阅读说明：本技术 一种脱细胞脐带补片及其制备方法和应用 (Acellular umbilical cord patch and preparation method and application thereof ) 是由 赵金忠 王立人 康育豪 鄢和新 朱雪晶 刘博武 蒋佳 于 2021-01-19 设计创作，主要内容包括：本发明公开了一种脱细胞脐带补片及其制备方法和应用。本发明的制备方法以人脐带组织制作新型脱细胞细胞外基质补片,对人脐带组织进行脱细胞处理,再经冷冻干燥成型后制备了具有三维多孔海绵样支架的脱细胞脐带补片。本发明的脱细胞脐带补片保留了一定的机械强度和细胞外基质成分,实现完全脱细胞和保留相对完整的胶原纤维结构；脱细胞脐带补片能够避免疾病的传播,同时在脱去细胞后,残留的细胞外基质结构仍富含大量生长因子,能够有效募集周围细胞定植分化。本发明的脱细胞脐带补片呈三维多孔网状结构,孔隙分布较均匀,材料具有一定的机械强度,适合作为促进慢性巨大肩袖损伤修复的新型组织工程材料。(The invention discloses a decellularized umbilical cord patch and a preparation method and application thereof. The preparation method of the invention prepares the novel acellular extracellular matrix patch by using human umbilical cord tissue, carries out acellular treatment on the human umbilical cord tissue, and prepares the acellular umbilical cord patch with the three-dimensional porous sponge-like scaffold after freeze drying and molding. The decellularized umbilical cord patch disclosed by the invention reserves certain mechanical strength and extracellular matrix components, realizes complete decellularization and reserves a relatively complete collagen fiber structure; the acellular umbilical cord patch can avoid the spread of diseases, and meanwhile, after the cells are removed, the residual extracellular matrix structure is still rich in a large number of growth factors, so that the peripheral cells can be effectively recruited for colonization and differentiation. The acellular umbilical cord patch disclosed by the invention is of a three-dimensional porous net structure, the pores are uniformly distributed, and the material has certain mechanical strength and is suitable for serving as a novel tissue engineering material for promoting the repair of chronic huge rotator cuff injury.)

1. A preparation method of the acellular umbilical cord patch is characterized by comprising the following steps:

step one, immersing the pretreated umbilical cord segment into TBS buffer solution, and placing the umbilical cord segment on a decoloring shaking table to vibrate for 12-24h at room temperature; discarding the TBS buffer solution, then adding a 1% Triton X-100 hypertonic solution, and placing on a decoloring shaking table and shaking at room temperature for 12-24 h; then discarding the hypertonic solution, adding TBS buffer solution containing 0.03% SDS, placing on a decoloring shaking table, and continuing shaking for 12-24h at room temperature;

step two, discarding the TBS buffer solution containing 0.03% SDS, washing the umbilical cord fragment by using the TBS buffer solution, shaking the umbilical cord fragment for 10-15 minutes at room temperature, repeating the shaking for 3-5 times, adding a new TBS buffer solution, and placing the umbilical cord fragment on a decoloring shaking table to continue shaking for 12-24 hours at room temperature; discarding the TBS buffer solution, adding double distilled water to wash the umbilical cord segments, shaking the umbilical cord segments for 10-15 minutes at room temperature, repeating the steps for 3-5 times, and collecting the cleaned umbilical cord segments;

step three, placing the cleaned umbilical cord segments in a low-temperature refrigerator at the temperature of-20 ℃ for storage; pre-cooling for 30min by using a freeze dryer, and then vacuumizing and freeze-drying the frozen umbilical cord segments for 12-24 h; packaging and sealing to obtain the acellular umbilical cord patch.

2. The method for preparing a decellularized umbilical cord patch according to claim 1, further comprising a quality inspection step:

performing fluorescence staining on cell nuclei of the umbilical cord segments obtained in the step two, namely the acellular extracellular matrix patch materials, and determining whether residual cell nuclei exist under a fluorescence microscope;

cutting the acellular extracellular matrix patch material without residual cell nucleuses after detection into sheets of 40-50 mu m, spraying gold on the surfaces of the sheets, observing whether the collagen structures of the sheets are damaged or not under a scanning electron microscope, and if the collagen structures of the sheets are not damaged, proving that the acellular umbilical cord patch with the three-dimensional porous sponge-like scaffold is successfully prepared.

3. The method for preparing the acellular umbilical cord patch according to claim 1, characterized in that in the first step, the pretreatment comprises the following specific steps: taking a fresh umbilical cord, quickly placing the umbilical cord in a sterile centrifuge tube, transferring the umbilical cord to a sterile operating platform, and cleaning the umbilical cord by using a 1xPBS buffer solution containing 1% of double antibodies until the surface of the umbilical cord is free from obvious blood stains; cutting the cleaned umbilical cord into small segments with the length of 4-6cm by using a scalpel, splitting the umbilical cord along a long axis, and carefully removing 2 umbilical arteries and 1 umbilical vein; and (3) unfolding the small segment of umbilical cord into a sheet shape, and then cleaning residual blood stain by using the 1xPBS buffer solution to obtain an umbilical cord fragment.

4. The method for preparing a decellularized umbilical cord patch according to claim 1, wherein the TBS buffer is 0.01mol/LTris-HCl, pH 8.0 buffer.

5. The method for preparing the decellularized umbilical cord patch according to claim 1, wherein the decellularized umbilical cord patch packaged in a sealing manner is stored in a refrigerator at 0-4 ℃ and is sterilized by irradiation.

6. The method of preparing a decellularized umbilical cord patch according to claim 5, wherein the irradiation conditions of the irradiation sterilization are 25k Gray.

7. An acellular umbilical cord patch prepared by the preparation method according to any one of claims 1 to 6.

8. The decellularized umbilical cord patch of claim 7, wherein a plurality of holes are uniformly perforated on the surface of the decellularized umbilical cord patch before use.

9. The decellularized umbilical cord patch of claim 8, wherein 9 circular holes with a diameter of 0.5mm need to be uniformly punched on the surface of the decellularized umbilical cord patch before use.

10. Use of the decellularized umbilical cord patch of any one of claims 7 to 9 in the repair of chronic giant rotator cuff injury.

Technical Field

The invention relates to the field of tissue engineering materials, in particular to a decellularized umbilical cord patch as well as a preparation method and application thereof.

Background

Rotator cuff tears are one of the most common causes of shoulder joint pain in elderly patients. Once the rotator cuff is torn, the rotator cuff tear is continuously enlarged without timely surgical intervention, so that the tendon-bone interface is damaged, tendon tissue degeneration disappears, and muscle tissue is fatted. The rotator cuff tear range of more than 3cm accounts for 20% of all rotator cuff injuries and 80% of postoperative re-tear cases, and is called chronic giant rotator cuff injury. Chronic massive rotator cuff injury can lead to severe shoulder joint dysfunction in patients, such as inability to pronate, lift and raise their arms backwards. And the surgical repair effect of the chronic huge rotator cuff injury is poor, and the long-term prognosis result is unpredictable, so that the patch has a reliable prospect of repairing the injured part.

The rotator cuff patch is mainly placed at the position of a large-area rotator cuff injury, and plays a role in recovering the mechanical conduction of the rotator cuff. The rotator cuff patch constructed by the high-molecular non-degradable material has good mechanical property, and has good mechanical property in the initial stage, but the non-degradable material gradually generates creep deformation along with the prolonging of time, so that the repair failure is caused. The degradable rotator cuff patch can play a role in recruiting cells while degrading, and promotes cell proliferation, tissue regeneration and tendon-bone interface repair, thereby achieving the function of promoting the regeneration of the injured rotator cuff.

The umbilical cord tissue has low immunity and is rich in a large amount of cytokines, the patch has a natural extracellular matrix structure, can provide a proper growth structure for recruited cells and induce the directional differentiation of the cells, but has poor mechanical properties and risks of disease transmission.

Disclosure of Invention

The invention aims to provide a decellularized umbilical cord patch and a preparation method and application thereof, aiming at the defects of the conventional umbilical cord tissue patch.

In order to achieve the purpose, the invention adopts the technical scheme that:

the first aspect of the invention provides a preparation method of a decellularized umbilical cord patch, which comprises the following steps:

step one, immersing the pretreated umbilical cord segment into TBS buffer solution, and placing the umbilical cord segment on a decoloring shaking table to vibrate for 12-24h at room temperature; discarding the TBS buffer solution, then adding a 1% Triton X-100 hypertonic solution, and placing on a decoloring shaking table and shaking at room temperature for 12-24 h; then discarding the hypertonic solution, adding TBS buffer solution containing 0.03% SDS, placing on a decoloring shaking table, and continuing shaking for 12-24h at room temperature;

step two, discarding the TBS buffer solution containing 0.03% SDS, washing the umbilical cord fragment by using the TBS buffer solution, shaking the umbilical cord fragment for 10-15 minutes at room temperature, repeating the shaking for 3-5 times, adding a new TBS buffer solution, and placing the umbilical cord fragment on a decoloring shaking table to continue shaking for 12-24 hours at room temperature; discarding the TBS buffer solution, adding double distilled water to wash the umbilical cord segments, shaking the umbilical cord segments for 10-15 minutes at room temperature, repeating the steps for 3-5 times, and collecting the cleaned umbilical cord segments;

step three, placing the cleaned umbilical cord segments in a low-temperature refrigerator at the temperature of-20 ℃ for storage; pre-cooling for 30min by using a freeze dryer, and then vacuumizing and freeze-drying the frozen umbilical cord segments for 12-24 h; packaging and sealing to obtain the acellular umbilical cord patch.

Further, the preparation method also comprises the quality inspection step:

performing fluorescence staining on cell nuclei of the umbilical cord segments obtained in the step two, namely the acellular extracellular matrix patch materials, and determining whether residual cell nuclei exist under a fluorescence microscope;

cutting the acellular extracellular matrix patch material without residual cell nucleuses after detection into sheets of 40-50 mu m, spraying gold on the surfaces of the sheets, observing whether the collagen structures of the sheets are damaged or not under a scanning electron microscope, and if the collagen structures of the sheets are not damaged, proving that the acellular umbilical cord patch with the three-dimensional porous sponge-like scaffold is successfully prepared.

Further, in the first step, the pretreatment specifically comprises the following steps: taking a fresh umbilical cord, quickly placing the umbilical cord in a sterile centrifuge tube, transferring the umbilical cord to a sterile operating platform, and cleaning the umbilical cord by using a 1xPBS buffer solution containing 1% of double antibodies until the surface of the umbilical cord is free from obvious blood stains; cutting the cleaned umbilical cord into small segments with the length of 4-6cm by using a scalpel, splitting the umbilical cord along a long axis, and carefully removing 2 umbilical arteries and 1 umbilical vein; and (3) unfolding the small segment of umbilical cord into a sheet shape, and then cleaning residual blood stain by using the 1xPBS buffer solution to obtain an umbilical cord fragment.

Further, the TBS buffer was 0.01mol/LTris-HCl, pH 8.0 buffer.

Further, the sealed and packaged decellularized umbilical cord patch is stored in a refrigerator at 0-4 ℃ and is subjected to irradiation sterilization.

Further preferably, the irradiation conditions of the irradiation sterilization are 25k Gray.

The second aspect of the invention provides a decellularized umbilical cord patch prepared by the preparation method.

Further, several holes need to be uniformly punched on the surface of the decellularized umbilical cord patch before use.

Further preferably, before use, the surface of the decellularized umbilical cord patch needs to be uniformly perforated with 9 circular holes with the diameter of 0.5 mm.

In a third aspect, the invention provides the use of the above decellularized umbilical cord patch in the repair of chronic giant rotator cuff injury.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention relates to a method for preparing a novel acellular extracellular matrix patch by using human umbilical cord tissue, which comprises the steps of carrying out acellular treatment on the human umbilical cord tissue, and then preparing the acellular umbilical cord patch with a three-dimensional porous sponge-like scaffold after freeze drying and forming.

The decellularized umbilical cord patch disclosed by the invention reserves certain mechanical strength and extracellular matrix components, realizes complete decellularization and reserves a relatively complete collagen fiber structure; the acellular umbilical cord patch can avoid the spread of diseases, and meanwhile, after the cells are removed, the residual extracellular matrix structure is still rich in a large number of growth factors, so that the peripheral cells can be effectively recruited for colonization and differentiation.

The acellular umbilical cord patch disclosed by the invention is of a three-dimensional porous net structure, the pores are uniformly distributed, and the material has certain mechanical strength and is suitable for serving as a novel tissue engineering material for promoting the repair of chronic huge rotator cuff injury.

Drawings

FIG. 1 is an electron microscope image of the coronal plane of the decellularized umbilical cord patch of the present invention;

FIG. 2 is an electron microscope image of the membrane surface of the decellularized umbilical cord patch of the invention;

FIG. 3 is an electron microscope image of the Huatong glue surface of the decellularized umbilical cord patch of the invention.

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

Example 1

Preparation of a decellularized umbilical cord patch comprising the steps of:

taking a fresh umbilical cord, quickly placing the umbilical cord in a sterile centrifuge tube, transferring the umbilical cord to a sterile operating platform, and cleaning the umbilical cord by using a 1xPBS buffer solution containing 1% of double antibodies until the surface of the umbilical cord is free from obvious blood stains; cutting the cleaned umbilical cord into small segments with the length of 4-6cm by using a scalpel, splitting the umbilical cord along a long axis, and carefully removing 2 umbilical arteries and 1 umbilical vein; spreading the small section of umbilical cord into a sheet shape, and then cleaning residual blood stain by using the 1xPBS buffer solution to obtain an umbilical cord segment;

immersing the pretreated umbilical cord segment into TBS buffer solution, and placing on a decoloring shaking table to shake for 12-24h at room temperature; the TBS buffer (0.01mol/LTris-HCl, pH 8.0) was discarded, then 1% Triton X-100 hypertonic solution was added and placed on a decolorizing shaker for 24h at room temperature; then removing the hypertonic solution, adding TBS buffer solution containing 0.03% SDS, placing on a decoloring shaking table, and continuing shaking for 24 hours at room temperature;

step two, discarding the TBS buffer solution containing 0.03% SDS, adding 50mL of TBS buffer solution into a 15cm dish to wash the umbilical cord segments, shaking the umbilical cord segments for 15 minutes at room temperature, repeating the shaking for 3 times, adding new 50mL of TBS buffer solution, and placing the umbilical cord segments on a decoloring shaking bed to continue shaking for 12 to 24 hours at room temperature; discarding the TBS buffer solution, adding 50ml of double distilled water into a 15cm dish to wash the umbilical cord fragments, shaking the umbilical cord fragments for 15 minutes at room temperature, repeating the steps for 3 times, and collecting the cleaned umbilical cord fragments;

step three, transferring the cleaned umbilical cord segments into a culture dish with the thickness of 100mm, and storing the umbilical cord segments in a low-temperature refrigerator at the temperature of-20 ℃ (the storage time is not more than one week); precooling for 30min by using a freeze dryer, putting the frozen umbilical cord segments and a 100mm culture dish on a tray in the freeze dryer, vacuumizing, and freeze-drying for 24h in the freeze dryer; packaging and sealing to obtain the desired acellular umbilical cord patch.

The sealed and packaged decellularized umbilical cord patch needs to be stored in a refrigerator at 0-4 ℃ and is subjected to radiation sterilization, wherein the radiation condition of the radiation sterilization is 25k Gray.

The embodiment also provides a quality inspection method of the decellularized umbilical cord patch, which comprises the following steps:

performing fluorescence staining on cell nuclei of the umbilical cord segments obtained in the second step, namely the acellular extracellular matrix patch materials, and determining whether residual cell nuclei exist under a fluorescence microscope;

and cutting the acellular extracellular matrix patch material without residual cell nucleuses after detection into sheets of 40-50 mu m, spraying gold on the surfaces of the sheets, observing whether the collagen structures of the sheets are damaged or not under a scanning electron microscope, and if the collagen structures of the sheets are not damaged, proving that the acellular umbilical cord patch with the three-dimensional porous sponge-like scaffold is successfully prepared (as shown in figures 1-3).

Application example

The application of the decellularized umbilical cord patch in repairing chronic giant rotator cuff injury comprises the following steps:

(1) uniformly punching 9 circular holes with the diameter of 0.5mm on the surface of the decellularized umbilical cord patch, so that bone marrow mesenchymal cells excited by medulla on the humeral head can be conveniently recruited to the patch, and the regeneration of the rotator cuff is promoted;

(2) taking sand beach chair position for patient; before operation, marking out the bony marks of the shoulder joints by using a marking pen, sterilizing by a conventional operation, and paving a towel;

(3) a puncture cone enters the subacromial gap through an outer access to establish a working channel, the arthroscope enters the subacromial gap forwards along the rear lateral edge of the subacromial gap, and the adhesive band in the subacromial gap is separated bluntly; clear the synovium and the adhesive tape of the hyperplasia and congestion of the subacromial space by using a planer tool, a radio frequency and other ion tools;

(4) performing medullary excitation operation on humeral head to promote bone marrow mesenchymal cells to be recruited to the decellularized umbilical cord patch;

(5) selecting a proper acellular extracellular matrix patch according to the size of the injury; the grasping forceps grasp the edge of the rotator cuff tissue, fully release the rotator cuff tissue and ensure that the rotator cuff tissue completely covers tendon insertion points; arranging the inner row of the ground anchors from front to back, arranging the tail ends of the ground anchors out of the body, taking two ground anchors in the inner row, and enabling one tail of each ground anchor to penetrate through the fixing hole in the outer row; similarly, the remaining two tail wires pass through the other outer row of the anchor bolts; respectively drilling a hole near the outer side edge of the shoulder sleeve dead center of the greater tubercle humerus, placing an anchor nail, placing the acellular umbilical cord patch on the outer side of the shoulder sleeve through a grasper, tensioning a suture, crossing to form a net, pressing the tendon end on a footprint area, and tightly fixing the patch and a shoulder sleeve tissue; the arthroscope is withdrawn and the wound is sutured.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.