that the treatment effect is influenced.

(3) Anti-inflammatory drugs: is applied to anti-inflammatory and anti-adhesion after tendon injury operation. Because inflammatory reaction is the inducing factor of adhesion after tendon injury, the clinical application of anti-inflammatory drugs achieves the effect of inhibiting adhesion. However, the oral preparation needs large dose and belongs to systemic administration, the gastrointestinal tract reaction is large, the local drug concentration is low, and the clinical treatment effect is controversial.

(4) Adhesion loosening operation: the peritendinous adhesion tissue is excised by a surgical method, and the tendon and the peritendinous tissue are artificially separated to restore the sliding function of the tendon. However, the method is a selection of other treatment methods after failure, belongs to the last step of clinical treatment, and due to the characteristic that the tendon is easy to be fibrously healed after being injured, the operation often falls into the treatment cycle of adhesion-release-re-adhesion, and the physical and mental health of a patient is seriously influenced.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method of an aponeurosis scaffold, which is simple and rapid, has low cost and does not relate to harmful chemical substances.

The second purpose of the invention is to provide an aponeurosis scaffold which has good biological activity, no immunological rejection and good flexibility, can effectively prevent cells from permeating and avoid tendon adhesion.

The third purpose of the invention is to provide the application of the aponeurosis bracket in preparing products for preventing tendon adhesion.

In order to solve the technical problems, the following technical scheme is adopted:

in a first aspect, the invention provides a method for preparing an aponeurosis scaffold, comprising the following steps: and (3) breaking the aponeurosis cells, then breaking cell nuclei, removing deoxyribonucleic acid by using DNase, and finally slicing to obtain the aponeurosis scaffold.

As a further technical scheme, the method also comprises a first rinsing before the rupture of the aponeurosis cells;

preferably, the rinsing liquid of the first rinsing comprises a buffer solution containing an anticoagulant, wherein the anticoagulant is heparin, and the buffer solution is PBS buffer solution;

preferably, the time of the first rinsing is 20-40min, preferably 30 min.

As a further technical scheme, the rupture of the aponeurosis cells is that aponeurosis is placed in a surfactant solution and is shaken;

preferably, the surfactant solution comprises NP-40 solution or Triton X-100 solution, preferably Triton X-100 solution;

preferably, the concentration of the Triton X-100 solution is 1-3% (w/v), preferably 2% (w/v);

preferably, the temperature of the shaking is 2-10 ℃, preferably 4 ℃;

preferably, the time of the oscillation is 18 to 30 hours, preferably 24 hours.

As a further technical scheme, the cell nucleus is broken by placing the aponeurosis in a surfactant solution and shaking;

preferably, the surfactant solution comprises an SDS solution, a sodium glycocholate solution, or a sodium taurocholate solution, preferably an SDS solution;

preferably, the concentration of the SDS solution is 0.1-0.5% (w/v), preferably 0.25% (w/v);

preferably, the temperature of the shaking is 2-10 ℃, preferably 4 ℃;

preferably, the time of the oscillation is 18 to 30 hours, preferably 24 hours.

As a further technical scheme, the step of removing deoxyribonucleic acid by DNase comprises the steps of putting the aponeurosis into DNase solution for digestion;

preferably, the concentration of the DNase solution is 0.5-2g/L, preferably 1 g/L;

preferably, the temperature of the digestion is 36-38 ℃, preferably 37 ℃;

preferably, the time of digestion is 0.5-1.5h, preferably 1 h.

As a further technical scheme, the section is to cut the aponeurosis to the thickness of 150-250 μm, preferably to the thickness of 200 μm.

As a further technical scheme, the step of breaking the aponeurosis cells and breaking the space between cell nuclei further comprises a second rinsing;

preferably, a third rinsing is further included between the disruption of the cell nuclei and the removal of the deoxyribonucleic acid by DNase;

preferably, the removing of deoxyribonucleic acid by DNase further comprises a fourth rinsing between the sections;

preferably, a fifth rinse is also included after the slicing.

As a further technical scheme, the rinsing solutions of the second rinsing, the third rinsing, the fourth rinsing and the fifth rinsing respectively and independently comprise a buffer solution, and the buffer solution is preferably a PBS buffer solution;

preferably, the time of the second rinsing, the third rinsing and the fourth rinsing is 20-40min, preferably 30 min;

preferably, the time of the fifth rinsing is 5-15min, preferably 10 min.

In a second aspect, the invention provides an aponeurosis scaffold.

In a third aspect, the invention provides the use of an aponeurosis scaffold in the manufacture of a product for preventing tendon adhesion.

Compared with the prior art, the aponeurosis scaffold provided by the invention and the preparation method and application thereof have the following beneficial effects:

the preparation method of the aponeurosis bracket provided by the invention takes the aponeurosis tissue of the animal as the raw material, firstly, the aponeurosis cell is broken, and the cell membrane is removed; then breaking the cell nucleus and removing the nuclear membrane and most of nucleic acid; residual deoxyribonucleic acid is removed through DNA enzyme, so that immunological rejection caused by contact of exogenous deoxyribonucleic acid or protein and the wound is avoided; and finally, cutting the aponeurosis into slices to obtain the aponeurosis bracket with good performance. The preparation method is simple and quick, has low cost and does not relate to harmful chemical substances.

The aponeurosis scaffold is an animal-derived biological material, has good biological activity, no immunological rejection and good flexibility, can effectively prevent cells from permeating and avoid tendon adhesion.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram of porcine aponeurosis provided in example 1 of the present invention and a porcine aponeurosis scaffold of the present invention;

fig. 2A is a hematoxylin-eosin (H & E) staining chart of the pig tendon membrane and the pig tendon membrane stent of the present invention provided in experimental example 1 of the present invention, with an upward magnification of 100X and a downward magnification of 200X;

fig. 2B is a Masson trichrome staining chart of the pig tendon membrane and the pig tendon membrane stent of the present invention provided in experimental example 2 of the present invention, with an upward magnification of 100X and a downward magnification of 200X;

FIG. 3 is a crystal violet staining pattern provided in test example 3 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In a first aspect, the invention provides a method for preparing an aponeurosis scaffold, comprising the following steps: and (3) breaking the aponeurosis cells, then breaking cell nuclei, removing deoxyribonucleic acid by using DNase, and finally slicing to obtain the aponeurosis scaffold.

The aponeurosis of the invention are derived from animal aponeurosis tissue, such as porcine aponeurosis tissue, ovine aponeurosis tissue, bovine mucosal tissue, and the like. The preparation method of the aponeurosis bracket of the invention takes aponeurosis tissues of animals as raw materials, firstly, the aponeurosis cells are crushed, and the cell membranes are removed; then breaking the cell nucleus and removing the nuclear membrane and most of nucleic acid; residual deoxyribonucleic acid is removed through DNA enzyme, so that immunological rejection caused by contact of exogenous deoxyribonucleic acid or protein and the wound is avoided; and finally, cutting the aponeurosis into slices to obtain the aponeurosis bracket with good performance. The preparation method is simple and quick, has low cost and does not relate to harmful chemical substances.

In some preferred embodiments, the disrupting of the aponeurosis cells further comprises a first rinsing. The surface of fresh animal aponeurosis tissue is stained, therefore, the aponeurosis tissue is firstly cleaned to remove the blood stain on the surface of the aponeurosis tissue.

Preferably, the rinsing solution of the first rinsing comprises a buffer containing an anticoagulant, preferably heparin, and the buffer is preferably a PBS buffer.

In the present invention, in order to protect the biological activity of the aponeurosis tissue from being destroyed, it is necessary to maintain the aponeurosis in a suitable environment during the preparation process. Anticoagulants aid in the removal of blood stains, and therefore, the present invention employs a buffer containing an anticoagulant at a pH of 7.2-7.4 to rinse the aponeurosis tissue. Anticoagulants include, but are not limited to, heparin, or other anticoagulants well known to those skilled in the art; buffers include, but are not limited to, PBS buffer, or other buffers known to those skilled in the art that can be used with biological materials.

Preferably, the time of the first rinsing is 20-40min, preferably 30 min.

In the present invention, the time of the first rinsing is typically, but not limited to, 20min, 24min, 28min, 32min, 36min or 40min, preferably 30 min.

By further optimization and adjustment of anticoagulant, buffer and rinsing time, blood stains of aponeurosis tissue are sufficiently removed.

In some preferred embodiments, the disrupting of the aponeurosis cells is by shaking the aponeurosis into a surfactant solution. The surfactant can dissolve lipid on the surface of the cell membrane and change the permeability of the cell membrane, so that the purpose of breaking cells is achieved, and compared with other cell breaking methods, the method for breaking cells by using the surfactant is mild.

Preferably, the surfactant solution includes, but is not limited to NP-40 solution or Triton X-100 solution, or other milder surfactants known to those skilled in the art, preferably Triton X-100 solution. The aponeurosis is biological material with bioactivity, so that a surfactant with small destructiveness is adopted when the cell membrane is crushed, and the bioactivity of the aponeurosis is protected as much as possible.

Preferably, the concentration of the Triton X-100 solution is typically, but not limited to, 1% (w/v), 1.4% (w/v), 1.8% (w/v), 2.2% (w/v), 2.6% (w/v), or 3% (w/v), preferably 2% (w/v).

Preferably, the temperature of the shaking is typically, but not limited to, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, preferably 4 ℃. Helping to maintain the biological activity of the aponeurosis under cryogenic conditions.

Preferably, the time of the oscillation is typically, but not limited to, 18h, 20h, 22h, 24h, 26h, 28h or 30h, preferably 24 h.

Through further optimization and adjustment of the operation conditions of the broken cells, the cells are sufficiently broken and the cell membranes and the matrix are removed without destroying the precursors of the biological activity of the aponeurosis.

In some preferred embodiments, the disrupting cell nuclei is shaking the aponeurosis in a surfactant solution. In the invention, the nuclear membrane is also broken by adopting the surfactant, and the nuclear membrane is more difficult to break compared with the cell membrane, so the surfactant with better membrane breaking effect is needed.

Preferably, the surfactant solution includes, but is not limited to, SDS solution, sodium glycocholate solution, or sodium taurocholate solution, or a surfactant solution known to those skilled in the art that can be used to disrupt cell nuclei, preferably SDS solution.

Preferably, the concentration of the SDS solution is typically, but not limited to, 0.1% (w/v), 0.2% (w/v), 0.3% (w/v), 0.4% (w/v), or 0.5% (w/v), preferably 0.25% (w/v).

Preferably, the temperature of the shaking is typically, but not limited to, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, preferably 4 ℃.

Preferably, the time of the oscillation is typically, but not limited to, 18h, 20h, 22h, 24h, 26h, 28h or 30h, preferably 24 h.

Through further optimization and adjustment of the operation conditions for breaking cell nucleus, the cell nucleus is broken sufficiently, and most of nucleic acid is removed.

In some preferred embodiments, the removing deoxyribonucleic acid by dnase is digesting the aponeurosis in a dnase solution. After the cell membrane and cell nucleus are broken, a small amount of deoxyribonucleic acid remains on the aponeurosis, and the deoxyribonucleic acid needs to be removed. In the present invention, these remaining deoxyribonucleic acids are degraded and removed by DNase.

Preferably, the concentration of the DNase solution is typically, but not limited to, 0.5g/L, 0.6g/L, 0.8g/L, 1g/L, 1.2g/L, 1.4g/L, 1.6g/L, 1.8g/L or 2g/L, preferably 1 g/L.

Preferably, the temperature of the digestion is typically, but not limited to, 36 ℃, 36.4 ℃, 36.8 ℃, 37.2 ℃, 37.6 ℃ or 38 ℃, preferably 37 ℃.

Preferably, the time of digestion is typically, but not limited to, 0.5h, 0.7h, 0.9h, 1.1h, 1.3h or 1.5h, preferably 1 h.

Through further optimization and adjustment of the operating conditions for removing the residual deoxyribonucleic acid, the residual deoxyribonucleic acid on the aponeurosis can be sufficiently removed.

In some preferred embodiments, the sectioning is to section the aponeurosis to a thickness of 150-250 μm, preferably 200 μm. In the present invention, the aponeurosis thickness is typically, but not limited to, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm or 250 μm. The aponeurosis with the thickness has good flexibility, and can effectively prevent cells from permeating.

In some preferred embodiments, the disrupting aponeurosis cells and disrupting the spaces between the cell nuclei further comprises a second rinse.

Preferably, a third rinsing is further included between the disruption of the cell nuclei and the removal of the deoxyribonucleic acid by DNase;

preferably, the removing of deoxyribonucleic acid by DNase further comprises a fourth rinsing between the sections;

preferably, a fifth rinse is also included after the slicing.

In the invention, different reagents are adopted in three steps of breaking aponeurosis cells, breaking cell nuclei and removing deoxyribonucleic acid by DNase, so that in order to avoid the influence of the reagent in the previous step on the next step and fully exert the function of the reagent, a second rinsing, a third rinsing and a fourth rinsing are required to be added to remove the reagent residues in the previous step; to preserve the bioactivity of the post-sectioning aponeurosis, debris is removed and a fifth rinse is required after sectioning.

In some preferred embodiments, the rinsing solutions of the second rinsing, the third rinsing, the fourth rinsing and the fifth rinsing independently include but are not limited to a buffer solution, and the buffer solution is preferably a PBS buffer solution. To avoid changes in the biological activity of the aponeurosis, the buffer is used to rinse the aponeurosis.

Preferably, the time of the second rinsing, the third rinsing and the fourth rinsing is respectively 20min, 24min, 28min, 32min, 36min or 40min, and preferably 30 min.

By further optimizing and adjusting the time of the second rinsing, the third rinsing and the fourth rinsing, the reagent residues in the previous step are sufficiently removed.

Preferably, the time of the fifth rinsing is typically, but not limited to, 5min, 7min, 9min, 11min, 13min or 15min, preferably 10 min. After slicing, the aponeurosis is rinsed and wetted by buffer solution, which is helpful for preservation of aponeurosis.

In a second aspect, the invention provides an aponeurosis scaffold.

The aponeurosis bracket of the invention is an animal-derived biological material, has good biological activity, no immunological rejection, good flexibility, can be directly stored at minus 80 ℃ and is convenient to store.

In a third aspect, the invention provides the use of an aponeurosis scaffold in the manufacture of a product for preventing tendon adhesion.

The aponeurosis scaffold can effectively prevent cells from permeating and can be applied to preparation of products for preventing tendon adhesion.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.