WO2023093908A1 - 可吸收生物膜、制法及其应用 - Google Patents

可吸收生物膜、制法及其应用 Download PDF

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WO2023093908A1
WO2023093908A1 PCT/CN2022/135032 CN2022135032W WO2023093908A1 WO 2023093908 A1 WO2023093908 A1 WO 2023093908A1 CN 2022135032 W CN2022135032 W CN 2022135032W WO 2023093908 A1 WO2023093908 A1 WO 2023093908A1
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absorbable
biofilm
cross
parts
collagen
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PCT/CN2022/135032
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English (en)
French (fr)
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范代娣
康华平
段志广
古娟
史静静
严建亚
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陕西巨子生物技术有限公司
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Publication of WO2023093908A1 publication Critical patent/WO2023093908A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • the application relates to the technical field of medical devices, in particular to an absorbable biofilm, its preparation method and its application.
  • Oral implant restoration technology is one of the important revolutions in the development of stomatology in the last century. Because of its good retention and realistic aesthetic effects, it has greatly improved the quality of life and mental state of patients with tooth defects.
  • the long-term success rate of dental implants depends on many factors, including the choice of implant site, the state of soft and hard tissue morphology, whether the bone quality around the implant is intact, the amount of bone meal implanted, and so on.
  • Dental implant technology has become a common method for tooth loss restoration. Clinically, due to the physiological bone resorption after tooth loss and the bone tissue defect in traumatic tooth loss, some alveolar ridges are often too low, too narrow or have local depressions. In most cases, 40% to 80% of patients have insufficient bone mass, and lateral perforation often occurs during implantation, resulting in implant failure.
  • absorbable biofilms are surgically placed between oral soft tissues and bone defects to create a biological barrier to create a relatively closed environment for bone regeneration and selectively block fibroblasts and epithelial cells that migrate faster A biocompatible material that allows cells to enter the bone defect without hindering the natural healing of the wound.
  • absorbable biofilms can be divided into collagen membranes, polymer membranes, tissue membranes, chitosan membranes and other types of membranes.
  • these types of membranes have more or less certain defects, such as slow degradation, expensive prices, or too hard materials for clinical use.
  • Membranes composed of a single type of material cannot fully achieve the effect of membrane repair guidance, so compound blending of multiple materials to achieve better therapeutic effects has become a research hotspot in recent years.
  • Pan SX et al. combined L-polylactide, N-methylpyrrolidone, trimethylene carbonate and their copolymers to form a film to repair mandibular bone damage in sheep. This material can promote the degradation of the film, avoid secondary surgery, and reduce Pain and financial burden for patients.
  • Bio-Guide has been registered for clinical use in China. Bio-Gide biofilms have stable effects but are relatively expensive.
  • Bote Medical Collagen Membrane produced by Fujian Boyuan Co., Ltd. is an earlier clinical oral repair membrane independently developed by China.
  • Haiao Oral Restoration Membrane produced by Yantai Zhenghai Biological Co., Ltd. was originally used to repair the oral mucosa. Because it is easy to warp around the mouth after implantation, the risk after use is increased. The emergence of resorbable biofilm has greatly promoted the development of restorative dentistry. It not only solves the problem of bone defects in oral restoration, but also meets the requirements of oral aesthetics.
  • the purpose of this application is to provide an absorbable biofilm, which is obtained by twice crosslinking with the first crosslinking agent and the second crosslinking agent, has strong mechanical properties, soft texture and toughness, and It has a certain viscosity, can be effectively attached to the application site, has a strong isolation effect, and can selectively block non-osteoblasts from the bone defect area, allowing osteoblasts to enter the bone defect area smoothly, and the absorbable
  • the biofilm can promote the proliferation of capillaries, make the capillaries grow into the bone defect area, promote the further anastomosis of the new blood vessels with the blood vessels around the bone defect area, and finally form a complete blood supply system in the bone defect area. Absorbed biofilms can be degraded in vivo, have low immune rejection, have the effects of accelerating growth and actively inducing differentiation, and can promote the repair of bone defect areas.
  • An absorbable biofilm obtained by crosslinking collagen, cellulose derivatives and other polysaccharides with a crosslinking agent.
  • the collagen is 1-10 parts, preferably 1-5 parts, more preferably 1-2.5 parts; the cellulose 1-5 parts of derivatives, preferably 1-4 parts, more preferably 1-2.5 parts; said other polysaccharides are 1-15 parts, preferably 1-10 parts, more preferably 1-5 parts .
  • cellulose derivative is selected from one of carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose One or two or more, preferably carboxymethyl cellulose.
  • the cross-linking agent comprises a first cross-linking agent and a second cross-linking agent, preferably, the first cross-linking agent is selected from From one or more of carbodiimide, glutaraldehyde, genipin and N-hydroxysulfosuccinimide, preferably carbodiimide;
  • the second crosslinking agent is a divalent metal salt other than magnesium and mercury, preferably calcium chloride, zinc chloride, manganese chloride or ferrous chloride, more preferably calcium chloride.
  • the mass concentration of the crosslinking agent is 1-10%, preferably 1-6%, more preferably 2-6%.
  • a method for preparing an absorbable biofilm according to any one of items 1-8 comprising the steps of:
  • the freeze-dried product is soaked in the first cross-linking agent for cross-linking, then placed in the second cross-linking agent for cross-linking, then freeze-dried, and then compressed to obtain an absorbable biofilm.
  • a method for repairing alveolar bone defect or alveolar ridge reconstruction which comprises preparing the absorbable biofilm described in any one of items 1-8 or the method described in any one of items 9-12 The absorbable biofilm is placed at the defect site of the patient.
  • a method for filling a bone defect comprising preparing the absorbable biofilm according to any one of claims 1-8 or the method according to any one of claims 9-12 Place where the patient needs it.
  • the absorbable biofilm described in this application is obtained by double cross-linking technology, has the advantages of strong mechanical properties, soft texture, toughness, low immunogenicity, and controllable degradation, and has a certain viscosity, which can be effectively attached to
  • the use site is widely used in the fields of oral medicine such as periodontics, oral implantology and alveolar surgery, and the absorbable biofilm can stabilize the bone graft particles, avoid the displacement of bone graft particles, improve the quality of bone formation, and greatly improve the quality of bone grafting. Implant survival rate.
  • the present application provides an absorbable biofilm, which is obtained by crosslinking collagen, cellulose derivatives and other polysaccharides with a crosslinking agent.
  • the collagen is 1-10 parts, preferably 1-5 parts, more preferably 1-2.5 parts;
  • the cellulose derivative is 1-5 parts, preferably 1-4 parts, more preferably 1-2.5 parts;
  • said other polysaccharides are 1-15 parts, preferably 1-10 parts, more preferably 1-5 parts.
  • the collagen is 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc.;
  • the cellulose derivatives are 1 part, 2 parts, 3 parts, 4 parts, 5 parts, etc.;
  • the other polysaccharides are 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 copies etc.
  • the other polysaccharides refer to polysaccharides other than cellulose derivatives, for example, the other polysaccharides are selected from chitosan, alginic acid, chitooligosaccharides, chitosan and chitin One or more of them, preferably alginic acid.
  • the chitosan is the product of N-deacetylation of chitin, which is replaced by an amino group at the C2 position, has many unique properties such as biodegradability, cell affinity and biological effects, and contains free amino groups, It is the only basic polysaccharide among natural polysaccharides, and the amino group in the chitosan structure is more reactive than the acetamido group in the chitin molecule, which makes the polysaccharide have excellent biological selenium function and can perform chemical modification reactions , therefore, is considered as a functional biomaterial with greater application potential than cellulose.
  • the alginic acid refers to a kind of natural polyuronic acid existing in the cell walls of brown algae such as kelp and giant algae, which is composed of ⁇ -D-mannuronic acid (M) and ⁇ -L-guluronic acid (G).
  • M ⁇ -D-mannuronic acid
  • G ⁇ -L-guluronic acid
  • Alginic acid exists in the cytoplasm in its natural state and plays a role in strengthening the cell wall. Alginic acid combines with various cations in seawater to form various alginates.
  • the extract obtained from seaweed is usually sodium alginate.
  • Sodium alginate has the characteristics of thickening, suspending, emulsifying, stabilizing, forming gel, forming film and spinning fiber. It has a long and wide range of uses in food, paper and cosmetic industries, especially in the field of biomedical engineering in recent years. found to be useful.
  • the chitooligosaccharide refers to a class of water-soluble amino sugar compounds with a low degree of polymerization produced after hydrolysis of chitin and chitosan, and is the product of chitin oligomers and chitosan oligomers. general term.
  • the chitosan is a deacetylated product of chitin, also known as chitin and chitin, with a chemical formula of (C 6 H 11 NO 4 ) n , which is produced after deacetylation of chitin by concentrated alkaline water
  • the water-soluble product of the scientific name is (1,4)-2-amino-2-deoxy- ⁇ -D-glucan.
  • Chitosan is a cationic polymer with good chemical stability, decomposed at about 185°C, non-toxic, insoluble in water and lye, soluble in sulfuric acid, organic acid (such as 1% acetic acid solution) and weak acid aqueous solution.
  • Soluble in dilute acid to form a viscous and transparent colloidal solution of chitosan salt the H+ in the solution combines with the amino group in the molecule to form a positively charged polymer substance, which can undergo acylation, carboxylation, and hydroxylation , Alkylation, esterification (sulfation), aldimination, azidation, salification, hydrolysis, chelation, oxidation, chlorination, grafting and crosslinking reactions.
  • the chitin also known as chitin, is a structurally homopolysaccharide formed by the polymerization of N-acetylglucosamine through ⁇ linkages. It is widely present in the shells of crustaceans, the carapaces of insects, and the cell walls of fungi. It also exists in In some green algae, it is mainly used to support the body skeleton and protect the body.
  • the collagen is animal-derived collagen and/or Fan's recombinant humanized collagen, preferably Fan's recombinant humanized collagen.
  • the Fan's recombinant humanized collagen is generally invented by Professor Fan Daidi of Northwest University in China, so it is also called Fan's Human-like Collagen or Fan's Human-like Collagen (FHLC).
  • the Fan's recombinant humanized collagen refers to the recombinant collagen described in claim 1 of Chinese Patent Application Publication CN1371919A, which has a three-chain, triple-helical structure, which can be disclosed in, for example, the Chinese Patent Application Publication CN1371919A Genetic engineering expression method to prepare.
  • the cellulose derivative is selected from one or more of carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose, preferably carboxymethyl cellulose.
  • the crosslinking agent includes a first crosslinking agent and a second crosslinking agent, preferably, the first crosslinking agent is selected from carbodiimide, glutaraldehyde, genipin and N- One or more than two kinds of hydroxysulfosuccinimide, preferably carbodiimide;
  • the second crosslinking agent is a divalent metal salt other than magnesium and mercury, preferably calcium chloride, zinc chloride or ferrous chloride, more preferably calcium chloride.
  • N-hydroxybenzotriazole or N-hydroxysuccinimide is often added, which is generally prepared by thiourea dehydration or urea dehydration, and hydrolyzed to obtain urea derivatives.
  • the genipin refers to the product of geniposide hydrolyzed by ⁇ -glucosidase, which is an excellent natural biological cross-linking agent, which can be cross-linked with protein, collagen, gelatin and chitosan to make biological materials ,, such as artificial bones, wound dressing materials, etc., its toxicity is much lower than glutaraldehyde and other commonly used chemical cross-linking agents. It can also be used to treat liver disease, lower blood pressure, and laxative.
  • N-hydroxyl sulfosuccinimide is a cross-linking agent, which can form a stable active ester intermediate for the preparation of hydrophilic active esters.
  • sulfo-NHS the efficiency of EDC-mediated coupling can be increased.
  • the absorbable biofilm obtained by cross-linking collagen, cellulose derivatives and other polysaccharides twice through two sets of cross-linking agents has strong mechanical properties, soft texture, toughness and certain viscosity. , can be effectively attached to the site of use, and has a strong isolation effect. While playing the role of a mechanical barrier, it can guide the regeneration of tissues and capillaries, promote the further anastomosis of new blood vessels with the surrounding blood vessels in the bone defect area, and improve the hardness of new bone and its The amount of regeneration is widely used in the field of oral medicine such as periodontics, oral implantation and groove surgery.
  • the mass concentration of the first crosslinking agent is 0.1-10%, preferably 0.1-5%, more preferably 0.5-3%; the mass concentration of the second crosslinking agent is 1-10%. 10%, preferably 1-6%, more preferably 2-6%.
  • the mass concentration of the first crosslinking agent is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.;
  • the mass concentration of the second crosslinking agent is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and so on.
  • the absorbable biofilm has a thickness of 0.1-1 mm.
  • the thickness of the absorbable biofilm is 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm and so on.
  • the application provides a method for preparing the above-mentioned absorbable biofilm, which comprises the following steps:
  • the freeze-dried product is soaked in the first cross-linking agent for cross-linking, then placed in the second cross-linking agent for cross-linking, then freeze-dried, and then compressed to obtain an absorbable biofilm.
  • the freezing temperature is -80 to -30°C, and preferably, the freezing time is 2-6 hours.
  • the freezing temperature can be -80°C, -70°C, -60°C, -50°C, -40°C, -30°C, etc.; the freezing time can be 2h, 3h, 4h, 5h, 6h, etc.
  • the mixed liquid before freeze-drying, it further includes putting the cross-linked product into a mold for standing, the standing temperature is 2-8°C, and the standing time is 4-12h.
  • the drying time is 24-48 hours.
  • the drying time can be 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, etc.
  • the crosslinking is performed in the first crosslinking agent for 24-48 hours, preferably, the crosslinking is performed in the second crosslinking agent for 0.5-6 hours.
  • it can be crosslinked in the first crosslinker for 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours etc.; can be crosslinked in the second crosslinking agent for 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, etc.;
  • the present application does not make any restrictions, which can be determined according to needs, for example, the freeze-dried product can be soaked in the first cross-linking agent and the second cross-linking agent.
  • Cross-linking agent for cross-linking is
  • the solution B is added to the solution A, and the solution is continuously stirred and dispersed for 0.5-1 h under the condition of 40-50° C., and the stirring speed is 100-500 revolutions per minute.
  • water for injection is used to dissolve collagen to obtain a second solution.
  • the present application does not make any restriction, and it can be selected as required to make it meet the requirements.
  • water for injection is mixed with cellulose derivatives and other polysaccharides, and swollen overnight to obtain a solution.
  • the absorbable biofilm prepared by using the method described in this application because the collagen, cellulose derivatives and other polysaccharides are cross-linked by the double cross-linking technology, the obtained absorbable biofilm It has strong mechanical properties, soft texture, toughness, and certain viscosity. It can be effectively attached to the application site, has a strong isolation effect, can guide the regeneration of tissues and capillaries, and promotes the further anastomosis of new blood vessels and blood vessels around bone defects. It can be widely used in the field of oral medicine such as periodontics, oral implantology and alveolar surgery to improve the hardness and production of new bone.
  • the application provides the application of the above-mentioned absorbable biofilm or the absorbable biofilm prepared by the above-mentioned method in the field of stomatology, preferably in periodontology, oral implantology or alveolar surgery.
  • the present application provides a method for repairing alveolar bone defect or alveolar ridge reconstruction, which includes placing the above-mentioned absorbable biofilm or the absorbable biofilm prepared by the above-mentioned method at the defect position of the patient .
  • the absorbable biofilm is used in combination with bone meal.
  • the bone meal is a commonly used bone meal in the field.
  • the alveolar bone defect refers to the tissue loss in the upper and lower jaws surrounding the teeth, and placing the absorbable biofilm described in this application at the defect position of the patient can repair the alveolar bone defect ;
  • the alveolar ridge is the alveolar bone where the original teeth are located after the tooth falls off or is missing in the oral cavity.
  • Putting the absorbable biofilm described in the present application at the defect position of the patient can repair the tooth. Groove crest.
  • the present application provides a method for filling a bone defect, which includes placing the above-mentioned absorbable biofilm or the absorbable biofilm prepared by the above-mentioned method at the desired position of the patient.
  • the bone defect is a bone defect caused by extraction socket, cyst excision or stump extraction.
  • the absorbable biofilm is used in combination with bone meal.
  • the tooth extraction socket refers to a cavity left on the gum wound after tooth extraction
  • the cyst refers to a cystic skin lesion containing fluid or viscous matter and cellular components.
  • Oral cysts include two types: soft tissue cysts and jaw cysts.
  • Common soft tissue cysts include salivary gland cyst, sebaceous cyst, dermoid or epidermoid cyst, thyroglossal duct cyst, branchial cleft cyst, etc.
  • Surgical excision and complete excision are recommended for all types of soft tissue cysts to prevent recurrence due to residual tissue, and some salivary gland cysts require partial or complete excision.
  • Jaw cysts include odontogenic jaw cysts, nonodontogenic jaw cysts, and extravasation cysts, and usually require surgical treatment. For some localized odontogenic cysts, it can be performed under local anesthesia.
  • the cyst excision refers to excision of cystic skin lesions containing fluid or viscous and cellular components.
  • the residual root refers to the complete loss of the crown part due to various reasons, and only the tooth root remains, which may or may not be seen clinically under the gums.
  • the most important reason for the residual root is dental caries. From the beginning, a cavity in the crown may gradually develop into a residual crown. The residual crown continues to caries and gradually becomes a residual root. This is the main reason. teeth. Most of the crown defect due to caries and other reasons is called the residual crown, while the crown is basically missing, and only the remaining tooth root is called the residual root. Once the residual crown and residual root are formed, the pulp cavity and root canal of the tooth are exposed to the bacterial environment of the oral cavity. It can also cause other diseases in the whole body, so the stump roots need to be pulled out.
  • the size of the defect was measured with a sterilized alumina sheet or a periodontal probe, and the absorbable biofilm was trimmed into a suitable shape in a dry state and placed on the bone graft site in a dry state. Only relying on the blood infiltration of the operation area, it is usually best to cover with absorbable biofilm after implantation of bone powder particles. Due to the good hydrophilicity of the absorbable biofilm, it can be closely adhered and fitted to the bone graft material and the surrounding bone wall, so no fixation is required. If the defect area is large, absorbable biofilm can be fixed with sutures or titanium nails.
  • % means wt%, ie weight percentage.
  • the reagents or instruments used, whose manufacturers are not indicated, are commercially available conventional reagent products, wherein, Table 1 is the raw material information used in the examples and comparative examples.
  • Examples 3-21 used the amount of raw materials described in Table 2, and used the method described in Example 1 to prepare absorbable biofilms.
  • the growth of the OD value reflects the increase of living cells, and the relative growth rate (RGR) of the calculated cells is greater than 85%.
  • RGR relative growth rate
  • the ISO10993 cytotoxic reaction grade the The materials have no toxicity to mouse fibroblasts, and are rated as grade 0 or grade 1, that is, qualified and non-cytotoxic. This shows that this application has good biocompatibility.
  • the absorbable biofilm described in the freeze-dried embodiment 1-21 is cut into strips of 70mm*20mm, the tensile testing machine moves at a speed of 100 ⁇ 10mm, and detects the tensile strength according to the standard of GB/T 1040.3-2006 , and the results are shown in Table 4.

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Abstract

本申请公开了一种可吸收生物膜、制法及其应用。所述可吸收生物膜通过使用交联剂将胶原蛋白、纤维素类衍生物和其他多糖类物质交联得到。所述的可吸收生物膜，是利用双交联技术得到，机械性能强、质地柔软、有韧性，免疫原性低，降解可控等优点，且具有一定的粘性，可有效贴附于使用部位，广泛应用于牙周科、口腔种植及牙槽外科等口腔医学领域，并且所述的可吸收生物膜可稳定植骨颗粒，避免植骨颗粒的移位，提高成骨质量，大大提高种植体的生存率。

Description

可吸收生物膜、制法及其应用 技术领域
本申请涉及医疗器械技术领域，尤其涉及一种可吸收生物膜、制法及其应用。
背景技术
口腔种植修复技术是上世纪口腔医学发展的重要革命之一，其因良好的固位、逼真的美观效果，极大提高了牙缺损患者的生活质量和精神状态。种植牙的长期成功率依赖于诸多因素，其中包括种植位点的选择、软硬组织形态的状态、种植体周边骨质是否完好、骨粉种植量等等。牙种植技术已成为目前牙缺失修复的常用手段，临床上由于缺牙后的生理性骨吸收以及外伤性缺牙时的骨组织缺损，常有一些牙槽嵴过低、过窄或局部有凹陷的病例，40％～80％的患者都存在骨量不足，在种植过程中常发生侧方穿孔，导致种植失败。
随着引导性骨再生技术在临床上的应用，上述问题将得以解决。可吸收生物膜是利用外科手术的方式将膜置于口腔软组织与骨缺损之间建立生物屏障，以此创造一个相对封闭的骨再生环境，选择性地阻挡迁移速度较快的成纤维细胞和上皮细胞进入骨缺损区，而同时又不妨碍伤口自然愈合的一种生物相容性材料。目前可吸收生物膜可分为胶原膜、高分子聚合膜、组织膜、壳聚糖膜和其他种类膜。但这些类型的膜或多或少都存在一定的缺陷，或降解速度慢、或价格昂贵、或材料过硬不利用临床使用等。单一类型材料组成的膜不能完全达到膜修复引导的效果，因此多种材料的复合共混来达到更优的治疗效果是近几年研究的热点。Pan SX等将左旋聚丙交酯、N-甲基吡咯烷酮与三亚甲基碳酸酯及其共聚物复合成膜，修复羊的下颔骨损伤，该材料可促进膜的降解，避免二次手术，降低了患者疼痛及经济负担。目前进口胶原膜中只有Bio-Guide在中国取得临床使用注册，Bio-Gide生物膜效果稳定但是价格较高。福建博远公司生产的博特医用胶原膜是中国自主开发研制的较早用于临床的口腔修复膜，由于其工艺简单价格便宜，在临床上得到了广 泛的应用，但是对温度要求高，需要在2～5℃的低温条件下保存。烟台正海生物公司生产的海奥口腔修复膜本来是应用于口腔黏膜的修复，由于其植入口腔后四周易翘起，增加了其使用后的风险。可吸收生物膜的出现极大地推动了口腔修复医学的发展，它不仅解决了口腔修复中的骨缺损问题；而且还满足了口腔美学的要求。但可吸收生物膜在临床使用中仍存在膜暴露及感染、膜塌陷及移位、软组织感染、抗原性、不可吸收膜的体内置留时间、可吸收膜的体内维持时间等问题，故研究一种安全有效，同时价格更为低廉的可吸收生物膜迫在眉睫。
发明内容
本申请的目的在于提供一种可吸收生物膜，所述的可吸收生物膜通过使用第一交联剂和第二交联剂两次交联得到，机械性能强，质地柔软、有韧性，且具有一定的粘性，可有效贴附于使用部位，隔离效果强，能够选择性地将非成骨细胞阻挡于骨缺损区以外，使成骨细胞顺利地进入骨缺损区，并且所述的可吸收生物膜可促进毛细血管的增生，使得毛细血管向骨缺损区长入，促使新生血管与骨缺损区周围血管进一步吻合，最终在骨缺损区形成完整的供血系统，此外，本申请所述的可吸收生物膜可在体内降解，免疫排斥反应低，具有加速生长和主动诱导分化的作用，可促进骨缺损区的修复。
本申请具体技术方案如下：
1.一种可吸收生物膜，所述可吸收生物膜通过使用交联剂将胶原蛋白、纤维素类衍生物和其他多糖类物质交联得到。
2.根据项1所述的可吸收生物膜，其中，按重量份计，所述胶原蛋白为1-10份，优选为1-5份，进一步优选为1-2.5份；所述纤维素类衍生物为1-5份，优选为1-4份，进一步优选为1-2.5份；所述其他多糖类物质为1-15份，优选为1-10份，进一步优选为1-5份。
3.根据项1或2所述的可吸收生物膜，其中，所述胶原蛋白为动物源胶原蛋白和/或范氏重组人源化胶原蛋白，优选为范氏重组人源化胶原蛋白。
4.根据项1-3中任一项所述的可吸收生物膜，其中，所述纤维素类衍生物选自羧甲基纤维素、羟乙基纤维素和羟丙基纤维素中的一种或两种以上，优选为羧甲基纤维素。
5.根据项1-4中任一项所述的可吸收生物膜，其中，所述其他多糖类物 质选自壳聚糖、海藻酸、甲壳低聚糖、几丁聚糖和几丁质中的一种或两种以上，优选为海藻酸。
6.根据项1-5中任一项所述的可吸收生物膜，其中，所述交联剂包括第一交联剂和第二交联剂，优选的，所述第一交联剂选自碳化二亚胺、戊二醛、京尼平和N-羟基硫代琥珀酰亚胺的一种或两种以上，优选为碳化二亚胺；
优选的，所述第二交联剂为除镁和汞之外的二价金属盐，优选为氯化钙、氯化锌、氯化锰或氯化亚铁，进一步优选为氯化钙。
7.根据项6所述的可吸收生物膜，其中，所述第一交联剂的质量浓度为0.1-10％，优选为0.1-5％，进一步优选为0.5-3％；所述第二交联剂的质量浓度为1-10％，优选为1-6％，进一步优选为2-6％。
8.根据项1-7中任一项所述的可吸收生物膜，其中，所述可吸收生物膜的厚度为0.1-1mm。
9.一种制备项1-8中任一项所述可吸收生物膜的方法，其包括下述步骤：
将纤维素类衍生物和其他多糖类物质溶解得到甲溶液；
将胶原蛋白溶解得到乙溶液，并将乙溶液加入到甲溶液中得到混合溶液，接着冷冻干燥得到冻干产物；
将所述冻干产物浸泡在第一交联剂中交联，然后再放入第二交联剂中进行交联，然后进行冷冻干燥，接着进行压制得到可吸收生物膜。
10.根据项9所述的方法，其中，冷冻温度为-30至-80℃，优选的，冷冻时间为2-6h。
11.根据项9或10所述的方法，其中，干燥时间为24-48小时。
12.根据项9-11中任一项所述的方法，其中，在第一交联剂中交联24-48小时，优选的，在第二交联剂中交联0.5-6小时。
13.项1-8中任一项所述的可吸收生物膜或者项9-12中任一项所述的方法制备得到的可吸收生物膜在口腔医学领域中的应用，优选在牙周科、口腔种植科或牙槽外科中的应用。
14.一种牙槽骨缺损或牙槽嵴重建的修复方法，其包括将项1-8中任一项所述的可吸收生物膜或者项9-12中任一项所述的方法制备得到的可吸收生物膜置于患者的缺损位置处。
15.根据权利要求14所述的修复方法，其中，所述可吸收生物膜与骨粉联合使用。
16.一种骨缺损的填充方法，其包括将权利要求1-8中任一项所述的可吸收生物膜或者权利要求9-12中任一项所述的方法制备得到的可吸收生物膜置于患者需要的位置处。
17.根据项16所述的填充方法，其中，所述骨缺损是由拔牙窝、囊肿切除或残根拔除引起的骨缺损。
18.根据项16或17所述的填充方法，其中，所述可吸收生物膜与骨粉联合使用。
发明的效果
本申请所述的可吸收生物膜，是利用双交联技术得到，机械性能强、质地柔软、有韧性，免疫原性低，降解可控等优点，且具有一定的粘性，可有效贴附于使用部位，广泛应用于牙周科、口腔种植及牙槽外科等口腔医学领域，并且所述的可吸收生物膜可稳定植骨颗粒，避免植骨颗粒的移位，提高成骨质量，大大提高种植体的生存率。
具体实施方式
下面所描述的实施方式对本申请做以详细说明。虽然显示了本申请的具体实施例，然而应当理解，可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本申请，并且能够将本申请的范围完整的传达给本领域的技术人员。
需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
本申请提供了一种可吸收生物膜，所述可吸收生物膜通过使用交联剂将胶原蛋白、纤维素类衍生物和其他多糖类物质交联得到。
在一个实施方案中，按重量份计，所述胶原蛋白为1-10份，优选为1-5 份，进一步优选为1-2.5份；所述纤维素类衍生物为1-5份，优选为1-4份，进一步优选为1-2.5份；所述其他多糖类物质为1-15份，优选为1-10份，进一步优选为1-5份。
例如，按重量份计，所述胶原蛋白为1份、2份、3份、4份、5份、6份、7份、8份、9份、10份等；
所述纤维素类衍生物为1份、2份、3份、4份、5份等；
所述其他多糖类物质为1份、2份、3份、4份、5份、6份、7份、8份、9份、10份、11份、12份、13份、14份、15份等。
所述其他多糖类物质指的是除了纤维素类衍生物以外的多糖物质，例如，所述其他多糖物质选自壳聚糖、海藻酸、甲壳低聚糖、几丁聚糖和几丁质中的一种或两种以上，优选为海藻酸。
所述壳聚糖是甲壳素N-脱乙酰基的产物，其在C2位上被一个氨基所代替，具有生物降解性、细胞亲和性和生物效应等许多独特的性质，其含有游离氨基，是天然多糖中唯一的碱性多糖，并且壳聚糖结构中的氨基基团比甲壳素分子中的乙酰氨基基团反应活性更强，使得该多糖具有优异的生物硒功能并能进行化学修饰反应，因此，被认为是比纤维素具有更大应用潜力的功能性生物材料。
所述海藻酸指的是存在海带、巨藻等褐藻细胞壁中的一种天然多糖醛酸，是由β-D-甘露糖醛酸(M)和α-L-古罗糖醛酸(G)经过1,4-键合形成的线型共聚物，G和M在海藻酸中的含量对纤维的成胶性能有明显的影响。海藻酸在自然状态下存在于胞质中，起着强化细胞壁的作用。海藻酸与海水中各种阳离子结合成为各种海藻酸盐。从海藻中得到的提取物通常是海藻酸钠。海藻酸钠具有增稠、悬浮、乳化、稳定、形成凝胶、形成薄膜和纺制纤维的特性，在食品、造纸及化妆等工业有悠久及广泛的用途，特别是近年来在生物医学工程领域发现有重要用途。
所述甲壳低聚糖指的是由甲壳素和壳聚糖经水解后产生的一类低聚合度可溶于水的氨基糖类化合物，是甲壳素低聚物和壳聚糖低聚物的总称。
所述几丁聚糖是几丁质的脱乙酰基产物，又名甲壳素、甲壳质，化学式为(C 6H 11NO 4) n，是几丁质经浓碱水脱去乙酰基后生成的水溶性产物，学名为(1，4)-2-氨基-2-脱氧-β-D-葡聚糖。几丁聚糖为阳离子聚合物，化学稳定性好，约185℃分解，无毒，不溶于水和碱液，可溶解于硫酸、有机酸 (如1％醋酸溶液)及弱酸水溶液。溶于稀酸生成粘稠透明的几丁聚糖盐胶体溶液，此时溶液中的H+即与分子中的氨基结合，生成带正电荷的高分子物质，可发生酰化、羧基化、羟基化、烷化、酯化(硫酸酯化)、醛亚胺化、叠氮化、成盐、水解、螯和、氧化、氯化、枝接与交连等反应。
所述几丁质又称壳多糖，是N-乙酰葡糖胺通过β连接聚合而成的结构同多糖，广泛存在于甲壳类动物的外壳、昆虫的甲壳和真菌的胞壁中，也存在于一些绿藻中，主要是用来作为支撑身体骨架以及对身体起保护的作用。
在一个实施方案中，以重量份计，当加入1-10份，优选为1-5份，进一步优选为1-2.5份的胶原蛋白时，需要加入1-5份，优选为1-4份，进一步优选为1-2.5份的纤维素类衍生物以及加入1-15份，优选为1-10份，进一步优选为1-5份的其他多糖类物质。
在一个实施方案中，所述胶原蛋白为动物源胶原蛋白和/或范氏重组人源化胶原蛋白，优选为范氏重组人源化胶原蛋白。
所述范氏重组人源化胶原蛋白一般是指中国西北大学的范代娣教授发明的，因此又称作范氏重组胶原蛋白或范氏重组人源化胶原蛋白(Fan’s Human-like Collagen，FHLC)。所述的范氏重组人源化胶原蛋白是指中国专利申请公开CN1371919A的权利要求1所述的重组胶原蛋白，其具有三链、三螺旋结构，其可以采用例如该中国专利申请公开CN1371919A中公开的基因工程表达方法来制备。
在一个实施方案中，所述纤维素类衍生物选自羧甲基纤维素、羟乙基纤维素和羟丙基纤维素中的一种或两种以上，优选为羧甲基纤维素。
在一个实施方案中，所述交联剂包括第一交联剂和第二交联剂，优选的，所述第一交联剂选自碳化二亚胺、戊二醛、京尼平和N-羟基硫代琥珀酰亚胺的一种或两种以上，优选为碳化二亚胺；
优选的，所述第二交联剂为除镁和汞之外的二价金属盐，优选为氯化钙、氯化锌或氯化亚铁，进一步优选为氯化钙。
所述碳化二亚胺是指含有N＝C＝N官能团的一类常用的失水剂，主要用作多肽、蛋白质、核苷酸合成中的脱水剂，主要用于活化羧基，促使酰胺和酯的生成。反应中常加入N-羟基苯并三氮唑或N-羟基琥珀酰亚胺，一般由硫脲失硫化氢或脲失水制备，水解得到脲衍生物。
所述京尼平指的是栀子苷经β-葡萄糖苷酶水解后的产物，是一种优良 的天然生物交联剂，可以与蛋白质、胶原、明胶和壳聚糖等交联制作生物材料，，如人造骨骼、伤口包扎材料等，其毒性远低于戊二醛和其他常用化学交联剂。也可用于治疗肝脏疾病、降压、通便等。
所述N-羟基硫代琥珀酰亚胺是一种交联剂，可形成稳定的活泼酯中间体，用于制备亲水性活泼酯。在存在磺基-NHS的情况下，可使EDC介导的偶联效率增加。
本申请通过两组交联剂将胶原蛋白、纤维素类衍生物和其他多糖类物质进行两次交联所得到的可吸收生物膜机械性能强、质地柔软，有韧性，且具有一定的粘性，可有效贴附于使用部位，隔离效果强，在起到机械屏障作用的同时，可引导组织、毛细血管的再生，促使新生血管与骨缺损区周围血管进一步吻合，提高新骨的硬度及其再生量，广泛应用于牙周科、口腔种植即压槽外科等口腔医学领域。
在一个实施方案中，所述第一交联剂的质量浓度为0.1-10％，优选为0.1-5％，进一步优选为0.5-3％；所述第二交联剂的质量浓度为1-10％，优选为1-6％，进一步优选为2-6％。
例如，所述第一交联剂的质量浓度为0.1％、0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％等；
所述第二交联剂的质量浓度为1％、2％、3％、4％、5％、6％、7％、8％、9％、10％等。
在一个实施方案中，所述可吸收生物膜的厚度为0.1-1mm。
例如，所述可吸收生物膜的厚度为0.1mm、0.2mm、0.3mm、0.4mm、0.5mm、0.6mm、0.7mm、0.8mm、0.9mm、1mm等。
本申请提供了一种制备上述所述可吸收生物膜的方法，其包括下述步骤：
将纤维素类衍生物和其他多糖类物质溶解得到甲溶液；
将胶原蛋白溶解得到乙溶液，并将乙溶液加入到甲溶液中得到混合溶液，接着冷冻干燥得到冻干产物；
将所述冻干产物浸泡在第一交联剂中交联，然后再放入第二交联剂中进行交联，然后进行冷冻干燥，接着进行压制得到可吸收生物膜。
在一个实施方案中，冷冻温度为-80至-30℃，优选的，冷冻时间为2-6h。
例如，冷冻温度可以为-80℃、-70℃、-60℃、-50℃、-40℃、-30℃等；冷冻时间可以为2h、3h、4h、5h、6h等。
在一个实施方案中，在得到混合液之后，在冷冻干燥之前还包含将交联后的产物放入模具中进行静置，静置温度为2-8℃，静置时间为4-12h。
在一个实施方案中，干燥时间为24-48小时。
例如，干燥时间可以为24小时、26小时、28小时、30小时、32小时、34小时、36小时、38小时、40小时、42小时、44小时、46小时、48小时等。
在一个实施方案中，在第一交联剂中交联24-48小时，优选的，在第二交联剂中交联0.5-6h。
例如，可以在第一交联剂中交联24小时、26小时、28小时、30小时、32小时、34小时、36小时、38小时、40小时、42小时、44小时、46小时、48小时等；在第二交联剂中可以交联0.5小时、1小时、2小时、3小时、4小时、5小时、6小时等；
对于第一交联剂和第二交联剂与冻干产物的用量关系，本申请不作任何限制，其可以根据需要进行确定，例如，可以将冻干产物浸泡在第一交联剂和第二交联剂中以进行交联。
在一个实施方案中，将乙溶液加入到甲溶液中，在40-50℃的条件下持续搅拌分散0.5-1h，搅拌速度为每分钟100-500转。
在一个实施方案中，使用注射用水溶解胶原蛋白得到乙溶液。
对于注射用水的用量，本申请不作任何限制，其可以根据需要进行选择使其符合要求。
在一个实施方案中，使用注射用水和纤维素类衍生物以及其他多糖类混合，并过夜溶胀得到甲溶液。
对于注射用水的用量，本申请不作任何限制，本领域技术人员可以根据需要进行确定。
本申请通过使用所述的方法所制得的可吸收生物膜，由于采用两次交联技术将胶原蛋白、纤维素类衍生物以及其他多糖类物质进行交联，所得到的可吸收生物膜的机械性能强、质地柔软，有韧性，且具有一定的粘性，可有效贴附于使用部位，隔离效果强，可引导组织、毛细血管的再生，促进新生血管与骨缺损区周围血管进一步吻合，提高新骨的硬度及其生成量，可广泛应用于牙周科、口腔种植及牙槽外科等口腔医学领域。
本申请提供了上述所述的可吸收生物膜或者上述所述的方法制备得到 的可吸收生物膜在口腔医学领域中的应用，优选在牙周科、口腔种植科或牙槽外科中的应用。
本申请提供了一种牙槽骨缺损或牙槽嵴重建的修复方法，其包括将上述所述的可吸收生物膜或者上述所述的方法制备得到的可吸收生物膜置于患者的缺损位置处。在一个实施方案中，所述可吸收生物膜与骨粉联合使用。
在本申请中，所述骨粉是本领域常用的骨粉。
在本申请中，所述牙槽骨缺损是指上下颌骨包围着牙齿的部位出现了组织缺失，将本申请所述的可吸收生物膜置于患者的缺损位置处，能够修复牙槽骨缺损；
在本申请中，所述牙槽嵴是口腔内牙齿脱落或缺失后，原来牙齿所在位置的牙槽骨处，将本申请所述的可吸收生物膜置于患者的缺损位置处，能够修复牙槽嵴。
本申请提供了一种骨缺损的填充方法，其包括将上述所述的可吸收生物膜或者上述所述的方法制备得到的可吸收生物膜膜置于患者的需要的位置处。在一个实施方案中，所述骨缺损是由拔牙窝、囊肿切除或残根拔除引起的骨缺损。在一个实施方案中，所述可吸收生物膜与骨粉联合使用。
在本申请中，所述拔牙窝指的是拔牙后牙龈创面留下的一个凹洞；
所述囊肿指的是含有液体或黏稠物及细胞成分的囊性皮损，口腔囊肿包括两种类型：软组织囊肿和颌骨囊肿。常见的软组织囊肿，包括唾液腺囊肿、皮脂腺囊肿、皮样或表皮样囊肿、甲状舌管囊肿、鳃裂囊肿等。所有类型的软组织囊肿都建议手术切除并完全切除，以防止残余组织引起的复发，一些唾液腺囊肿需要部分或完全切除。颌骨囊肿包括牙源性颌骨囊肿、非牙源性颌骨囊肿和外渗性囊肿，通常需要手术治疗。对于一些局限性牙源性囊肿，可以在局部麻醉下进行。如果感染伴有抗生素，应在控制炎症后进行外科治疗。同时，应该进行诸如牙根管治疗的治疗，并且应该在全身麻醉下进行广泛的治疗。所述囊肿切除指的是切除含有液体或黏稠物及细胞成分的囊性皮损。
所述残根是指因为各种原因造成牙冠部的全部缺失,只保留了牙根,临床上在龈下可能会看到,也可能看不到牙根。对于残根最主要的原因是龋齿,从一开始牙冠的一个龋洞,慢慢发展可能变成了残冠,残冠继续进行龋坏,慢慢就变成了残根,这是主要原因牙齿。由于龋坏等原因而致使牙冠的大部分缺损， 称为残冠，而牙冠基本缺失，仅剩余牙根，称为残根。一旦形成了残冠、残根，牙齿的髓腔、根管就暴露于口腔的有菌环境之中，细菌可以通过根管而到达根尖，形成根尖周围炎，使牙齿成为病灶牙，进一步还可以引起全身的其它疾病，因此，需要将残根拔除。
在本申请中，用消毒后的氧化铝片或牙周探针测量缺损部位大小，可吸收生物膜在干燥状态下修剪成合适的形状，在干燥状态下放置于植骨位点，就位后仅仅依靠术区的血液浸润，通常骨粉颗粒植入后最好覆盖可吸收生物膜联合使用。由于可吸收生物膜具有良好的亲水性，可以和植骨材料以及周围骨壁紧密粘附并贴合，故无需固定。若缺损区域较大，可通过缝合或钛钉固定可吸收生物膜。
实施例
本申请对试验中所用到的材料以及试验方法进行一般性和/或具体的描述，在下面的实施例中，如果无其他特别的说明，％表示wt％，即重量百分数。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品，其中，表1是实施例和对比例所用到的原料信息。
表1 原料信息
  纯度 生产厂家
范氏重组人源化胶原蛋白 >90％ 西安巨子生物基因技术有限公司
羧甲基纤维素 / 安徽山河药用辅料股份有限公司
海藻酸 / 青岛明月海藻集团有限公司
碳化二亚胺 >99％ 苏州昊帆生物股份有限公司
羟丙基纤维素 / 武汉拉那白医药化工有限公司
壳聚糖 / 浙江金壳药业股份有限公司
戊二醛 / 阿拉丁试剂(上海)有限公司
羟乙基纤维素 / 武汉拉那白医药化工有限公司
几丁质 / 阿拉丁试剂(上海)有限公司
几丁聚糖 / 阿拉丁试剂(上海)有限公司
甲壳低聚糖 / 浙江金壳药业股份有限公司
京尼平 / 上海源叶生物科技有限公司
N-羟基硫代琥珀酰亚 / 阿拉丁试剂(上海)有限公司
动物源胶原蛋白 >90％ 浙江金壳药业股份有限公司
实施例1可吸收生物膜的制备
(1)分别称取羧甲基纤维素1g、海藻酸1g，加入50ml注射用水，过 夜溶胀，形成甲溶液。
(2)称取1.2g范氏重组人源化胶原蛋白，加入50ml注射用水溶解，全部加入到上述甲溶液中，在45℃的条件下持续搅拌分散0.5h，搅拌速度为每分钟100转，得到混合溶液，然后将混合溶液灌装到模具中，置于2-8℃冰箱静置10h后转移到冰箱中冷冻，冷冻温度为-80℃，冷冻时间为2h，接着放入冷冻干燥机中干燥28小时，得到冻干产物。
(3)将上述冻干产物浸泡在浓度为2％的碳化二亚胺中交联30h，然后转移至浓度为3％的氯化钙中浸泡2h进行交联，交联后转移至冰箱中冷冻，冷冻温度为-80℃，冷冻时间为2h，接着放入冷冻干燥机中干燥28小时，然后利用压膜机进行压制得到厚度为0.213mm的可吸收生物膜。
实施例2可吸收生物膜的制备
(1)分别称取羟丙基纤维素0.8g、壳聚糖1g，加入50ml注射用水50ml，过夜溶胀，形成甲溶液。
(2)称取0.8g范氏重组人源化胶原蛋白，加入50ml注射用水溶解，全部加入到上述甲溶液中，在40℃的条件下持续搅拌分散0.6h，搅拌速度为每分钟120转，得到混合溶液，然后将混合溶液灌装到模具中，置于2-8℃冰箱静置12h后转移到冰箱中冷冻，冷冻温度为-80℃，冷冻时间为2h，接着放入冷冻干燥机中干燥30小时，得到冻干产物。
(3)将上述冻干产物浸泡在浓度为3％的戊二醛中交联30h，然后转移至浓度为4％的氯化锰中浸泡3h进行交联，交联后转移至冰箱中冷冻，冷冻温度为-80℃，冷冻时间为2h，接着放入冷冻干燥机中干燥30小时，然后利用压膜机进行压制得到厚度为0.123mm的可吸收生物膜。
实施例3-21可吸收生物膜的制备
实施例3-21使用表2中所述的原料用量，并使用实施例1所述的方法进行制备得到可吸收生物膜。
对比例1
(1)分别称取羧甲基纤维素1g、海藻酸1g，加入50ml注射用水，过夜溶胀，形成甲溶液。
(2)称取1.2g范氏重组人源化胶原蛋白，加入50ml注射用水溶解，全部加入到上述甲溶液中，在45℃的条件下持续搅拌分散0.5h，搅拌速度为每分钟100转，得到混合溶液，然后将混合溶液灌装到模具中，置于2-8℃冰箱静置10h后转移到冰箱中冷冻，冷冻温度为-80℃，冷冻时间为2h，接着放入冷冻干燥机中干燥28小时，得到冻干产物，其由于不能交联而得不到膜材料。
表2 实施例所用到的原料用量表
Figure PCTCN2022135032-appb-000001
Figure PCTCN2022135032-appb-000002
实验例1生物相容性
使用实施例1-21所得到的可吸收生物膜进行生物相容性实验，采用的是检测细胞毒性的MTT法，测定可吸收生物膜浸提液对小鼠纤维细胞成活率的影响，其结果如表3所示。
表3 可吸收生物膜MTT法测试结果
Figure PCTCN2022135032-appb-000003
Figure PCTCN2022135032-appb-000004
从表3可以看出，在2、4、7天后，OD值的增长即反应活细胞的增多，另计算所得细胞的相对增值率(RGR)均大于85％，根据ISO10993细胞毒性反应等级，该材料对小鼠成纤维细胞没有毒性，均评级为0级或1级，即合格、无细胞毒性。这说明本申请具有良好的生物相容性。
实验例2抗张强度
将冷冻干燥后的实施例1-21所述的可吸收生物膜裁剪成70mm*20mm的条形，拉力试验机以100±10mm速度移动，按照GB/T 1040.3-2006的标准，检测抗张强度，其结果如表4所示。
表4 可吸收生物膜拉伸强度测试结果
Figure PCTCN2022135032-appb-000005
从表4可以看出，交联之后的可吸收生物膜具有更好的拉伸强度。
实验例3降解时间检测
准确称取0.02g的实施例1-21的膜材料置于盛有10ml PBS缓冲液的试管中，并加入2mg的胶原酶，置于37℃水浴中，定时观察，3组平行实验，记录样品完全澄清所需的时间，取平均值，其结果如表5所示。
表5 可吸收生物膜降解时间测试结果
Figure PCTCN2022135032-appb-000006
从表5可以看出，本申请所得到的可吸收生物膜具有较好的抗降解能力。
由于可吸收生物膜的拉伸强度和抗降解能力是评价该产品发挥临床作用的主要指标，从表4、表5可以看出本申请得到的产品具有更好的拉伸强度和抗降解能力，更有利于临床操作、更好的发挥对软组织中成纤维细胞的阻挡作用和引导骨再生。
以上所述，仅是本申请的较佳实施例而已，并非是对本申请作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更 或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容，依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本申请技术方案的保护范围。

Claims (15)

  1. 一种可吸收生物膜，所述可吸收生物膜通过使用交联剂将胶原蛋白、纤维素类衍生物和其他多糖类物质交联得到。
  2. 根据权利要求1所述的可吸收生物膜，其中，按重量份计，所述胶原蛋白为1-10份，优选为1-5份，进一步优选为1-2.5份；所述纤维素类衍生物为1-5份，优选为1-4份，进一步优选为1-2.5份；所述其他多糖类物质为1-15份，优选为1-10份，进一步优选为1-5份。
  3. 根据权利要求1或2所述的可吸收生物膜，其中，所述胶原蛋白为动物源胶原蛋白和/或范氏重组人源化胶原蛋白，优选为范氏重组人源化胶原蛋白。
  4. 根据权利要求1-3中任一项所述的可吸收生物膜，其中，所述纤维素类衍生物选自羧甲基纤维素、羟乙基纤维素和羟丙基纤维素中的一种或两种以上，优选为羧甲基纤维素；
    优选的，所述其他多糖类物质选自壳聚糖、海藻酸、甲壳低聚糖、几丁聚糖和几丁质中的一种或两种以上，优选为海藻酸。
  5. 根据权利要求1-4中任一项所述的可吸收生物膜，其中，所述交联剂包括第一交联剂和第二交联剂，优选的，所述第一交联剂选自碳化二亚胺、戊二醛、京尼平和N-羟基硫代琥珀酰亚胺的一种或两种以上，优选为碳化二亚胺；
    优选的，所述第二交联剂为除镁和汞之外的二价金属盐，优选为氯化钙、氯化锌、氯化锰或氯化亚铁，进一步优选为氯化钙；
    优选的，所述第一交联剂的质量浓度为0.1-10％，优选为0.1-5％，进一步优选为0.5-3％；所述第二交联剂的质量浓度为1-10％，优选为1-6％，进一步优选为2-6％；
    优选的，所述可吸收生物膜的厚度为0.1-1mm。
  6. 一种制备权利要求1-5中任一项所述可吸收生物膜的方法，其包括下述步骤：
    将纤维素类衍生物和其他多糖类物质溶解得到甲溶液；
    将胶原蛋白溶解得到乙溶液，并将乙溶液加入到甲溶液中得到混合溶液， 接着冷冻干燥得到冻干产物；
    将所述冻干产物浸泡在第一交联剂中交联，然后再放入第二交联剂中进行交联，然后进行冷冻干燥，接着进行压制得到可吸收生物膜。
  7. 根据权利要求6所述的方法，其中，冷冻温度为-30至-80℃，优选的，冷冻时间为2-6h。
  8. 根据权利要求6或7所述的方法，其中，干燥时间为24-48小时。
  9. 根据权利要求6-8中任一项所述的方法，其中，在第一交联剂中交联24-48小时，优选的，在第二交联剂中交联0.5-6小时。
  10. 权利要求1-5中任一项所述的可吸收生物膜或者权利要求6-9中任一项所述的方法制备得到的可吸收生物膜在口腔医学领域中的应用，优选在牙周科、口腔种植科或牙槽外科中的应用。
  11. 一种牙槽骨缺损或牙槽嵴重建的修复方法，其包括将权利要求1-5中任一项所述的可吸收生物膜或者权利要求6-9中任一项所述的方法制备得到的可吸收生物膜置于患者的缺损位置处。
  12. 根据权利要求11所述的修复方法，其中，所述可吸收生物膜与骨粉联合使用。
  13. 一种骨缺损的填充方法，其包括将权利要求1-5中任一项所述的可吸收生物膜或者权利要求6-9中任一项所述的方法制备得到的可吸收生物膜置于患者需要的位置处。
  14. 根据权利要求13所述的填充方法，其中，所述骨缺损是由拔牙窝、囊肿切除或残根拔除引起的骨缺损。
  15. 根据权利要求13或14所述的填充方法，其中，所述可吸收生物膜与骨粉联合使用。
PCT/CN2022/135032 2021-11-29 2022-11-29 可吸收生物膜、制法及其应用 WO2023093908A1 (zh)

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