CN114306755B - Biological patch for posterior scleral reinforcement and preparation method thereof - Google Patents
Biological patch for posterior scleral reinforcement and preparation method thereof Download PDFInfo
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Abstract
The invention provides a biological patch for posterior sclera reinforcement surgery and a preparation method thereof. The biological patch for posterior scleral reinforcement provided by the invention takes the dermal reticular layer and the small intestine submucosa as the base materials, and the spongy small intestine submucosa matrix and the dermal reticular matrix with the compact three-dimensional reticular structure are compounded together, so that the biological patch for posterior scleral reinforcement, which has good biocompatibility, is convenient for cell to grow in and has better mechanical property, can achieve the purposes of fusion with the original scleral tissue and thickening of the scleral tissue in the clinical application of posterior scleral reinforcement.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a biological patch for posterior sclera reinforcement and a preparation method thereof.
Background
High myopia is generally considered to be myopia with a degree of myopia greater than 600, and the axis of the eye for high myopia is usually continuously elongated, causing progressive increases in the degree of myopia.
In recent years, the proportion of people with high myopia has increased rapidly, and high myopia is mainly caused by scleral damage and scleral local dilation; at present, the clinical treatment means of myopia mainly adopts posterior scleral reinforcement; the posterior sclera reinforcement technology is that a certain reinforcing material is placed on the sclera of the posterior pole part of the eyeball to support the posterior pole part of the eyeball, and through a series of biological reactions, the sclera which is weak at the back part of the eyeball with high myopia is reinforced, the strength of the posterior sclera is improved, the deformation resistance of the sclera is enhanced, the axial of the eye is prevented from being further prolonged, and the visual function of the eye with high myopia is prevented from being further deteriorated.
In posterior scleral reinforcement, the selection, preparation and use of the reinforcement material has a significant impact on the effectiveness of the procedure and the healing process. In order to obtain a better treatment effect, the reinforcing material is required to have good biocompatibility, flexibility and strong tensile resistance. Clinical reinforcement materials can be divided into two categories, biological materials and non-biological materials, wherein biological materials are widely favored due to good biocompatibility; at present, the small intestine submucosa is a biological material commonly used for posterior scleral reinforcement, but the small intestine submucosa has a compact structure, cells are difficult to grow in, and the application of the small intestine submucosa in the posterior scleral reinforcement is limited.
Disclosure of Invention
The invention solves the problem that the small intestine submucosa is difficult to grow into cells when used for posterior sclera reinforcement.
In order to solve the above problems, the present invention provides a biological patch for posterior scleral reinforcement, which is formed by compounding a spongy intestinal submucosa matrix and a dermal reticular matrix having a dense three-dimensional reticular structure.
Optionally, a microporous structure is disposed on the dermal mesh matrix.
Another object of the present invention is to provide a method for preparing the biological patch for posterior scleral reinforcement surgery as described above, comprising the steps of:
s1: selecting a dermal reticular layer and a small intestine submucosa of an animal, and respectively carrying out degreasing treatment on the dermal reticular layer and the small intestine submucosa;
s2: respectively carrying out acellular treatment on the degreased dermal reticular layer and the degreased small intestine submucosa to obtain a dermal reticular layer matrix and a small intestine submucosa matrix;
s3: shearing the small intestine submucosa matrix, putting the small intestine submucosa matrix into an acetic acid aqueous solution containing pepsin, and stirring to obtain a small intestine submucosa matrix solution;
s4: soaking the dermal reticular matrix in the small intestine submucosa matrix solution, and freeze-drying to obtain a composite material;
s5: and carrying out crosslinking treatment on the composite material to obtain the biological patch for posterior scleral reinforcement.
Optionally, the step S1 of performing the degreasing treatment on the dermal reticular layer and the small intestine submucosa respectively comprises: respectively placing the dermal reticular layer and the small intestine submucosa in a degreasing solution for soaking for 6-12 h; the degreasing solution is at least one selected from acetone, absolute ethyl alcohol, ethylene glycol ethyl ether and isopropanol.
Optionally, the decellularizing the defatted dermal reticular layer and the defatted small intestine submucosa respectively comprises:
s21: respectively placing the degreased dermal reticular layer and the degreased small intestine submucosa in a cell removing solution, shaking for 20-24 h in a shaking table at room temperature, taking out, placing in sterile PBS, shaking for 2-3 h in the shaking table, and taking out, wherein the dermal reticular layer and the small intestine submucosa are respectively marked as a first dermal reticular layer and a first small intestine submucosa;
s22: respectively putting the first dermal reticular layer and the first small intestine submucosa into an enzyme solution, soaking for 20-24 h at 1-10 ℃, taking out, putting into sterile PBS, shaking for 2-3 h in a shaking table, and taking out to respectively obtain the dermal reticular layer matrix and the small intestine submucosa matrix.
Optionally, the cell-removing solution in step S21 is selected from a Triton X-100 solution with a volume fraction of 0.5% or an SDS solution with a mass volume fraction of 0.1%.
Optionally, the enzyme solution in step S22 is a trypsin solution with a mass volume fraction of 0.25% or a Dispasel enzyme solution with a concentration of 2.5U/mL.
Optionally, in the acetic acid aqueous solution containing pepsin in step S3, the mass fraction of pepsin is 0.1% -0.5%, and the volume fraction of acetic acid is 1% -5%; and in the step S3, the mass fraction of the small intestine submucosa matrix in the small intestine submucosa matrix solution is 1-5%.
Optionally, the cross-linking treatment of the composite material comprises: and (3) placing the composite material in a crosslinking treatment solution, shaking the composite material in a shaking table for 1 to 5 days at the temperature of between 15 and 25 ℃, and then freezing and drying the composite material to obtain the biological patch for posterior scleral reinforcement.
Optionally, the composite material is added into the crosslinking treatment liquid in a ratio of 0.05 g/mL-0.2 g/mL; the solvent of the crosslinking treatment liquid is alcohol with the concentration range of 40-95%, the crosslinking agent is one of genipin, glutaraldehyde or EDC, the mass volume fraction range of the crosslinking agent is 0.2-2%, and the pH value of the crosslinking treatment liquid is 5.0-7.0.
Optionally, before the degreasing treatment of the dermal reticular layer in step S1, the method further comprises: and perforating the dermis reticulation layer.
Compared with the prior art, the biological patch for posterior scleral reinforcement provided by the invention has the following advantages:
the biological patch for posterior scleral reinforcement provided by the invention takes the dermal reticular layer and the small intestine submucosa as the base materials, and the spongy small intestine submucosa matrix and the dermal reticular matrix with the compact three-dimensional reticular structure are compounded together, so that the biological patch for posterior scleral reinforcement, which has good biocompatibility, is convenient for cell to grow in and has better mechanical property, can achieve the purposes of fusion with the original scleral tissue and thickening of the scleral tissue in the clinical application of posterior scleral reinforcement.
Drawings
FIG. 1 is a microscopic topography of a spongy small intestine submucosa matrix in accordance with the present invention;
FIG. 2 is a microscopic topography of the dermal mesh matrix of the present invention;
FIG. 3 is a microscopic topography of the biological patch for posterior scleral reinforcement prepared in example 1 of the present invention.
Detailed Description
The following describes in detail embodiments of the present invention. The embodiments described below with reference to the drawings are exemplary and intended to be illustrative of the present invention and should not be construed as limiting the present invention, and all other embodiments that can be obtained by one skilled in the art based on the embodiments of the present invention without inventive efforts shall fall within the scope of protection of the present invention.
In order to solve the problem that cells are difficult to grow into the intestinal submucosa when the intestinal submucosa is used for posterior scleral reinforcement surgery, the invention provides a preparation method of a biological patch for the posterior scleral reinforcement surgery, which comprises the following steps:
s1: selecting a dermis reticular layer and a small intestine submucosa of an animal, and respectively carrying out degreasing treatment on the dermis reticular layer and the small intestine submucosa;
s2: respectively carrying out acellular treatment on the degreased dermal reticular layer and the degreased small intestine submucosa to obtain a dermal reticular layer matrix and a small intestine submucosa matrix;
s3: shearing the small intestine submucosa matrix, putting the small intestine submucosa matrix into an acetic acid aqueous solution containing pepsin, and stirring for 24-96 hours to obtain a small intestine submucosa matrix solution;
s4: soaking the dermal reticular matrix in the small intestine submucosa matrix solution, and freeze-drying to obtain the composite material;
s5: and carrying out crosslinking treatment on the composite material to obtain the biological patch for posterior scleral reinforcement.
The application preferably selects mature dermis reticular layer and small intestine submucosa of pig, sheep or cattle; the dermal reticular layer matrix and the small intestine submucosa matrix are respectively obtained after the degreasing treatment and the decellularization treatment are respectively carried out on the dermal reticular layer and the small intestine submucosa.
The prepared biological patch for posterior scleral reinforcement surgery has good biocompatibility, and can promote proliferation and growth of fibroblasts and vascular endothelial cells, and promote proliferation of collagen and formation of blood vessels; the small intestine submucosa matrix is directly used for the biological patch, and cells are difficult to grow in the patch because the small intestine mucosa has a compact structure and the thickness of the small intestine submucosa is only 25-100 mu m; in addition, the dermal reticular layer is thicker in collagen fiber and more in elastic fiber, and thick collagen fiber bundles are crisscrossed to form a compact three-dimensional reticular structure and have stronger toughness and elasticity;
the application preferably uses sterile scissors to cut the obtained small intestine submucosa matrix into small intestine submucosa matrix solution; soaking the dermal reticular matrix layer in small intestine submucosa matrix solution, and freeze drying to obtain spongy composite material composed of small intestine submucosa matrix and dermal reticular matrix layer; the temperature of freeze drying is preferably-60 ℃, and the pressure is 1Pa; and further performing cross-linking treatment on the composite material to combine the spongy small intestine submucosa matrix and the dermal reticular matrix layer into an integral structure to obtain the biological patch for posterior scleral reinforcement.
In the present application, the procedure of the degreasing treatment and the decellularization treatment of the dermal reticular layer and the small intestine submucosa are the same, but the processes of the degreasing treatment and the decellularization treatment of the dermal reticular layer and the small intestine submucosa are separately performed.
The invention provides a preparation method of a biological patch for posterior scleral reinforcement, which takes a dermal reticular layer and a small intestine submucosa as base materials, and compounds a spongy small intestine submucosa matrix and a dermal reticular matrix with a compact three-dimensional reticular structure, thereby preparing the biological patch for the posterior scleral reinforcement, which has good biocompatibility, is convenient for cell ingrowth and simultaneously has good mechanical property, and in the clinical application of the posterior scleral reinforcement, the biological patch can achieve the purposes of fusion with the original scleral tissue and scleral tissue thickening.
The application preferably selects the dermis reticular layer of the animal, comprises selecting the animal skin of a healthy mature animal, carrying out unhairing and disinfection treatment on the animal skin, and cutting the dermis reticular layer matrix with the thickness of 0.1-2 mm by using a drum type skin taking machine; when a drum-type dermatome is used for cutting the dermal reticular layer matrix, firstly, subcutaneous tissues are cut off, the thickness of the animal skin is 1-4 mm, and then, the epidermis and the dermal papilla layer are cut off, so that the dermal reticular layer matrix with the thickness of 0.1-2 mm is prepared; this application is through with subcutaneous tissue, epidermis and the whole excision of dermis papilla layer, only leave dermis reticular layer, thereby the operation of having avoided getting rid of the epidermis and the unable thorough problem that leads to taking off the cell of getting rid of the epidermis totally, simultaneously, dermis reticular layer is main dense connective tissue composition in the dermis layer, its collagen fiber is thicker, elastic fiber is more, the cell composition is less, thick collagen fiber bundle vertically and horizontally staggered is dense three-dimensional network structure, and have stronger toughness and elasticity, consequently after taking off cell processing, still can keep better mechanical properties and its three-dimensional network structure, thereby can obtain the better biological sclera support who satisfies the cell dermis reticular layer matrix that takes off of clinical demand.
Specifically, the step S1 of the present application of performing the degreasing treatment on the dermal reticular layer and the small intestine submucosa respectively includes: respectively placing the dermal reticular layer and the small intestine submucosa in a degreasing solution for soaking for 6-12 h; wherein the degreasing solution for degreasing the reticular dermis and the small intestine submucosa can be the same or different; in the present application, the degreasing solution for degreasing the dermal reticular layer and the small intestine submucosa is preferably at least one selected from the group consisting of acetone, absolute ethyl alcohol, ethylene glycol ethyl ether, and isopropyl alcohol.
Similarly, the procedure for decellularizing the defatted dermal reticular layer and the defatted small intestine submucosa may be the same or different; in order to simplify the preparation process, the two decellularization processes are preferably the same, but the two decellularization processes are performed separately; specifically, it is preferable that the decellularization treatment of each of the defatted reticular dermis and the defatted submucosa of small intestine includes:
s21: respectively placing the degreased dermal reticular layer and the degreased small intestine submucosa in a cell removal solution, shaking for 20-24 h at room temperature in a shaking table, taking out, placing in sterile PBS (phosphate buffer solution), shaking for 2-3 h in the shaking table, taking out, and respectively marking as a first dermal reticular layer and a first small intestine submucosa;
s22: respectively putting the first dermal reticular layer and the first small intestine submucosa into an enzyme solution, soaking for 20-24 h at 1-10 ℃, taking out, putting into sterile PBS, shaking for 2-3 h in a shaking table, and taking out to respectively obtain a dermal reticular layer matrix and a small intestine submucosa matrix.
In the application, the cell-removing solution in the step S21 is preferably Triton X-100 solution with volume fraction of 0.5% (v/v) or SDS solution with mass volume fraction of 0.1% (w/v); the enzyme solution in the step S22 is trypsin solution with the mass volume fraction of 0.25% or dispase enzyme solution with the concentration of 2.5U/mL.
In the application, preferably, in the acetic acid aqueous solution containing pepsin in the step S3, the mass fraction of the pepsin is 0.1-0.5%, and the volume fraction of the acetic acid is 1-5%; and in the small intestine submucosa matrix solution prepared in the step S3, the mass fraction of the small intestine submucosa matrix is 1-5%.
The application prefers that the crosslinking treatment of the composite material comprises: and (3) placing the composite material in a crosslinking treatment solution, shaking the composite material in a shaking table for 1 to 5 days at the temperature of between 15 and 25 ℃, and then freezing and drying the composite material to obtain the biological patch for posterior scleral reinforcement.
Specifically, in the crosslinking treatment process, the composite material is added into the crosslinking treatment solution in a ratio of 0.05 g/mL-0.2 g/mL; the solvent of the crosslinking treatment liquid is preferably 40-95% of alcohol, the crosslinking agent is one of genipin, glutaraldehyde or EDC, the mass volume fraction range of the crosslinking agent is 0.2-2%, and the PH of the crosslinking treatment liquid is 5.0-7.0.
In addition, because the dermal reticular layer is the main dense connective tissue component in the dermal layer, the structure is compact, and the dermal reticular layer lacks a micropore structure, so that the dermal reticular layer is not beneficial to the growth of fibroblasts and vascular endothelial cells, and the fusion with the original scleral tissue and the formation of collagen are influenced; therefore, the method for preparing the biological patch for posterior scleral reinforcement provided by the present application may further include, before the step S1 of performing the degreasing treatment on the dermal reticular layer: perforating the dermis reticular layer; the dermal reticular layer is perforated to increase the aperture inside the dermal reticular layer and allow fibroblasts and vascular endothelial cells to grow in, so that the goal of fusion with the original scleral tissue and thickening of the original scleral tissue is achieved; the drilling method in the application can be mechanical drilling or laser drilling.
Further, the preparation method of the biological patch for posterior scleral reinforcement provided by the present application, after the cross-linking treatment, may further include:
s6: cutting the biological patch for posterior scleral reinforcement surgery obtained in the step S5 into a strip-shaped biological patch with the length of 40-80 mm and the width of 5-20 mm;
s7: and (5) sealing and packaging the strip-shaped biological patch obtained in the step (S6), and sterilizing.
The sterilization method can be cobalt 60 radiation, electron beam irradiation or ethylene oxide sterilization, and the biological patch for posterior sclera reinforcement can be obtained after sterilization.
Another object of the present invention is to provide a biological patch for posterior scleral reinforcement, which is prepared by the method for preparing the biological patch for posterior scleral reinforcement as described above.
The biological patch for posterior sclera reinforcement provided by the invention takes the dermal reticular layer and the small intestine submucosa as the base materials, and the spongy small intestine submucosa matrix and the dermal reticular matrix with the compact three-dimensional reticular structure are compounded together, so that the biological patch for posterior sclera reinforcement, which has good biocompatibility, is convenient for the cells to grow in, and simultaneously has good mechanical property, can achieve the purposes of fusing with the original scleral tissues and thickening the scleral tissues in the clinical application of the posterior sclera reinforcement.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Example 1
The embodiment provides a preparation method of a biological patch for posterior scleral reinforcement, which comprises the following steps:
s1: selecting pigskin of healthy mature pig, depilating and sterilizing the pigskin, firstly cutting subcutaneous tissue by a drum type skin taking machine to ensure that the thickness of the pigskin is 2.5mm, and then cutting off epidermis and dermal papilla layer to prepare a dermal reticular layer with the thickness of 1 mm;
soaking the dermal reticular layer in acetone for 9h to obtain a defatted dermal reticular layer;
selecting a small intestine jejunum part of a mature pig, repeatedly washing and disinfecting, longitudinally splitting, and scraping a mucous membrane layer, a serous membrane layer and a muscular layer of the small intestine to obtain a small intestine submucosa layer;
soaking the small intestine submucosa in acetone for 9h to obtain degreased small intestine submucosa;
s2: placing the degreased dermal reticular layer in a Triton X-100 solution with the volume fraction of 0.5%, shaking for 22h at room temperature in a shaking table, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 2.5h in the shaking table, taking out, and marking as a first dermal reticular layer;
soaking the first dermal reticular layer in 0.25% trypsin solution at 5 deg.C for 22h, taking out, placing in sterile PBS, shaking in a shaker for 2.5h, and taking out to obtain dermal reticular layer matrix;
placing the degreased small intestine submucosa in a Triton X-100 solution with the volume fraction of 0.5%, shaking for 22h at room temperature in a shaking table, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 2.5h in the shaking table, taking out, and marking as a first small intestine submucosa;
soaking the first small intestine submucosa in 0.25% trypsin solution at 5 deg.c for 22 hr, taking out, adding into sterile PBS, shaking in shaking table for 2.5 hr, and taking out to obtain small intestine submucosa matrix;
s3: cutting the small intestine submucosa matrix with sterile scissors, and placing into acetic acid water solution containing pepsin, wherein the mass fraction of the pepsin is 0.3%, and the volume fraction of the acetic acid is 3%; stirring for 60h to obtain a small intestine submucosa matrix solution with the mass fraction of 3%;
s4: pouring the small intestine submucosa matrix solution into a culture dish, soaking the dermal reticular matrix obtained in the step S2 into the small intestine submucosa matrix solution, and carrying out freeze drying at-60 ℃ and 1Pa to obtain a spongy composite material consisting of the small intestine submucosa matrix and the dermal reticular matrix;
s5: placing the composite material in a crosslinking treatment solution with the solvent of alcohol, the crosslinking agent of genipin and the mass volume fraction of 1.1% and the pH of 6.0 at the temperature of 20 ℃, oscillating the composite material in a shaking table for 3 days, performing crosslinking treatment on the composite material, and finally performing freeze drying to obtain a biological patch for posterior scleral reinforcement surgery;
s6: cutting the biological patch for posterior scleral reinforcement surgery obtained in the step S5 into a strip-shaped biological patch with the length of 60mm and the width of 13 mm;
s7: and (4) hermetically packaging the strip-shaped biological patch obtained in the step (S6), and performing sterilization treatment through cobalt 60 radiation to obtain the biological patch for posterior sclera reinforcement surgery for clinical use.
Referring to fig. 1 to 3, the test was performed on the individual spongy intestinal submucosa matrix, the individual dermal network matrix, and the biological patch for posterior scleral augmentation prepared in this example, and it can be seen from the figures that the biological patch for posterior scleral augmentation prepared in this example is formed by compounding the spongy intestinal submucosa matrix and the dermal network matrix having a dense three-dimensional network structure.
The biological patch prepared by the embodiment has good biocompatibility, is convenient for cell ingrowth, has good mechanical property, and can achieve the purposes of fusion with the original scleral tissue and scleral tissue thickening in the clinical application of posterior scleral reinforcement.
Example 2
The embodiment provides a preparation method of a biological patch for posterior scleral reinforcement, which comprises the following steps:
s1: selecting cattle hide of healthy mature cattle, performing depilation and disinfection treatment on the cattle hide, firstly cutting off subcutaneous tissues by using a drum-type skin taking machine to ensure that the thickness of the cattle hide is 4mm, and then cutting off epidermis and a dermal papilla layer to prepare a dermal reticular layer with the thickness of 2 mm;
soaking the dermal reticular layer in isopropanol for 12h to obtain a defatted dermal reticular layer;
selecting a small intestine jejunum part of a mature cow, repeatedly washing and disinfecting, longitudinally splitting, and scraping a mucous membrane layer, a serous membrane layer and a muscular layer of the small intestine to obtain a small intestine submucosa layer;
soaking the small intestine submucosa in isopropanol for 12h to obtain degreased small intestine submucosa;
s2: placing the degreased dermal reticular layer in an SDS solution with the mass volume fraction of 0.1%, shaking for 24h at room temperature in a shaking table, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 3h in the shaking table, taking out, and marking as a first dermal reticular layer;
soaking the first dermal reticular layer in a dispase enzyme solution with the concentration of 2.5U/mL for 24 hours at 10 ℃, taking out, placing in sterile PBS, shaking for 3 hours in a shaking table, and taking out to obtain a dermal reticular layer matrix;
placing the degreased small intestine submucosa in an SDS solution with the mass volume fraction of 0.1%, shaking for 24 hours at room temperature in a shaking table, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 3 hours in the shaking table, taking out, and marking as a first small intestine submucosa;
soaking the first small intestine submucosa in 2.5U/mL Dispasel enzyme solution at 10 ℃ for 24h, taking out, placing in sterile PBS, shaking for 3h in a shaking table, and taking out to obtain a small intestine submucosa matrix;
s3: cutting the small intestine submucosa matrix with sterile scissors, and placing into an acetic acid aqueous solution containing pepsin, wherein the mass fraction of the pepsin is 0.5%, and the volume fraction of the acetic acid is 5%; stirring for 96h to obtain a small intestine submucosa matrix solution with the mass fraction of 5%;
s4: pouring the small intestine submucosa matrix solution into a culture dish, soaking the dermal reticular matrix obtained in the step S2 into the small intestine submucosa matrix solution, and carrying out freeze drying at-60 ℃ and 1Pa to obtain a spongy composite material consisting of the small intestine submucosa matrix and the dermal reticular matrix;
s5: placing the composite material in a crosslinking treatment solution at the temperature of 25 ℃, wherein the solvent is alcohol, the crosslinking agent is glutaraldehyde, the mass volume fraction of the glutaraldehyde is 2%, and the pH is 7.0, shaking the table for 5 days, performing crosslinking treatment on the composite material, and finally performing freeze drying to obtain a biological patch for posterior scleral reinforcement;
s6: cutting the biological patch for posterior scleral reinforcement surgery obtained in the step S5 into a strip-shaped biological patch with the length of 80mm and the width of 20 mm;
s7: and (5) sealing and packaging the strip-shaped biological patch obtained in the step (S6), and performing sterilization treatment through electron beam irradiation to obtain the biological patch for posterior scleral strengthening surgery for clinical use.
The detection process and the detection result of the embodiment are referred to the relevant contents in embodiment 1.
Example 3
The embodiment provides a preparation method of a biological patch for posterior scleral reinforcement, which comprises the following steps:
s1: selecting sheep skin of healthy mature sheep, performing unhairing and disinfection treatment on the sheep skin, cutting subcutaneous tissues by using a drum type skin taking machine to enable the thickness of the sheep skin to be 1mm, and then cutting an epidermis and a dermis papilla layer to prepare a dermis reticular layer with the thickness of 0.1 mm;
soaking the dermal reticular layer in isopropanol for 6h to obtain a defatted dermal reticular layer;
selecting a small intestine jejunum part of a mature cow, repeatedly washing and disinfecting, longitudinally splitting, and scraping a mucous membrane layer, a serous membrane layer and a muscular layer of the small intestine to obtain a small intestine submucosa layer;
soaking the small intestine submucosa in isopropanol for 6h to obtain degreased small intestine submucosa;
s2: placing the degreased dermal reticular layer in an SDS solution with the mass volume fraction of 0.1%, shaking for 20h at room temperature, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 2h, taking out, and marking as a first dermal reticular layer;
soaking the first dermal reticular layer in a dispase enzyme solution with the concentration of 2.5U/mL for 20 hours at the temperature of 1 ℃, taking out, placing in sterile PBS, shaking for 2 hours in a shaking table, and taking out to obtain a dermal reticular layer matrix;
placing the degreased small intestine submucosa in an SDS solution with the mass volume fraction of 0.1%, shaking for 20h at room temperature in a shaking table, taking out, placing in sterile PBS (phosphoric acid buffer solution), shaking for 2h in the shaking table, taking out, and marking as a first small intestine submucosa;
soaking the first small intestine submucosa in 2.5U/mL Dispasel enzyme solution at 1 ℃ for 20h, taking out, placing in sterile PBS, shaking for 2h in a shaking table, and taking out to obtain a small intestine submucosa matrix;
s3: cutting the small intestine submucosa matrix with sterile scissors, and placing into acetic acid water solution containing pepsin, wherein the mass fraction of the pepsin is 0.1%, and the volume fraction of the acetic acid is 1%; stirring for 24h to obtain a small intestine submucosa matrix solution with the mass fraction of 1%;
s4: pouring the small intestine submucosa matrix solution into a culture dish, soaking the dermal reticular matrix obtained in the step S2 into the small intestine submucosa matrix solution, and carrying out freeze drying at-60 ℃ and 1Pa to obtain a spongy composite material consisting of the small intestine submucosa matrix and the dermal reticular matrix;
s5: placing the composite material in a crosslinking treatment solution at the temperature of 15 ℃, wherein the solvent is alcohol, the crosslinking agent is glutaraldehyde, the mass volume fraction of the glutaraldehyde is 0.2%, and the pH is 5.0, shaking the composite material in a shaking table for 1 day, performing crosslinking treatment on the composite material, and finally performing freeze drying to obtain the biological patch for posterior scleral reinforcement;
s6: cutting the biological patch for posterior scleral reinforcement surgery obtained in the step S5 into a strip-shaped biological patch with the length of 40mm and the width of 5 mm;
s7: and (5) hermetically packaging the strip-shaped biological patch obtained in the step (S6), and performing sterilization treatment through electron beam irradiation to obtain the biological patch for posterior sclera reinforcement surgery for clinical use.
The detection process and the detection result of this embodiment are described in example 1.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.
Claims (8)
1. A biological patch for posterior scleral reinforcement is characterized by being formed by compounding a spongy small intestine submucosa matrix and a dermal reticular matrix with a compact three-dimensional reticular structure; the dermal reticular matrix is provided with a microporous structure;
the preparation method of the biological patch for posterior scleral reinforcement surgery comprises the following steps:
s1: selecting a dermis reticular layer and a small intestine submucosa of an animal, and respectively carrying out degreasing treatment on the dermis reticular layer and the small intestine submucosa;
s2: performing acellular treatment on the degreased dermal reticular layer and the degreased small intestine submucosa respectively to obtain a dermal reticular layer matrix and a small intestine submucosa matrix;
s3: shearing the small intestine submucosa matrix, putting the small intestine submucosa matrix into an acetic acid aqueous solution containing pepsin, and stirring to obtain a small intestine submucosa matrix solution;
s4: soaking the dermal reticular matrix in the small intestine submucosa matrix solution, and freeze-drying to obtain a composite material;
s5: performing crosslinking treatment on the composite material to obtain a biological patch for posterior scleral reinforcement;
before the degreasing treatment of the dermis reticular layer in the step S1, the method further comprises the following steps: and perforating the dermis reticulation layer.
2. The biological patch for posterior scleral reinforcement surgery according to claim 1, wherein the degreasing treatment of the dermal reticular layer and the small intestine submucosa in step S1 respectively comprises: respectively placing the dermal reticular layer and the small intestine submucosa in a degreasing solution to soak for 6-12 h; the degreasing solution is at least one selected from acetone, absolute ethyl alcohol, ethylene glycol ethyl ether and isopropanol.
3. The biological patch for posterior scleral reinforcement according to claim 1, wherein the decellularizing the defatted dermal network and the defatted small intestine submucosa, respectively, comprises:
s21: respectively placing the degreased dermal reticular layer and the degreased small intestine submucosa in a cell removing solution, shaking for 20-24 h in a shaking table at room temperature, taking out, placing in sterile PBS, shaking for 2-3 h in the shaking table, and taking out, wherein the dermal reticular layer and the small intestine submucosa are respectively marked as a first dermal reticular layer and a first small intestine submucosa;
s22: respectively putting the first dermal reticular layer and the first small intestine submucosa into an enzyme solution, soaking for 20-24 h at 1-10 ℃, taking out, putting into sterile PBS, shaking for 2-3 h in a shaking table, and taking out to respectively obtain the dermal reticular layer matrix and the small intestine submucosa matrix.
4. The biological patch for posterior scleral reinforcement surgery according to claim 3, wherein the decellularization solution in the step S21 is selected from the group consisting of a Triton X-100 solution having a volume fraction of 0.5% and an SDS solution having a mass volume fraction of 0.1%.
5. The biological patch for posterior scleral reinforcement surgery according to claim 3, wherein the enzyme solution in the step S22 is trypsin solution with a mass volume fraction of 0.25% or Dispasel enzyme solution with a concentration of 2.5U/mL.
6. The biological patch for posterior scleral reinforcement surgery according to claim 1, wherein in the acetic acid aqueous solution containing pepsin in step S3, the mass fraction of pepsin is 0.1% to 0.5%, and the volume fraction of acetic acid is 1% to 5%; the mass fraction of the small intestine submucosa matrix in the small intestine submucosa matrix solution in the step S3 is 1-5%.
7. The biological patch for posterior scleral reinforcement surgery according to any one of claims 1 to 6, wherein the cross-linking treatment of the composite material comprises: and (2) placing the composite material in a crosslinking treatment solution, shaking the composite material for 1 to 5 days in a shaking table at the temperature of between 15 and 25 ℃, and then freezing and drying the composite material to obtain the biological patch for posterior sclera reinforcement.
8. The biological patch for posterior scleral reinforcement surgery according to claim 7, wherein the composite material is added to the cross-linking treatment solution in a ratio of 0.05g/mL to 0.2 g/mL; the solvent of the crosslinking treatment liquid is alcohol with the concentration range of 40-95%, the crosslinking agent is one of genipin, glutaraldehyde or EDC, the mass volume fraction range of the crosslinking agent is 0.2-2%, and the pH value of the crosslinking treatment liquid is 5.0-7.0.
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| CN104958791B (en) * | 2015-07-29 | 2017-12-08 | 东莞博与再生医学有限公司 | A kind of glaucoma surgery compound bio matrix and preparation method thereof |
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