A decellularized oral collagen plug and its preparation method

Abstract

This invention relates to a decellularized oral collagen plug and its preparation method. The plug comprises an upper decellularized matrix membrane and a lower decellularized sponge plug. The preparation method is as follows: pretreated fresh animal-derived membrane material is treated with trypsin solution and sodium hydroxide solution, washed with purified water to obtain a decellularized matrix membrane; a portion of the obtained decellularized matrix membrane is placed in a low-temperature tissue ball mill, pulverized, homogenized, filtered, and washed with purified water to obtain a decellularized collagen solution; the decellularized collagen solution is poured into a freeze-drying mold, defoamed under low-temperature vacuum, covered with the decellularized matrix membrane, and after vacuum defoaming, freeze-dried and vacuum thermally cross-linked to obtain the finished collagen plug. This invention's decellularized oral collagen plug is suitable for filling and covering wounds after tooth extraction. The upper decellularized matrix membrane effectively prevents fibroblasts and epithelial cells from invading bone defect areas, while the lower decellularized sponge plug stabilizes blood clots, provides a biological scaffold, reduces postoperative complications, and promotes wound healing.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a decellularized oral collagen plug and its preparation method. Background Technology

[0002] The healing process of a tooth extraction wound is lengthy and complex. It begins with the formation of a blood clot, which then organizes and gradually forms granulation tissue. After about 20 days, the granulation tissue is gradually replaced by mature connective tissue, initiating early bone resorption and reconstruction. Immature bone tissue slowly transforms into mature bone tissue, and only then is the tooth extraction wound considered healed. Postoperative complications such as bleeding, swelling, pain, and dry socket are common after tooth extraction, accompanied by alveolar ridge resorption, which is detrimental to later implant restoration.

[0003] Most existing products use biofilms to cover the extraction socket to aid in its recovery. However, no product on the market can simultaneously fill the extraction socket and cover the extraction area. In other words, no product can both provide physical isolation to prevent fibroblasts and epithelial cells from invading the bone defect area and stabilize the blood clot, provide a biological scaffold, reduce postoperative complications, and promote wound healing.

[0004] Chinese patent application CN110559486A discloses a composite collagen membrane for bone grafting in alveolar bone defect areas. It comprises a double-layer structure consisting of a dense collagen fiber membrane layer and a collagen sponge layer. The collagen fiber membrane layer is composed of bovine collagen fibers, and the collagen sponge layer is composed of bovine collagen fibers and hyaluronic acid. It is evident that the composite collagen membrane disclosed in this patent has a layered structure, which still cannot achieve the functions of stabilizing blood clots in extraction sockets, providing a biological scaffold, reducing postoperative complications, and promoting wound healing. Commercially available collagen sponges for filling extraction sockets all employ collagen extraction and purification processes, resulting in long production cycles, rapid degradation, easy expansion upon contact with water, and residual chemical cross-linking agents. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a decellularized oral collagen plug that can simultaneously fill the extraction socket and cover the extraction wound. It not only provides physical isolation to prevent fibroblasts and epithelial cells from invading the bone defect area, but also stabilizes the blood clot, provides a biological scaffold, reduces postoperative complications, and promotes wound healing.

[0006] Another objective of this invention is to provide a method for preparing decellularized oral collagen plugs.

[0007] The technical problem solved by this invention is achieved through the following technical solution:

[0008] A decellularized oral collagen plug, comprising:

[0009] The decellularized matrix membrane above is used to cover the tooth extraction socket and shield the soft tissue from invading the bone defect area;

[0010] The decellularized sponge plug below is used to fill the extraction socket to prevent and alleviate postoperative complications and promote wound healing.

[0011] The decellularized matrix membrane can be a square, circular, or elliptical sheet-like structure, with a thickness of 0.1-1 mm and a coverage area of ​​300-1200 mm². 2 The decellularized sponge plug has a circular or elliptical cross-section with a cross-sectional area of ​​30-100 mm². 2 The longitudinal height of the decellularized sponge plug is 10-20 mm.

[0012] Furthermore, the decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar, spherical, or ellipsoidal three-dimensional structure, and the decellularized matrix membrane covers the upper surface of the decellularized sponge plug, with the decellularized matrix membrane and the upper surface of the decellularized sponge plug being cross-linked into one.

[0013] Furthermore, the lower surface of the decellularized sponge plug is a smooth surface, which can be a plane, a sphere, or an ellipsoid.

[0014] A method for preparing a decellularized oral collagen plug, the method comprising the following steps:

[0015] Step 1) Preparation of decellularized matrix membrane:

[0016] A. Material collection and pretreatment: Fresh animal-derived membrane materials are collected, blood, fat and impurities are removed, and they are cut into flat standard sizes to obtain processed animal-derived membrane materials;

[0017] B. Preparation of decellularized matrix membrane: The animal-derived membrane material processed in step A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane.

[0018] Step 2) Preparation of decellularized collagen solution: Take a portion of the prepared decellularized matrix membrane, crush, homogenize, filter and remove processing aids to obtain decellularized collagen solution;

[0019] Step 3) Shaping of oral collagen plugs:

[0020] A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam under low temperature vacuum for 30 min-1 h, and then cover it with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 30 min-1 h.

[0021] B. Freeze-dry for 35-48 hours;

[0022] C. Physical cross-linking of the freeze-dried sample: Electron beam radiation or vacuum thermal cross-linking is used to obtain a collagen plug product with a decellularized matrix membrane on the top and a decellularized sponge plug on the bottom.

[0023] D. Cut the cross-linked sample according to the usage specifications and pack it in double-layer sealed packaging.

[0024] E. Sterilization: The packaged samples are sent for irradiation sterilization.

[0025] Moreover, the specific steps for material collection and pretreatment in step 1)A are as follows: fresh animal-derived membrane materials are collected from the slaughterhouse. The animal-derived membrane materials are pig pericardium or pig peritoneum or bovine pericardium or bovine peritoneum. They are washed with cold water until there is no blood color. The fat and impurities on the rough surface are removed by hand. Then, the pericardium or peritoneum is cut into flat membrane pieces with a size of 6×9cm~12×16cm, depending on the size and flatness of the animal-derived membrane materials.

[0026] Moreover, the specific steps for preparing the decellularized matrix membrane in step 1)B are as follows: the pretreated animal-derived membrane material is placed in a 0.05%~0.25% trypsin solution and shaken at 35-40℃ for 1-4 hours, then placed in a 0.5-1M sodium hydroxide solution and shaken for 1-4 hours, and finally washed with purified water until the conductivity of the washing solution differs from that of the purified water by ±2μS / cm, thus obtaining the decellularized matrix membrane.

[0027] Moreover, the specific steps for preparing the decellularized collagen solution in step 2) are as follows: take a portion of the prepared decellularized matrix membrane and put it into a low-temperature tissue ball mill, add 0.5M-2M acetic acid solution, grind and homogenize for 20-45 minutes, filter it with a 110-130 mesh filter, wash the filtered collagen solution with purified water until the pH value of the washing solution differs from that of the purified water by ±1, and the decellularized collagen solution is obtained.

[0028] Furthermore, the electron beam radiation is 5-10 kGy, and the vacuum thermal crosslinking parameters are: vacuum degree < 20 Pa, temperature 80~120℃, and vacuum thermal crosslinking time 4-8 h; the irradiation sterilization is performed using gamma ray sterilization or electron beam sterilization, with an irradiation dose of 15-25 kGy.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. This invention relates to a decellularized oral collagen plug, used for filling and covering tooth extraction sockets. It consists of two parts: the upper part is a decellularized matrix membrane that covers the extraction socket and has certain mechanical properties and degradation cycle, playing a role in physical isolation and stabilizing the microenvironment of the bone defect area, which is conducive to bone repair; the lower part is a plug-like sponge used for filling the extraction socket. It is designed according to the internal structure of the extraction socket and perfectly fits the extraction wound. It is prepared by crushing the decellularized matrix membrane and can reduce post-extraction complications and promote wound healing.

[0031] 2. The decellularized oral collagen plug of this invention has a decellularized matrix membrane on top, which acts as a barrier membrane to physically isolate the tooth and prevent the downward migration of gingival fibroblasts and epithelial cells. This allows slower-growing osteoblasts to proliferate, differentiate, and mineralize within the defect area, thereby achieving bone tissue regeneration and reducing alveolar ridge resorption. The lower plug-like collagen sponge, after being inserted into the extraction socket, can quickly absorb blood, stabilize blood clots, and prevent postoperative bleeding, dry socket, and other complications. Collagen is the main structural protein supporting tissues and can provide a biological scaffold for cell activities such as cell attachment, migration, and proliferation, promoting wound healing.

[0032] 3. The present invention relates to decellularized oral collagen plugs and their preparation method, which employs decellularization technology throughout the entire process rather than extraction and purification. The decellularization process removes cellular components from tissues or organs through physical, chemical, or enzymatic methods, while retaining the structure and bioactive components of the natural extracellular matrix (ECM). Products produced by the decellularization process have the characteristics of intact collagen fibers, high degree of cross-linking, resistance to degradation, and the ability to maintain volume stability after being wetted. Moreover, the decellularization process can be completed within 2 days, while the extraction and purification process involves enzymatic hydrolysis, salting out, dialysis, etc., which can take anywhere from 15 days to several months. Therefore, using decellularization technology to prepare sponges can significantly shorten the production cycle. Furthermore, the main component of sponges prepared by the decellularization method is still collagen, with a purity of up to 98% or more.

[0033] 4. The present invention relates to decellularized oral collagen plugs and their preparation method. The raw materials for the oral collagen plugs are porcine or bovine animal tissues, which are widely available and natural materials. The main components are type I collagen and type III collagen. They have low immunogenicity, high bioactivity, and are biodegradable, thus avoiding secondary removal or "occupancy" effects after tooth extraction wound healing.

[0034] 5. The present invention relates to decellularized oral collagen plugs and their preparation method. The preparation of oral collagen plugs mainly involves decellularization and physical cross-linking. Compared with commercially available extracted and purified collagen sponges, decellularized sponge plugs have a significantly shorter production cycle, higher product density, slower degradation, and higher water absorption rate, while maintaining stable volume. They do not swell after being wetted, which could lead to wound rupture or excessive wound pressure affecting blood supply. At the same time, physical cross-linking ensures good product shaping, high safety, and the absence of residual chemical cross-linking agents or other toxic substances.

[0035] 6. The decellularized oral collagen plug of the present invention and its preparation method are suitable for filling and covering wounds after tooth extraction. The upper barrier membrane can effectively block the invasion of bone defect areas by rapidly growing fibroblasts and epithelial cells, and create a closed and stable microenvironment for slower-growing osteoblasts. The lower plug-like sponge can stabilize blood clots, provide biological scaffolds, reduce postoperative complications, and promote wound healing.

[0036] 7. The decellularized oral collagen plug of the present invention has a decellularized matrix membrane on the upper part that can cover the tooth extraction socket, and a plug-like sponge on the lower part that can fill the tooth extraction socket. The two are tightly bonded (see electron micrograph). Even after being moistened with water, the two layers remain firmly bonded without delamination. This avoids the separation and relative movement of the two during product use, which would lead to uneven stress, lack of support, inability to stabilize blood clots, and is not conducive to wound healing. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the structure of the decellularized oral collagen plug of the present invention;

[0038] Figure 2 This is a half-sectional view of the decellularized oral collagen plug of the present invention;

[0039] Figure 3 Image a is a photograph of the decellularized oral collagen plug of the present invention, and image b is a photograph of the decellularized oral collagen plug of the present invention from another angle.

[0040] Figure 4 This is an electron micrograph of the decellularized oral collagen plug of the present invention;

[0041] Figure 5 Photo a shows the extraction socket immediately filled with collagen plugs according to Example 1, 14 days post-operation. Photo b shows the extraction socket without any filling material, only with cotton balls applied to stop bleeding. After hemostasis, the bone flap on the filling site was sutured and the wound was closed, 14 days post-operation.

[0042] Figure 6 Image a shows the histopathological images of the test sample group (using Example 1) one week after surgery, and image b shows the histopathological images of the blank group one week after surgery.

[0043] Figure 7 Image a shows the histopathological findings of the test sample group (using Example 1) 4 weeks post-surgery; image b shows the histopathological findings of the control group 4 weeks post-surgery.

[0044] Figure 8 Image a shows the histopathological images of the test sample group using Example 1 at 8 weeks post-operation, and image b shows the histopathological images of the blank group at 8 weeks post-operation.

[0045] Figure 9 This is the dry weight test data for oral collagen plugs and commercially available collagen sponges in Example 2;

[0046] Figure 10 This is the dry weight loss rate test data for oral collagen plugs and commercially available collagen sponges in Example 2.

[0047] Explanation of reference numerals in the attached figures:

[0048] 1-Decellularized matrix membrane; 2-Decellularized sponge plug. Detailed Implementation

[0049] The present invention will now be described in more detail through specific embodiments. These embodiments are intended to provide a further understanding and explanation of the present invention, and are for descriptive purposes only, and do not constitute any limitation on the scope of protection of the present invention.

[0050] A type of decellularized oral collagen plug, such as Figure 1 , Figure 2 As shown, it includes: an upper decellularized matrix membrane 1, used to cover the tooth extraction socket and shield soft tissue from invading the bone defect area; and a lower decellularized sponge plug 2, used to fill the tooth extraction socket to prevent and alleviate postoperative complications and promote wound healing.

[0051] The decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar, spherical, or ellipsoidal three-dimensional structure, with the decellularized matrix membrane covering the upper surface of the decellularized sponge plug, and the decellularized matrix membrane and the upper surface of the decellularized sponge plug are cross-linked together. The lower surface of the decellularized sponge plug is a smooth surface, which can be planar, spherical, or ellipsoidal.

[0052] Decellularized matrix membranes can be square, circular, or elliptical sheet-like structures, with a thickness of 0.1-1 mm and a coverage area of ​​300-1200 mm². 2 The decellularized sponge plug has a circular or elliptical cross-section with a cross-sectional area of ​​30-100 mm². 2 The longitudinal height of the decellularized sponge plug is 10-20 mm.

[0053] A method for preparing a decellularized oral collagen plug, the method comprising the following steps:

[0054] Step 1) Preparation of decellularized matrix membrane:

[0055] A. Material Collection and Pre-treatment: Fresh animal-derived membrane materials are collected, blood, fat, and impurities are removed, and the membranes are cut into flat, standard sizes to obtain processed animal-derived membrane materials. Specific steps are as follows: Fresh animal-derived membrane materials are collected from slaughterhouses. These materials are either pig pericardium or pig peritoneum, or bovine pericardium or bovine peritoneum. They are washed in cold water until no blood remains. Fat and impurities on the rough surfaces are gently removed by hand. The pericardium or peritoneum is then cut into flat sheets, with dimensions ranging from 6×9cm to 12×16cm, depending on the size and flatness of the animal-derived membrane material.

[0056] B. Preparation of decellularized matrix membrane: The animal-derived membrane material treated in step A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane. The specific steps are as follows: The pretreated animal-derived membrane material is placed in a 0.05%~0.25% trypsin solution and shaken at 35-40℃ for 1-4 hours, then placed in a 0.5-1M sodium hydroxide solution and shaken for 1-4 hours. Finally, it is washed with purified water until the conductivity of the washing solution differs from that of the purified water by ±2μS / cm to obtain a decellularized matrix membrane. For porcine peritoneum or bovine peritoneum, it is preferred to treat with 0.1% trypsin at 37℃ for 2 hours and shaken with 0.5M sodium hydroxide for 1 hour; for porcine pericardium or bovine pericardium, it is preferred to treat with 0.25% trypsin at 37℃ for 2 hours and shaken with 1M sodium hydroxide for 2 hours.

[0057] Step 2) Preparation of decellularized collagen solution: A portion of the prepared decellularized matrix membrane is pulverized, homogenized, filtered, and processing aids are removed to obtain decellularized collagen solution. Specifically, a portion of the prepared decellularized matrix membrane is placed in a low-temperature tissue ball mill, and 0.5M-2M acetic acid solution is added. The mixture is pulverized and homogenized for 20-45 minutes, then filtered through a 110-130 mesh filter. The filtered collagen solution is washed with purified water until the pH of the washing solution differs from that of the purified water by ±1. For porcine peritoneum or bovine peritoneum, a 0.5-1.5M acetic acid solution is preferred, and the mixture is pulverized and homogenized for 20-30 minutes. For bovine pericardium or bovine peritoneum, a 1.5-2M acetic acid solution is preferred, and the mixture is pulverized and homogenized for 30-40 minutes.

[0058] Step 3) Shaping of oral collagen plugs:

[0059] A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam under low temperature vacuum for 30 min-1 h, and then cover it with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 30 min-1 h.

[0060] B. Freeze-dry for 35-48 hours; when the longitudinal height of the decellularized sponge plug is 10-15 mm, freeze-drying for 35-40 hours is preferred; when the longitudinal height of the decellularized sponge plug is 15-20 mm, freeze-drying for 40-48 hours is preferred.

[0061] C. Physically cross-link the lyophilized sample: Electron beam radiation or vacuum thermal cross-linking is used to obtain a collagen plug product with a decellularized matrix membrane on top and a decellularized sponge plug on the bottom; Electron beam radiation is 5-10 kGy, and if vacuum thermal cross-linking is used, the parameters are: vacuum degree < 20 Pa, temperature 80~120℃, and vacuum thermal cross-linking time is 4-8 h.

[0062] D. Cut the cross-linked sample according to the usage specifications and pack it in double-layer sealed packaging.

[0063] E. Sterilization: The packaged samples are sent for irradiation sterilization. Irradiation sterilization is carried out by gamma ray sterilization or electron beam sterilization, and the irradiation dose is 15-25 kGy.

[0064] Example 1

[0065] A type of decellularized oral collagen plug, such as Figure 3 a, Figure 3 As shown in b, the structure includes: an upper decellularized matrix membrane to cover the extraction socket and shield the bone defect area from soft tissue invasion; and a lower decellularized sponge plug to fill the extraction socket, prevent and alleviate postoperative complications, and promote wound healing. The decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar structure, with the decellularized matrix membrane covering the upper surface of the decellularized sponge plug. The decellularized matrix membrane and the upper surface of the decellularized sponge plug are cross-linked together.

[0066] In this embodiment, the decellularized matrix membrane can be a square sheet structure with a thickness of 0.5 mm; its coverage area is 15*20 mm = 300 square millimeters; the decellularized sponge plug has a columnar structure with a smooth lower surface, which in this embodiment is spherical. The transverse cross-section of the decellularized sponge plug is circular with a transverse diameter of 7 mm and a cross-sectional area of ​​38.5 mm². 2 The longitudinal height of the decellularized sponge plug is 15 mm.

[0067] The preparation method of decellularized oral collagen plugs in this embodiment includes the following steps:

[0068] Step 1) Preparation of decellularized matrix membrane:

[0069] A. Material Collection and Pre-treatment: Fresh pig peritoneum is collected, and blood, fat, and impurities are removed. It is then cut into flat, standard sizes to obtain processed animal-derived membrane materials. Specific steps are as follows: Fresh pig peritoneum is collected from the slaughterhouse, washed with cold water until no blood remains, and the rough surfaces are gently removed by hand to remove fat and impurities. The peritoneum is then cut into flat sheets, each 10×10cm in size, depending on the actual size and flatness of the peritoneum.

[0070] B. Preparation of decellularized matrix membrane: The porcine peritoneum treated in step 1) A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane. The specific steps are as follows: the pretreated porcine peritoneum is placed in a 0.1% trypsin solution and shaken at 37°C for 2 hours, then placed in a 0.5M sodium hydroxide solution and shaken for 1 hour, and finally washed with purified water until the conductivity of the washing solution differs from that of the purified water by 1.42 μS / cm to obtain the decellularized matrix membrane.

[0071] Step 2) Preparation of decellularized collagen solution: Take a portion of the prepared decellularized matrix membrane, pulverize, homogenize, filter and remove processing aids to obtain decellularized collagen solution; the specific steps are as follows: take a portion of the prepared decellularized matrix membrane and put it into a low-temperature tissue ball mill, add 1M acetic acid solution, pulverize and homogenize for 20 minutes, filter with a 120-mesh filter, wash the filtered collagen solution with purified water until the pH value of the washing solution differs from that of the purified water by 0.76, and the decellularized collagen solution is obtained.

[0072] Step 3) Shaping of oral collagen plugs:

[0073] A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam at low temperature under vacuum for 30 minutes, then cover with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 30 minutes.

[0074] B. Freeze-dry for 35 hours;

[0075] C. Physical cross-linking of the freeze-dried sample: Vacuum thermal cross-linking was used to obtain a collagen plug product with a decellularized matrix membrane on the top and a decellularized sponge plug on the bottom; the vacuum thermal cross-linking parameters were: vacuum degree 5 Pa, temperature 105 ℃, and vacuum thermal cross-linking time 6 h.

[0076] D. According to the clinical usage specifications, cut the cross-linked sample according to the usage specifications and pack it in double-layer sealed packaging;

[0077] E. Sterilization: The packaged samples are sent for irradiation sterilization. Irradiation sterilization is carried out by gamma ray sterilization or electron beam sterilization, and the irradiation dose is 25 kGy.

[0078] like Figure 4 The image shown is an electron microscope image (longitudinal cross-section) of the sample of Example 1 of the decellularized oral collagen plug of the present invention. It can be seen that the two layers of decellularized matrix membrane and decellularized sponge plug are tightly bonded together.

[0079] The sample prepared in Example 1 was compared with commercially available collagen sponge products. The comparison items were water absorption rate, volume expansion rate, density, total protein content, and hydroxyproline content (related to collagen purity). The results showed that the sample prepared in this invention had similar water absorption rate and collagen purity to commercially available similar products, but had a low volume expansion rate and high density.

[0080]

[0081] The oral collagen plug prepared in Example 1 was used for filling and repairing extraction sockets in beagle dogs. The results showed that the oral collagen plug could effectively stop bleeding and promote wound healing. The specific experimental procedure is as follows:

[0082] Nine healthy adult beagle dogs were selected and randomly divided into three groups at weeks 1, 4, and 8. The bilateral mandibular third premolars were extracted. The left extraction socket was immediately packed with oral collagen plugs (test group), while the right extraction socket was left unpacked (blank group), with hemostasis achieved only by applying pressure with cotton balls. After hemostasis, the bone flaps over the packed areas were sutured, and the wounds were closed. Hemostasis time was recorded intraoperatively for each group. Postoperatively, routine clinical observation was performed, and histopathological evaluation was conducted at weeks 1, 4, and 8.

[0083] The results show that:

[0084] (1) The oral collagen plug test sample group of Example 1 of the present invention can effectively stop bleeding within 2 minutes after implantation, with an average hemostasis time of 1.55±0.30 min. The blank group can effectively stop bleeding within 5 minutes after applying gauze, with an average hemostasis time of 4.38±0.64 min. There is a significant difference between the two groups.

[0085] (2) Fourteen days post-surgery, the wounds in the control group were not completely closed and remained depressed, while the wounds in the implantation area of ​​the test group healed well, closed, and became flat. Figure 5 a, Figure 5 As shown in b.

[0086] (3) Pathological histological results at 1, 4 and 8 weeks postoperatively showed that, compared with the control group, the test sample group had faster growth of new blood vessels, woven bone and osteoblasts in the tooth extraction socket, which was conducive to wound healing. However, the control group lacked oral collagen plugs, biological scaffolds and barrier membranes, resulting in excessive soft tissue growth in the tooth extraction socket and slow bone tissue repair.

[0087] like Figure 6 a, Figure 6 As shown in b, one week post-surgery, in the test group: a large amount of implant residue was visible, with numerous fibroblasts and a small number of new blood vessels around the implant. In the control group: obvious fibrous tissue and a small number of new blood vessels were visible around the wound.

[0088] like Figure 7 a, Figure 7 As shown in b, 4 weeks post-surgery, in the test group: abundant woven bone and significant fibrous tissue were visible at the wound site, with obvious neovascularization and numerous osteoblasts at the alveolar bone margin. In the control group: numerous osteoblasts and a small amount of fibrous tissue were visible at the wound site.

[0089] like Figure 8 a, Figure 8 As shown in b, 8 weeks post-surgery, in the test group: Extensive woven bone and new bone formation were observed. The bone structure was intact, and the osteocyte morphology was normal. No connective tissue hyperplasia was observed. In the control group: A small amount of woven bone and abundant fibrous connective tissue were visible at the wound edge.

[0090] Example 2

[0091] A decellularized oral collagen plug includes: an upper decellularized matrix membrane for covering the extraction socket and shielding the bone defect area from soft tissue invasion; and a lower decellularized sponge plug for filling the extraction socket to prevent and alleviate postoperative complications and promote wound healing. The decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar structure, with the decellularized matrix membrane covering the upper surface of the decellularized sponge plug. The decellularized matrix membrane and the upper surface of the decellularized sponge plug are cross-linked together.

[0092] In this embodiment, the decellularized matrix membrane can be a circular sheet structure with a thickness of 1 mm and a diameter of 30 mm, covering an area of ​​706.5 square millimeters. The decellularized sponge plug has a columnar structure with a smooth lower surface; in this embodiment, the smooth lower surface of the decellularized sponge plug is spherical. The transverse cross-section of the decellularized sponge plug is circular with a transverse diameter of 8 mm and a cross-sectional area of ​​50.24 mm². 2 The longitudinal height of the decellularized sponge plug is 20 mm.

[0093] The preparation method of decellularized oral collagen plugs in this embodiment includes the following steps:

[0094] Step 1) Preparation of decellularized matrix membrane:

[0095] A. Material Collection and Pre-treatment: Fresh pig pericardium is collected, blood, fat, and impurities are removed, and it is cut into flat, standard sizes to obtain processed animal-derived membrane materials. Specific steps are as follows: Fresh pig pericardium is collected from the slaughterhouse, washed with cold water until no blood remains, and the rough surfaces are gently removed by hand to remove fat and impurities. The pericardium is then cut into flat sheets, 6×9cm in size, depending on the size and flatness of the pericardium.

[0096] B. Preparation of decellularized matrix membrane: The porcine pericardium treated in step A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane. The specific steps are as follows: the pretreated porcine pericardium is placed in a 0.25% trypsin solution and shaken at 37°C for 2 hours, then placed in a 1M sodium hydroxide solution and shaken for 2 hours, and finally washed with purified water until the conductivity of the washing solution differs from that of the purified water by 0.68 μS / cm to obtain the decellularized matrix membrane.

[0097] Step 2) Preparation of decellularized collagen solution: Take a portion of the prepared decellularized matrix membrane, pulverize, homogenize, filter and remove processing aids to obtain decellularized collagen solution; the specific steps are as follows: take a portion of the prepared decellularized matrix membrane and put it into a low-temperature tissue ball mill, add 2M acetic acid solution, pulverize and homogenize for 30 minutes, filter with a 120-mesh filter, wash the filtered collagen solution with purified water until the pH value of the washing solution differs from that of the purified water by 0.59, and the decellularized collagen solution is obtained.

[0098] Step 3) Shaping of oral collagen plugs:

[0099] A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam at low temperature under vacuum for 45 min, then cover with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 45 min;

[0100] B. Freeze-dry for 48 hours;

[0101] C. Physical cross-linking of the freeze-dried sample: Electron beam irradiation of 5 kGy was used to obtain a collagen plug product with a decellularized matrix membrane on the top and a decellularized sponge plug on the bottom.

[0102] D. According to the clinical usage specifications, cut the cross-linked sample according to the usage specifications and package it in a double-layer sealed package.

[0103] E. Sterilization: The packaged samples are sent for irradiation sterilization. Irradiation sterilization is carried out by gamma ray sterilization or electron beam sterilization, and the irradiation dose is 25 kGy.

[0104] To ensure the biosafety of the prepared product, the residual α-gal content and clearance rate, DNA residue, and in vitro cytotoxicity were tested. The results showed that the product prepared by this method had cleared the factors that cause immunogenicity and had high biosafety.

[0105]

[0106] In vitro degradation experiment:

[0107] This experiment used Sigma (CAT# C0130) collagenase, which is a mixture of enzymes secreted by Clostridium histolyticum and belongs to the matrix metalloproteinase (MMP) family.

[0108] Oral collagen plugs and commercially available collagen sponges were incubated at 37°C at a ratio of 2.5 U collagenase per 1 mg of sample. Samples were taken at 2 h, 4 h, 8 h, 16 h, 24 h, and 48 h for analysis. Excess water was drained, and the samples were centrifuged, filtered, and then freeze-dried to remove residual degradation. The dry weight was then measured. Figure 9 , Figure 10 The data shown in Example 2 are test data of oral collagen plugs and commercially available collagen sponges by dry weight and dry weight loss rate. The results show that the in vitro degradation of oral collagen plugs is significantly slower than that of commercially available collagen sponges.

[0109] Example 3

[0110] A decellularized oral collagen plug includes: an upper decellularized matrix membrane for covering the extraction socket and shielding the bone defect area from soft tissue invasion; and a lower decellularized sponge plug for filling the extraction socket to prevent and alleviate postoperative complications and promote wound healing. The decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar structure, with the decellularized matrix membrane covering the upper surface of the decellularized sponge plug. The decellularized matrix membrane and the upper surface of the decellularized sponge plug are cross-linked together.

[0111] In this embodiment, the decellularized matrix membrane can be a square sheet structure with a thickness of 0.1 mm; its coverage area is 30*40 mm = 1200 square millimeters; the decellularized sponge plug has a columnar structure with a smooth lower surface, which in this embodiment is spherical. The transverse cross-section of the decellularized sponge plug is circular with a transverse diameter of 11.3 mm and a cross-sectional area of ​​100 mm². 2 The longitudinal height of the decellularized sponge plug is 10 mm.

[0112] The preparation method of decellularized oral collagen plugs in this embodiment includes the following steps:

[0113] Step 1) Preparation of decellularized matrix membrane:

[0114] A. Material Collection and Pre-treatment: Fresh bovine pericardium is collected, blood, fat, and impurities are removed, and it is cut into flat, standard sizes to obtain processed animal-derived membrane materials. Specific steps are as follows: Fresh bovine pericardium is collected from the slaughterhouse, washed with cold water until no blood remains, and the rough surfaces are gently removed by hand to remove fat and impurities. The pericardium is then cut into flat sheets, each 12×16cm in size, depending on the size and flatness of the pericardium.

[0115] B. Preparation of decellularized matrix membrane: The bovine pericardium treated in step A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane. The specific steps are as follows: the pretreated bovine pericardium is placed in 0.05% trypsin solution and shaken at 35°C for 2 hours, then placed in 1M sodium hydroxide solution and shaken for 1 hour, and finally washed with purified water until the conductivity of the washing solution differs from that of the purified water by 1.35 μS / cm to obtain the decellularized matrix membrane.

[0116] Step 2) Preparation of decellularized collagen solution: Take a portion of the prepared decellularized matrix membrane, pulverize, homogenize, filter and remove processing aids to obtain decellularized collagen solution; the specific steps are as follows: take a portion of the prepared decellularized matrix membrane and put it into a low-temperature tissue ball mill, add 0.5M acetic acid solution, pulverize and homogenize for 45 min, filter with a 120 mesh filter, wash the filtered collagen solution with purified water until the pH value of the washing solution differs from that of the purified water by 0.76, and the decellularized collagen solution is obtained.

[0117] Step 3) Shaping of oral collagen plugs:

[0118] A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam at low temperature under vacuum for 1 hour, then cover with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 1 hour;

[0119] B. Freeze-dry for 40 hours;

[0120] C. Physical cross-linking of the freeze-dried sample: Electron beam radiation or vacuum thermal cross-linking is used to obtain a collagen plug product with a decellularized matrix membrane on the top and a decellularized sponge plug on the bottom. In this embodiment, vacuum thermal cross-linking is used. The vacuum thermal cross-linking parameters are: vacuum degree 18 Pa, temperature 120℃, and vacuum thermal cross-linking time 8 h.

[0121] D. According to the clinical usage specifications, cut the cross-linked sample according to the usage specifications and package it in a double-layer sealed package.

[0122] E. Sterilization: The packaged samples are sent for irradiation sterilization, which is carried out by gamma ray sterilization or electron beam sterilization, with an irradiation dose of 15 kGy.

[0123] The oral collagen plug of this invention is prepared using decellularization and physical cross-linking processes, resulting in a short production cycle, high biocompatibility, suitable degradation rate, rapid water absorption, and stable volume. After the extraction wound is covered with a decellularized matrix membrane, a biological barrier is established, creating a relatively closed bone regeneration environment. This selectively blocks rapidly migrating fibroblasts and epithelial cells from entering the bone defect area, while simultaneously not hindering natural wound healing, thus achieving tissue regeneration and targeted repair.

[0124] Currently, tooth extraction often results in cavity defects, leading to complications such as bleeding, infection, swelling, pain, and dry socket within two weeks. Furthermore, alveolar bone resorption is most pronounced within three months post-extraction, potentially causing insufficient alveolar ridge bone and implant failure during later implantation. This invention, a decellularized oral collagen plug, simultaneously fills the extraction socket and covers the extraction site. Its main component is collagen, exhibiting low immunogenicity and excellent biocompatibility. After the extraction socket is filled with the decellularized sponge plug, it prevents external bacteria or fungi from entering the wound and causing adverse reactions, maintaining a stable and healing-promoting internal environment within the socket. This alleviates early postoperative symptoms and reduces the occurrence of complications. The sponge plug itself has numerous dense micropores that rapidly absorb blood, forming a stable collagen matrix blood clot that guides autologous cell growth, accelerates soft tissue healing, reduces fascicular bone resorption, and provides a better soft and hard tissue foundation for the healed extraction socket. The shape of the sponge plug mimics the structure of the tooth extraction socket, which helps to provide effective support for the tissue that has lost the support of the tooth after extraction, prevents the tissue from shrinking and collapsing during the healing process, and promotes wound recovery.

[0125] Although the embodiments and drawings of the present invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments and drawings.

Claims

1. A decellularized oral collagen plug, characterized in that: include: The decellularized matrix membrane above is used to cover the tooth extraction socket and shield the soft tissue from invading the bone defect area; The decellularized sponge plug below is used to fill the tooth extraction socket to prevent and alleviate postoperative complications and promote wound healing; The decellularized matrix membrane has a square, circular, or elliptical sheet-like structure, with a thickness of 0.1-1 mm and a coverage area of ​​300-1200 mm². 2 The decellularized sponge plug has a circular or elliptical cross-section with a cross-sectional area of ​​30-100 mm². 2 The longitudinal height of the decellularized sponge plug is 10-20 mm. The method for preparing the decellularized oral collagen plug includes the following steps: Step 1) Preparation of decellularized matrix membrane: A. Material collection and pretreatment: Fresh animal-derived membrane materials are collected, blood, fat and impurities are removed, and they are cut into flat standard sizes to obtain processed animal-derived membrane materials; B. Preparation of decellularized matrix membrane: The animal-derived membrane material processed in step A is decellularized and the processing aids are removed to obtain a decellularized matrix membrane. Step 2) Preparation of decellularized collagen solution: Take a portion of the prepared decellularized matrix membrane, crush, homogenize, filter and remove processing aids to obtain decellularized collagen solution; Step 3) Shaping of oral collagen plugs: A. Pour the decellularized collagen solution prepared above into a freeze-drying mold, defoam under low temperature vacuum for 30 min-1 h, and then cover it with the decellularized matrix membrane obtained in step 1), and further defoam under vacuum for 30 min-1 h. B. Freeze-dry for 35-48 hours; C. Physical cross-linking of the freeze-dried sample: Electron beam radiation or vacuum thermal cross-linking is used to obtain a collagen plug product with a decellularized matrix membrane on the top and a decellularized sponge plug on the bottom. D. Cut the cross-linked sample according to the usage specifications and pack it in double-layer sealed packaging; E. Sterilization: The packaged samples are sent for irradiation sterilization.

2. The decellularized oral collagen plug according to claim 1, characterized in that: The decellularized matrix membrane has a sheet-like structure; the decellularized sponge plug has a columnar, spherical, or ellipsoidal three-dimensional structure, and the decellularized matrix membrane covers the upper surface of the decellularized sponge plug, with the decellularized matrix membrane and the upper surface of the decellularized sponge plug being cross-linked together.

3. The decellularized oral collagen plug according to claim 1, characterized in that: The lower surface of the decellularized sponge plug is a smooth surface, which can be a plane, a sphere, or an ellipsoid.

4. The decellularized oral collagen plug according to claim 1, characterized in that: In step 1)A The specific steps for material collection and pretreatment are as follows: Fresh animal-derived membrane materials are collected from the slaughterhouse. The animal-derived membrane materials are pig pericardium or pig peritoneum or bovine pericardium or bovine peritoneum. They are washed with cold water until there is no blood color. The fat and impurities on the rough surface are gently removed by hand. Then, the pericardium or peritoneum is cut into flat membrane pieces with a size of 6×9cm~12×16cm.

5. The decellularized oral collagen plug according to claim 1, characterized in that: The specific steps for preparing the decellularized matrix membrane in step 1)B are as follows: the pretreated animal-derived membrane material is placed in a 0.05%~0.25% trypsin solution and shaken at 35-40℃ for 1-4 hours, then placed in a 0.5-1M sodium hydroxide solution and shaken for 1-4 hours, and finally washed with purified water until the conductivity of the washing solution differs from that of the purified water by ±2μS / cm, thus obtaining the decellularized matrix membrane.

6. The decellularized oral collagen plug according to claim 1, characterized in that: The specific steps for preparing the decellularized collagen solution in step 2) are as follows: Take a portion of the prepared decellularized matrix membrane and place it in a low-temperature tissue ball mill. Add 0.5M-2M acetic acid solution, grind and homogenize for 20-45 minutes, filter it using a 110-130 mesh filter, and wash the filtered collagen solution with purified water until the pH value of the washing solution differs from that of the purified water by ±1, thus obtaining the decellularized collagen solution.

7. The decellularized oral collagen plug according to claim 1, characterized in that: The electron beam radiation is 5-10 kGy, and the vacuum thermal crosslinking parameters are: vacuum degree <20 Pa, temperature 80-120℃, and vacuum thermal crosslinking time 4-8 h; the irradiation sterilization is performed by gamma ray sterilization or electron beam sterilization, and the irradiation dose is 15-25 kGy.

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