WO2024032284A1 - 一种巩膜加固材料及其制备方法和用途 - Google Patents
一种巩膜加固材料及其制备方法和用途 Download PDFInfo
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- WO2024032284A1 WO2024032284A1 PCT/CN2023/105874 CN2023105874W WO2024032284A1 WO 2024032284 A1 WO2024032284 A1 WO 2024032284A1 CN 2023105874 W CN2023105874 W CN 2023105874W WO 2024032284 A1 WO2024032284 A1 WO 2024032284A1
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- Prior art keywords
- cross
- scleral
- preparation
- linking
- reinforcement material
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Links
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Classifications
-
- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
-
- 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
- A61L27/56—Porous materials, e.g. foams or sponges
-
- 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
Definitions
- the present invention relates to the technical field of medical biological materials, and in particular to a scleral reinforcement material and its preparation method and use.
- Scleral reinforcement is a preventive surgery in which reinforcing materials (such as patches) are placed in the posterior pole and macular area to closely fit with the recipient sclera. New blood vessels grow in to form a thickened new sclera, which causes It mechanically reinforces the eyeball wall and controls the expansion of the eyeball, thus relieving the pressure of the eyeball extending backward and achieving the purpose of stabilizing the diopter.
- Ophthalmic patches can be divided into synthetic materials, autologous materials, allogeneic materials and xenogeneic materials.
- Synthetic materials include artificial pericardial patches, polytetrafluoroethylene, etc. Although they have the characteristics of low elasticity, certain flexibility and plasticity, and stable physical and chemical properties, they have poor tissue compatibility and cannot fuse with the recipient tissue. It is difficult for blood vessels to grow in and cannot improve the blood supply of the recipient tissue. It will cause fluid accumulation and hematoma after surgery, and complications such as rejection, deformation, displacement and prolapse may occur.
- Autologous materials include autologous fascia lata, autologous dermis, etc.
- autologous materials have the advantages of good tissue compatibility, no rejection reaction and strong anti-infection ability, they have problems such as limited sources, additional trauma, and easy to cause intraoperative and postoperative complications.
- Alloplastic materials include allograft sclera, allograft dura mater, etc. Although it has the advantages of good tissue tolerance and mild postoperative reaction, it has problems such as difficulty in source, difficulty in preservation, easy rejection and infection, and easy transmission of diseases.
- Xenogeneic materials mainly include acellular dermal matrix, etc.
- Xenogeneic acellular dermal matrix uses physical and/or chemical methods to decellularize mammalian skin, removing the cellular components and skin accessory structures in the skin epidermis, dermal matrix, and retaining the dermal matrix with a three-dimensional scaffold structure.
- a natural biological material is a natural biological material.
- acellular dermal matrix materials have many advantages such as rich sources and good biocompatibility, due to the large elongation at break of traditional acellular dermal matrix materials, they cannot meet the needs of scleral reinforcement because they only have a relatively high stretch rate. Only small materials can strengthen the sclera and prevent it from extending. In addition, take off The cellular dermal matrix material degrades quickly and will be completely degraded in about 1 to 3 months, while scleral reinforcement requires a longer period of time to maintain the reinforcement effect. Therefore, existing acellular dermal matrix materials cannot be used as scleral reinforcement materials.
- One of the objects of the present invention is to provide a method for preparing a scleral reinforcement material that overcomes the above-mentioned defects in the prior art, which includes the following steps:
- the decellularized dermal tissue is cross-linked to have an elastic modulus of 15MPa to 50MPa, and a degradation rate of less than 20% within 6 to 12 months.
- the cross-linking treatment described in step (3) includes chemical cross-linking treatment and/or thermal cross-linking treatment.
- the cross-linking agent used in the chemical cross-linking treatment is selected from one or more of the following cross-linking agents: glutaraldehyde, genipin, carbodiimide, epoxy compound.
- the carbodiimide includes EDC (1-(3-dimethylaminopropyl)-3-ethyl) at a weight concentration of 1 to 10 wt% or 1 to 10 wt%. carbodiimide hydrochloride) aqueous solution.
- the temperature of chemical cross-linking is 0°C to 37°C or 4°C to 25°C, and the time of chemical cross-linking is 12 to 60 hours or 24 to 48 hours.
- the cross-linking method is soaking or fumigation with a cross-linking agent.
- a step of removing the cross-linking agent is also included after the chemical cross-linking treatment, for example, removing the cross-linking agent through one or more of water cleaning, ventilation drying, and high-temperature drying.
- the elastic modulus is 30 MPa to 50 MPa.
- the decellularized dermal tissue is cross-linked so that the degradation rate within 26 weeks is less than 5%.
- the decellularized dermal tissue is cross-linked to obtain an elongation at break of 20% to 60% or 20% to 30%.
- the animal skin tissue is derived from cattle, pigs, sheep or horses.
- the degreasing and decellularizing treatment in step (2) includes:
- salt solution alkali solution, acid solution, alcohol solution for treatment, among which,
- the salt is one or more combinations of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride and potassium chloride;
- the alkali is one or more combinations of sodium hydroxide, potassium hydroxide, calcium hydroxide and sodium carbonate;
- the acid is one or more combinations of hydrochloric acid, acetic acid and carbonic acid;
- the alcohol is one or more combinations of methanol, ethanol, ethylene glycol and isopropyl alcohol.
- a drying step and a sterilizing step are also included, wherein the drying step is one or more combinations of ventilation drying, air blast drying, organic solvent drying, and vacuum drying;
- the sterilization step is electron beam sterilization, radiation sterilization, ethylene oxide sterilization or low-temperature ion sterilization.
- the above preparation method also includes drying the acellular dermal tissue to obtain a dry sample, packaging the dry sample, and performing terminal sterilization.
- Another object of the present invention is to provide a scleral reinforcing material, which is prepared according to the above method.
- the scleral reinforcement material prepared according to the above preparation method of the present invention is white or yellowish flake-shaped, with the main component being collagen and containing trace amounts of glycosaminoglycans.
- the method of the present invention can remove most of the immune components such as residual cells of animals, so that the residual amount of DNA contained therein is less than 20ng/mg.
- Another object of the present invention is to provide the use of the above-mentioned scleral reinforcement material in scleral reinforcement surgery.
- the present invention has the following beneficial technical effects:
- the preparation method of the scleral reinforcement material of the present invention uses animal dermal tissue as raw material, and after degreasing, decellularization and cross-linking processing, it has mechanical properties suitable for scleral reinforcement.
- this material is used in scleral reinforcement surgery, its mechanical properties such as elastic modulus and stretch rate can achieve the effect of reinforcing the sclera and preventing scleral extension.
- the preparation method of the present invention makes the dermal tissue stronger, has a smaller stretch rate, and is not easy to stretch.
- the dermal tissue has a certain degree of softness after hydration, which is suitable for ocular scleral implantation and avoids the need for scleral implantation.
- Traditional dermal tissue lacks softness as its hardness increases. If sclera is implanted, it will cause eye discomfort.
- the scleral reinforcement material prepared by the present invention also has suitable degradation properties for scleral reinforcement. Compared with the degradation rate of traditional acellular dermal matrix (complete degradation in about 1 to 3 months), the scleral reinforcement material of the present invention has suitable degradation properties. It can last for 6 to 12 months in the scleral tissue before degrading and is absorbed by the human body. It will not cause a strong inflammatory reaction and will not degrade too quickly before new tissue is formed. Therefore, it can meet the needs of scleral reinforcement surgery for a long time. The need for time to act as a reinforcement can avoid the failure of early reinforcement due to too rapid degradation of the material; in addition, it can also avoid the failure of the scleral reinforcement due to too slow degradation of the scleral reinforcement material. There is long-term inflammatory reaction and obvious foreign body reaction.
- the scleral reinforcement material prepared by the present invention has a pore structure that is conducive to cell growth, so it has excellent biological fusion. After being implanted into the sclera, it can fuse with the scleral tissue within a certain period of time to generate a thickened tissue.
- the present invention uses animal dermal tissue as raw material to prepare scleral reinforcement materials, which have good biocompatibility and can be completely degraded by the human body into small molecule amino acids that can be absorbed by the human body.
- the decellularization method used in the present invention can remove immunogenic components and will not cause strong immune rejection after implantation into the human body. At the same time, the introduction of toxic and potentially immunogenic decellularization reagents during the decellularization process is avoided to ensure the biological safety of the scleral reinforcement material.
- the scleral reinforcement material provided by the present invention is soft in texture, does not affect eyeball movement after being used to reinforce the sclera, and has no foreign body sensation.
- the mechanical strength of the dermal tissue is enhanced through cross-linking processing, which can meet the needs of scleral reinforcement for different myopic people.
- the scleral reinforcement material provided by the present invention can realize standardized batch processing, which is conducive to the promotion of surgical procedures and benefits more patients.
- Figure 1 is an appearance picture of the scleral reinforcement material according to Embodiment 1 of the present invention.
- Figure 2 is an SEM morphology diagram of the scleral reinforcement material in Example 1 of the present invention.
- Figure 3 is a HE stained picture (400 ⁇ ) of the scleral reinforcement material after decellularization in Example 1 of the present invention.
- Figure 4 is a comparison chart of the residual amounts of cellular DNA between the dermal raw materials and the scleral reinforcement materials used in Example 1 of the present invention.
- Figure 5 is a graphical representation of the degradation rate of the scleral reinforcement materials prepared in Examples 1 to 6 of the present invention when enzymatically hydrolyzed in collagenase solution.
- Figure 6 is a HE staining image of tissue sections of rabbits after the scleral reinforcement material of Example 4 of the present invention was implanted subcutaneously at 1, 4, 8, 12 and 26 weeks.
- Figure 7 is a picture of the appearance of the scleral reinforcement material implanted into the sclera of a rabbit for 3 months according to Embodiment 4 of the present invention.
- step 2 Use 1wt% hydrochloric acid aqueous solution to soak the sheet tissue obtained in step 1 at 10°C to 25°C, stir for 5 hours, and then rinse with purified water for 4 hours;
- step 5 Place the sheet tissue obtained in step 5 into an EDC aqueous solution with a concentration of 1wt%, soak it in a 4°C environment for 48 hours, and rinse with purified water for 4 hours;
- step (3) Place the sheet tissue obtained in step (3) into a vacuum freeze dryer and freeze-dry for 48 hours;
- step (4) Pack the sheet tissue obtained in step (4) into a blister box and sterilize it with ethylene oxide to obtain the scleral reinforcement material.
- the sample number of this material is 2.
- Example 2 The difference between this embodiment and Example 1 is that the concentration of the cross-linking agent EDC aqueous solution is 2.5 wt%, and the other conditions are the same as Example 1.
- the sample number of the scleral reinforcement material obtained in Example 2 is 3.
- Example 1 The difference between this embodiment and Example 1 is that the concentration of the cross-linking agent EDC aqueous solution is 5 wt%, and the other conditions are the same as Example 1.
- the sample number of the scleral reinforcement material obtained in Example 3 is 4.
- step 2 Use 1wt% hydrochloric acid aqueous solution to soak the sheet tissue in step 1 at 10°C to 25°C, stir for 5 hours, and then rinse with purified water for 4 hours;
- step 5 Place the sheet tissue obtained in step 5 into an EDC aqueous solution with a concentration of 5wt%, soak it in a 25°C environment for 48 hours, and rinse with purified water for 4 hours;
- step (3) Place the sheet tissue obtained in step (3) into a vacuum freeze dryer and freeze-dry for 48 hours;
- step (4) Pack the sheet tissue obtained in step (4) into a blister box and sterilize it with electron beam irradiation to obtain the scleral reinforcement material.
- the sample number for this material is 7.
- Example 4 The difference between this embodiment and Example 4 is that the concentration of the cross-linking agent EDC aqueous solution is 2.5 wt%, and the other conditions are the same as Example 4.
- the sample number of the scleral reinforcement material obtained in Example 5 is 6.
- Example 4 The difference between this embodiment and Example 4 is that the concentration of the cross-linking agent EDC aqueous solution is 1 wt%, and the other conditions are the same as Example 4.
- the sample number of the scleral reinforcement material obtained in Example 6 is 5.
- Example number 1 The mechanical properties and degradation properties of the scleral reinforcement materials prepared in Examples 1-6 (corresponding to samples 2, 3, 4, 7, 6, and 5) were tested respectively below, and the dermis that was not cross-linked after decellularization was used. Material (sample number 1) was used as a control.
- the scleral reinforcement material obtained by cross-linking treatment of the present invention is compared with the control material without cross-linking treatment.
- the elastic modulus of the scleral reinforcement material of the present invention increases with the increase of cross-linking agent concentration and cross-linking temperature, and the elongation at break decreases with the increase of cross-linking agent concentration and cross-linking temperature.
- the scleral reinforcement materials prepared in Examples 1-6 of the present invention can meet the requirements of scleral reinforcement for mechanical properties.
- the elastic modulus of the scleral reinforcement material of the present invention reaches 30MPa to 50MPa, the scleral reinforcement material breaks It is especially suitable for scleral reinforcement when the elongation is 20% to 30%.
- the present invention conducted an accelerated enzymatic hydrolysis test in a collagenase solution.
- the enzymatic hydrolysis was accelerated in the collagenase solution for 1 day and 2 days. Afterwards, the degradation rates of the non-crosslinked control samples were as high as 87.56% and 99.54% respectively, while the degradation rates of the scleral reinforcement material samples of Examples 1-6 of the present invention were significantly reduced.
- the scleral reinforcement material of the present invention is used in the in vivo environment where scleral reinforcement is located, its degradation rate is much lower than the degradation rate of accelerated enzymatic hydrolysis by collagenase. It can be expected that the degradation rate of the scleral reinforcement material of the present invention when used for scleral reinforcement will also be The degradation rate will be significantly lower than that of the control sample, which will be further verified through animal subcutaneous implantation degradation tests below.
- the scleral reinforcement material prepared in Example 4 was used to conduct an animal subcutaneous implantation degradation test.
- the degradation test periods were 1 week, 4 weeks, 8 weeks, 12 weeks and 26 weeks, and 20 healthy rabbits were selected, including 3 rabbits for each test period.
- the rabbit was anesthetized by intraperitoneal injection of pentobarbital sodium at a dose of 40 mg/kg, and the anesthetic preparation mass concentration was 1%.
- pentobarbital sodium at a dose of 40 mg/kg
- the anesthetic preparation mass concentration was 1%.
- the experimental animals were fixed on the operating table, the hair on both sides of the spine was removed, and the skin in the hair removal area was routinely disinfected.
- the surgical area was approximately 4cm x 4cm.
- the implantation requirements make a skin incision at each implantation site on both sides of the animal's spine, about 1cm long, and the distance between points on one side is at least 1cm.
- the subcutaneous tissue was bluntly dissected, deep to the superficial fascial muscle layer, and four subcutaneous pouches were made on each side of each rabbit.
- the test object is implanted on one side. The incision was closed and the subcutaneous tissue and skin were sutured in layers. Disinfect the wound with iodophor.
- the test period is 1 week, 4 weeks, 8 weeks, 12 weeks and 26 weeks.
- the samples obtained at each time period were subjected to tissue sections to observe the degradation of the scleral reinforcement materials.
- the HE staining diagrams of tissue sections with test periods of 1 week, 4 weeks, 8 weeks, 12 weeks and 26 weeks are shown in Figure 6.
- the scleral reinforcement material of the present invention shows good anti-degradation ability in animals and meets the requirements of scleral reinforcement for degradation performance.
- the sclera reinforcement material prepared in Example 4 was used to conduct a rabbit sclera reinforcement test.
- the rabbit was fixed on the surgical board in a supine position, and the operation was performed under sterile conditions. Sumianxin injection was injected intramuscularly for anesthesia. Ophthalmic scissors were used to make a 360° incision at the edge of the cornea and conjunctiva, and an incision of about 6 mm in length was made at the upper and lower sides. Separate the conjunctival sac to fully expose the sclera. Take the lateral, superior and inferior rectus muscles with strabismus hooks, and make traction lines for the lateral, superior and inferior rectus muscles. Trim the scleral reinforcement material to 25mm ⁇ 8mm from the superior, lateral and inferior rectus muscles.
- the two ends of the strip are fixed to the nasal side of the superior and inferior rectus muscle insertions.
- the sutures are absorbable. Suture the upper and lower incisions of the conjunctiva and wrap the operative eye with sterile dressing.
- the animals were sacrificed by air embolization of the marginal ear vein.
- the eyeballs were removed aseptically, and the cornea, iris, lens, vitreous body, choroid and retinal tissue were removed.
- the scleral tissue was washed in physiological saline, and the experimental eye rim was reinforced. Cut the sclera and the attached reinforcement material into strips, take photos and observe the fusion of the sclera reinforcement material and the sclera as shown in Figure 7. It can be seen from Figure 7 that the scleral reinforcement material of the present invention has grown together with the sclera of the subject animal, and has good fusion. The strength of the fused tissue is increased compared with the sclera before reinforcement, and the scleral reinforcement effect can be achieved.
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Abstract
本发明涉及一种巩膜加固材料及其制备方法和用途，该方法包括如下步骤：（1）以动物皮肤组织为原材料，切取真皮组织；（2）将所述真皮组织进行脱脂和脱细胞处理；（3）对脱细胞处理后的真皮组织进行交联处理使其具有弹性模量15MPa至50MPa，并且在6至12个月内降解率低于20％。本发明的巩膜加固材料具有适用于巩膜加固的力学性能和降解性能。
Description
相关申请的交叉引用
本申请要求于2022年8月8日提交的申请号为202210946834.6、名称为“一种巩膜加固材料及其制备方法和用途”的中国专利申请的权益和优先权，该中国专利申请的全部内容通过引用并入本文。
本发明涉及医疗生物材料技术领域，具体涉及一种巩膜加固材料及其制备方法和用途。
巩膜加固术是一种预防性手术，是将加固材料(如补片)置于后极部及黄斑区，与受体巩膜密合在一起，新生血管长入，形成加厚的新巩膜，起到了机械加固眼球壁，控制眼球扩张的作用，从而缓解了眼球向后延伸的压力，达到稳定屈光度的目的。
近年来，随着技术的不断发展，巩膜加固材料也在不断的优化完善。眼科补片可以分为合成材料、自体材料、异体材料和异种材料。合成材料包括人工心包补片、聚四氟乙烯等，虽然它们具有低弹性、一定的柔韧度和易塑性、理化性质稳定等特点，但是它们的组织相容性差，不能和受体组织融合，新生血管不易长入，不能改善受体组织血供，术后会引起积液和血肿，发生排异、变形、移位及脱出等并发症。自体材料包括自体阔筋膜、自体真皮等。自体材料虽然具有组织相容性好，无排斥反应及抗感染能力强等优点，但是其存在来源有限、增加额外创伤、易引起术中、术后并发症等问题。异体材料包括异体巩膜、异体硬脑膜等。虽然其表现出组织耐受性好、术后反应轻等优点，但存在来源困难、不易保存、易产生排斥及感染、易传播疾病等问题。异种材料主要包括脱细胞真皮基质等。异种脱细胞真皮基质是采用物理和(或)化学方法将哺乳动物皮肤进行脱细胞处理，脱去皮肤表皮、真皮基质中的细胞成分及皮肤附属结构，保留了具有三维支架结构的真皮基质，是一种天然生物材料。
虽然脱细胞真皮基质材料具有来源丰富、生物相容性好等诸多优点，但是，由于传统的脱细胞真皮基质材料的断裂伸长率较大，无法满足巩膜加固的需求，因为只有拉伸率较小的材料才能起到巩膜加固和防止巩膜延伸的效果。此外，脱
细胞真皮基质材料的降解较快，大约1～3月会完全降解，而巩膜加固需要更长时间保持加固作用。因此，现有的脱细胞真皮基质材料无法适用于巩膜加固材料。
发明内容
本发明的目的之一在于提供一种克服上述现有技术中的缺陷的巩膜加固材料的制备方法，其包括如下步骤：
(1)以动物皮肤组织为原材料，切取真皮组织；
(2)将所述真皮组织进行脱脂和脱细胞处理；
(3)对脱细胞处理后的真皮组织进行交联处理使其具有弹性模量15MPa至50MPa，并且在6至12个月内降解率低于20％。
在本发明的一些实施方式中，步骤(3)中所述的交联处理包括化学交联处理和/或热交联处理。
在本发明的一些实施方式中，所述的化学交联处理采用的交联剂选自下列交联剂中的一种或多种：戊二醛，京尼平，碳二亚胺，环氧化合物。
在本发明的一些实施方式中，所述的碳二亚胺包括重量浓度为1wt％至10wt％或者1wt％至10wt％的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)水溶液。
在本发明的一些实施方式中，化学交联的温度为0℃至37℃或者4℃至25℃，化学交联时间为12至60小时或者24至48小时。
在本发明的一些实施方式中，交联方式为交联剂浸泡或者熏蒸。
在本发明的一些实施方式中，在化学交联处理之后还包括去除交联剂的步骤，例如，通过水清洗、通风干燥、高温干燥中的一种或者多种去除交联剂。
在本发明的一些实施方式中，所述弹性模量为所述弹性模量为30MPa至50MPa。
在本发明的一些实施方式中，对脱细胞处理后的真皮组织进行交联处理使其在26周内的降解率低于5％。
在本发明的一些实施方式中，对脱细胞处理后的真皮组织进行交联处理使其断裂伸长率为20％至60％或者20％至30％。
在本发明的一些实施方式中，所述动物皮肤组织来源于牛，猪，羊或者马。
在本发明的一些实施方式中，所述步骤(2)中的脱脂和脱细胞处理包括：
采用盐溶液、碱溶液、酸溶液、醇溶液进行处理，其中，
所述盐为磷酸氢二钠、磷酸氢二钾、氯化钠和氯化钾中的一种或者多种组合；
所述碱为氢氧化钠、氢氧化钾、氢氧化钙和碳酸钠中的一种或者多种组合；
所述酸为盐酸、醋酸和碳酸中的一种或者多种组合；
所述醇为甲醇、乙醇、乙二醇和异丙醇中的一种或多种组合。
在本发明的一些实施方式中，还包括干燥步骤和灭菌步骤，其中，所述干燥步骤为通风晾干、鼓风烘干、有机溶剂干燥、真空烘干中的一种或者多种组合；所述灭菌步骤为电子束灭菌、辐照灭菌、环氧乙烷灭菌或低温离子灭菌。
在本发明的一些实施方式中，上述制备方法还包括将脱细胞真皮组织经过干燥处理得到干态样品，并且将干态样品包装，终端灭菌处理。
本发明的另一目的在于提供一种巩膜加固材料，该加固材料根据上述方法制备而成。
在本发明的一些实施方式中，根据本发明上述制备方法制得的巩膜加固材料为白色或者微黄色片状，主要成分为胶原蛋白，并包含微量糖胺聚糖。本发明方法能够大部分去除动物残留细胞等免疫性成分，使其中所含的DNA残留量低于20ng/mg。
本发明的又一目的在于提供上述巩膜加固材料在巩膜加固手术中的用途。
本发明相对于现有技术具有如下有益技术效果：
(1)本发明的巩膜加固材料的制备方法采用动物真皮组织作为原料，经脱脂脱细胞和交联处理之后使其具备适用于巩膜加固的力学性能。将该材料用于巩膜加固手术时，其弹性模量和拉伸率等力学性能可达到巩膜加固和防止巩膜延伸的效果。具体而言，本发明的制备方法使得真皮组织更加坚固，具有较小的拉伸率，不易拉伸，同时使真皮组织在水化后具有一定的柔软度，适合眼部巩膜的植入，避免传统的真皮组织随着硬度增加而缺少柔软度，若植入巩膜会引起眼部不舒适的问题。
(2)本发明制备的巩膜加固材料还具有适合巩膜加固的降解性能，相对于传统的脱细胞真皮基质的降解速率(大约1～3月完全降解)，本发明的巩膜加固材料具有合适的降解速度，在巩膜组织处可维持6～12个月后降解，被人体吸收，不会引起强烈的炎性反应，在新生组织生成之前不会过快降解，因此，能够满足巩膜加固手术需要较长时间起加固作用的需求，可避免由于材料降解过快而导致前期的加固作用失败；此外，也可避免由于巩膜加固材料降解速度过慢引起巩膜
长期存在炎症反应、异物反应明显。
(3)本发明制备的巩膜加固材料具有有利于细胞长入的孔隙结构，从而具有优异的生物融合性，植入巩膜后可以在一定时间内与巩膜组织融合，生成加厚的组织。
(4)本发明采用动物真皮组织作为原料制备巩膜加固材料，具有较好的生物相容性，能够被人体完全降解为人体可吸收的小分子氨基酸。
本发明采用的脱细胞方法可以去除免疫原成分，植入人体后不会引起强烈的免疫排斥反应。同时，在脱细胞过程中避免引入有毒性和潜在引起免疫的脱细胞试剂，从而保证巩膜加固材料的生物安全性。
本发明提供的巩膜加固材料质地柔软，用于加固巩膜后不影响眼球活动，无异物感。通过交联处理增强了真皮组织的力学强度，能够满足不同近视人群巩膜加固的需求。
本发明提供的巩膜加固材料可实现标准化批量加工，有利于术式推广，使更多患者受益。
以下将结合附图和具体实施方式对本发明进行进一步详细说明。
图1为本发明实施例1的巩膜加固材料的外观图片。
图2为本发明实施例1的巩膜加固材料的SEM形貌图。
图3为本发明实施例1的巩膜加固材料的脱细胞后HE染色图(400×)。
图4为本发明实施例1中所采用的真皮原材料和巩膜加固材料的细胞DNA残留量对照图。
图5为本发明实施例1至6中制备的巩膜加固材料在胶原酶溶液中酶解的降解率图示。
图6为本发明实施例4的巩膜加固材料植入兔子皮下1周、4周、8周、12周和26周的组织切片HE染色图。
图7为本发明实施例4的巩膜加固材料植入兔子巩膜处3个月的外观图片。
下面将结合本发明的实施例和附图，对本发明的技术方案进行清楚、完整地
描述，显然，所描述的实施例仅用于举例说明的一部分实施例，而未穷尽全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，而不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有付出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
除非另有具体说明，否则在这些实施例中记载的数值不限制本发明的范围。对于相关领域普通技术人员已知的技术、方法可能不作详细讨论，但在适当情况下，所述技术、方法应当被视为说明书的一部分。在这里示出和讨论的所有示例中，任何具体值应被解释为仅仅是示例性的，而不是作为限制。因此，示例性实施例的其它示例可以具有不同的值。
实施例1
本实施例中的巩膜加固材料的制备方法包括如下步骤：
(1)以猪皮为原料，洗净后，自皮下筋膜层切取厚度均匀的片状组织，保留乳头层和网状层，片状组织厚度为1mm；
(2)将切取的真皮组织进行脱脂和脱细胞处理，具体如下：
①用0.5wt％K2HPO4-KH2PO4的盐溶液清洗真皮4h，然后使用4wt％氢氧化钠在10℃～25℃下浸泡，搅拌处理5h，然后纯化水清洗4h；
②使用1wt％盐酸水溶液在10℃～25℃下浸泡经步骤①处理得到的片状组织，搅拌处理5h，然后纯化水清洗4h；
③重复步骤①和步骤②2～4遍；
④使用无水乙醇清洗步骤③处理后的片状组织24h，纯化水清洗4h；
⑤将步骤④处理获得的片状组织冷冻干燥48h；
(3)对脱细胞处理后的真皮组织进行交联处理，具体如下：
将步骤⑤处理获得的片状组织置于1wt％浓度的EDC水溶液中，4℃环境下浸泡48h，纯化水清洗4h；
(4)将步骤(3)处理获得的片状组织置于真空冷冻干燥机中，冷冻干燥48h；
(5)将步骤(4)处理获得的片状组织包装在吸塑盒中，环氧乙烷灭菌，即可得到巩膜加固材料，该材料的样品序号为2。
本实施例制备得到的巩膜加固材料的外观图如图1所示，通过扫描电镜观察
到的形貌SEM图片如图2所示，脱细胞后HE染色图(400×)如图3所示。
本实施例所采用的真皮原材料和巩膜加固材料的细胞DNA残留量对照如图4所示，根据图4可以看出，本实施例中的真皮组织经过脱细胞处理之后的真皮组织中DNA残留量很少，脱细胞效率达到98％以上。此外，相比于常规真皮脱细胞方法采用表面活性剂法和酶法(表面活性剂如SDS毒性大，有遗传毒性，破坏材料结构物，不易去除；酶会破坏材料结构和强度，本身也是会引起免疫排斥反应)，本发明选用的盐法和酸碱法，没有强毒性，且非常容易去除，无化学残留，是一种安全的脱细胞方法。
实施例2
本实施例与实施例1的区别在于交联剂EDC水溶液的浓度为2.5wt％，其余条件均与实施例1相同。实施例2得到的巩膜加固材料的样品序号为3。
实施例3
本实施例与实施例1的区别在于交联剂EDC水溶液的浓度为5wt％，其余条件均与实施例1相同。实施例3得到的巩膜加固材料的样品序号为4。
实施例4
本实施例中的巩膜加固材料的制备方法包括如下步骤：
(1)以猪皮为原料，洗净后，自皮下筋膜层切取厚度均匀的片状组织，去掉真皮的乳头层，片状组织厚度为0.6mm；
(2)将切取的真皮组织进行脱脂和脱细胞处理，具体如下：
①用0.9wt％氯化钠溶液清洗真皮2h，然后使用4wt％氢氧化钠在10℃～25℃下浸泡，搅拌处理5h，然后纯化水清洗4h；
②使用1wt％盐酸水溶液在10℃～25℃下浸泡步骤①中的片状组织，搅拌处理5h，然后纯化水清洗4h；
③重复步骤①和步骤②2～4遍；
④使用无水乙醇清洗步骤③处理后的片状组织24h，纯化水清洗4h；
⑤将步骤④处理获得的片状组织冷冻干燥48h；
(3)对脱细胞处理后的真皮组织进行交联处理，具体如下：
将步骤⑤处理获得的片状组织置于5wt％浓度的EDC水溶液中，25℃环境下浸泡48h，纯化水清洗4h；
(4)将步骤(3)处理获得的片状组织置于真空冷冻干燥机中，冷冻干燥48h；
(5)将步骤(4)处理获得的片状组织包装在吸塑盒中，电子束辐照灭菌，即可得到巩膜加固材料。该材料的样品序号为7。
实施例5
本实施例与实施例4的区别在于交联剂EDC水溶液的浓度为2.5wt％，其余条件均与实施例4相同。实施例5得到的巩膜加固材料的样品序号为6。
实施例6
本实施例与实施例4的区别在于交联剂EDC水溶液的浓度为1wt％，其余条件均与实施例4相同。实施例6得到的巩膜加固材料的样品序号为5。
以下分别对实施例1-6制备得到的巩膜加固材料(对应样品2、3、4、7、6、5)的力学性能和降解性能进行检测，并以脱细胞处理之后未进行交联的真皮材料(样品序号为1)作为对照。
1.弹性模量和断裂伸长率的测试
将实施例1-6制备得到的巩膜加固材料裁剪成50mm×10mm的尺寸，用纯化水浸润样品，浸润10min。用测厚仪测量试样标距内三点厚度，记录并取试样厚度的平均值。将试样装入拉力机夹具上，试样呈伸展状态，测量上下夹具间的长度，拉力机速度设为25mm/min。检测得到的弹性模量与断裂伸长率的数值如表1。
表1
由表1可知，本发明经交联处理得到的巩膜加固材料与未经交联处理的对照
样品1相比，本发明的巩膜加固材料的弹性模量随着交联剂浓度增加和交联温度升高而提高，断裂伸长率则随着交联剂浓度增加和交联温度升高而下降，通过本发明的研究发现，本发明实施例1-6制备得到的巩膜加固材料均能满足巩膜加固对于力学性能的要求，当本发明的巩膜加固材料的弹性模量达到30MPa至50MPa，断裂伸长率20％至30％时，尤其适合巩膜加固。
2.体外降解性能测试
为了比较本发明实施例1-6的巩膜加固材料和未交联材料的降解率，本发明在胶原酶溶液中进行加速酶解试验。
将本发明实施例1-6的巩膜加固材料样品和未交联对照样品裁剪成1cm×1cm的尺寸，放入装有Ⅰ型胶原酶溶液的小瓶中，其中Ⅰ型胶原酶的浓度为100u/mL，并将小瓶放入37℃水浴中，分别降解1天和2天后将产品冻干称重，计算出各样品的在Ⅰ型胶原酶溶液中的降解率，结果如图5所示。由图5可以看出，本发明实施例1-6的巩膜加固材料的降解率明显低于未交联对照样品的降解率，具体而言，在胶原酶溶液中加速酶解1天和2天之后，未交联的对照样品的降解率分别高达87.56％和99.54％，而本发明实施例1-6的巩膜加固材料样品的降解率明显降低。当本发明的巩膜加固材料应用于巩膜加固所处的体内环境时，其降解率远低于胶原酶加速酶解的降解率，可以预期本发明的巩膜加固材料用于巩膜加固时的降解率也将明显低于对照样品的降解率，下文将通过动物皮下植入降解试验进行进一步验证。
3.动物皮下植入降解试验
采用实施例4制备得到的巩膜加固材料进行动物皮下植入降解试验。
降解试验周期为1周、4周、8周、12周和26周，选用健康兔子20只，其中每个试验周期取3只兔子。在进行皮下植入时，使用戊巴比妥钠腹腔注射麻醉兔子，剂量为40mg/kg，麻药配制质量比浓度为1％。麻醉时应注意实验动物的保温，防止因温度过低导致动物死亡。将实验动物在手术台上固定，除去脊柱两侧被毛，常规消毒去毛区皮肤，手术区域约4cm x 4cm。按植入要求，在动物脊柱两侧每一植入位点作一皮肤切口，长约1cm，单侧点间距离至少1cm。钝性分离皮下组织，深至浅筋膜肌层，每只兔子的每一侧作4个皮下囊。一侧植入试验物。关闭切口并分层缝合皮下组织和皮肤。碘伏消毒伤口。试验周期为1周、
4周、8周、12周和26周。对各时间段获取的样品进行组织切片观察其中的巩膜加固材料的降解情况，试验周期为1周、4周、8周、12周和26周的组织切片HE染色图如图6所示。
从图6可观察到植入1周、4周、8周、12周后，巩膜加固材料未发生降解，植入26周后，材料依然能保持完整的结构，其降解率低于5％，因此，本发明的巩膜加固材料在动物体内表现出良好的抗降解能力，满足巩膜加固对于降解性能的需求。
4.巩膜加固动物试验
采用实施例4制备得到的巩膜加固材料进行家兔巩膜加固试验。
将家兔仰卧固定于手术板，无菌条件下操作，速眠新注射液肌肉注射麻醉，眼科剪于角结膜缘处作360°切口，并于上、下方各作一长约6mm切口，钝性分离结膜囊，充分暴露巩膜，斜视钩钩取外、上、下直肌，并作外、上、下直肌牵引线，将修剪为25mm×8mm的巩膜加固材料从上、外、下直肌下穿过，适当调整脱细胞真皮条带位置，使条带中央部位于后极部，紧贴巩膜，将条带两端各固定于上、下直肌肉止端鼻侧，可吸收缝线缝合结膜上、下方切口无菌敷料包扎术眼。
于术后3个月，耳缘静脉空气栓塞法分别处死动物，无菌摘取眼球，去除角膜、虹膜、晶状体、玻璃体、脉络膜和视网膜组织，把巩膜组织于生理盐水内清洗，实验眼沿加固条带大小剪取巩膜及附着的加固材料，拍照观察，巩膜加固材料与巩膜的融合情况如图7所示。根据图7可以看出，本发明的巩膜加固材料与受试动物的巩膜已经长在一起，融合性好，融合后的组织比加固前的巩膜强度增加，能够实现巩膜加固的效果。
以上结合具体实施例对本发明进行了具体说明，这些具体实施例仅仅是示例性的，不能以此限定本发明的保护范围，本领域技术人员在不脱离本发明的技术构思的前提下可对本发明进行各种修改、变化或替换。因此，依照本发明所作的各种等同变化仍属于本发明所涵盖的范围。
Claims (11)
- 一种巩膜加固材料的制备方法，其特征在于，包括如下步骤：(1)以动物皮肤组织为原材料，切取真皮组织；(2)将所述真皮组织进行脱脂和脱细胞处理；(3)对脱细胞处理后的真皮组织进行交联处理使其具有弹性模量15MPa至50MPa，并且在6至12个月内降解率低于20％。
- 根据权利要求1所述的制备方法，其特征在于，步骤(3)中所述的交联处理包括化学交联处理和/或热交联处理。
- 根据权利要求2所述的制备方法，其特征在于，所述的化学交联处理采用的交联剂选自下列交联剂中的一种或多种：戊二醛，京尼平，碳二亚胺，环氧化合物。
- 根据权利要求3所述的制备方法，其特征在于，所述的碳二亚胺包括重量浓度为1wt％至10wt％或者1wt％至5wt％的EDC水溶液。
- 根据权利要求3或4所述的制备方法，其特征在于，化学交联的温度为0℃至37℃或者4℃至25℃，化学交联时间为12至60小时。
- 根据权利要求1所述的制备方法，其特征在于，所述弹性模量为30MPa至50MPa。
- 根据权利要求1所述的制备方法，其特征在于，对脱细胞处理后的真皮组织进行交联处理使其断裂伸长率为20％至60％或者20％至30％。
- 根据权利要求1所述的制备方法，其特征在于，所述步骤(2)中的脱脂和脱细胞处理包括：采用盐溶液、碱溶液、酸溶液、醇溶液进行处理，其中，所述盐为磷酸氢二钠、磷酸氢二钾、氯化钠和氯化钾中的一种或者多种组合；所述碱为氢氧化钠、氢氧化钾、氢氧化钙和碳酸钠中的一种或者多种组合；所述酸为盐酸、醋酸和碳酸中的一种或者多种组合；所述醇为甲醇、乙醇、乙二醇和异丙醇中的一种或多种组合。
- 根据权利要求1所述的制备方法，其特征在于，还包括干燥步骤和灭菌步骤，其中，所述干燥步骤为通风晾干、鼓风烘干、有机溶剂干燥、真空烘干中的一种或者多种组合；所述灭菌步骤为电子束灭菌、辐照灭菌、环氧乙烷灭菌或低温离子灭菌。
- 一种巩膜加固材料，按照权利要求1至9中任何一项所述的方法制备而成。
- 巩膜加固材料在巩膜加固手术中的用途，所述巩膜加固材料按照权利要求1至9中任何一项所述的方法制备而成。
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