技术领域technical field
本发明属于软骨修复技术领域，具体涉及一种可吸收软骨修复系统，该系统由可吸收软骨修复支架和自体血组成，在人体生理温度37℃下成凝胶状，修补于软骨缺损部位，可实现软骨组织的修复。The invention belongs to the technical field of cartilage repair, and specifically relates to an absorbable cartilage repair system. The system is composed of an absorbable cartilage repair support and autologous blood. It becomes gelatinous at a human physiological temperature of 37°C, and can be repaired at cartilage defects. To achieve the repair of cartilage tissue.
背景技术Background technique
骨关节炎为一种行退性病变，是由于多种因素引起的关节软骨退化损伤、关节边缘和软骨下骨反应性增生。临床表现为缓慢发展的关节疼痛、压痛、僵硬、关节肿胀、活动受限和关节畸形等。X线平片后期可见关节间隙狭窄，CT及MRI检查可在早起发现关节软骨及软骨下骨质的异常改变。目前，我国60岁以上老人中，有55%的人患有骨关节炎，全国约有1.2亿人患有此病。Osteoarthritis is a degenerative disease, which is caused by multiple factors causing degeneration of articular cartilage, joint edge and subchondral bone reactive hyperplasia. Clinical manifestations include slowly developing joint pain, tenderness, stiffness, joint swelling, limited mobility, and joint deformity. X-ray plain film shows narrow joint space in the late stage, and abnormal changes of articular cartilage and subchondral bone can be found in CT and MRI examinations in the early stage. At present, 55% of people over the age of 60 in my country suffer from osteoarthritis, and about 120 million people in the country suffer from this disease.
当前对骨关节炎的治疗包括以下方面：对于早期病例，可进行理疗、运动锻炼治疗、使镇痛药物治疗、关节腔内注射透明质酸类润滑剂，但是，这些治疗方法都只能在一定程度上缓解骨关节炎的症状，不能够逆转骨关节炎病情进展、彻底治愈该疾病；对于晚期病例，在全身情况能耐受手术的条件下，进行人工关节置换术，是目前公认的消除疼痛、矫正畸形、改善功能的有效方法，但是，其对患者年龄、身体状况有严格要求，术后并发症较多，如感染、深静脉栓塞等，而且每例人工关节仅材料费就需要4～6万元。The current treatment of osteoarthritis includes the following aspects: for early cases, physical therapy, exercise therapy, analgesic drug treatment, intra-articular injection of hyaluronic acid lubricants, but these treatments can only be used in certain cases. It can alleviate the symptoms of osteoarthritis to a certain extent, but cannot reverse the progression of osteoarthritis and completely cure the disease; for advanced cases, artificial joint replacement is currently recognized as a pain relief method under the condition that the general condition can tolerate surgery. It is an effective method to correct deformity and improve function. However, it has strict requirements on the patient's age and physical condition, and there are many postoperative complications, such as infection, deep vein thrombosis, etc., and the material cost of each artificial joint needs 4 ~ 60,000 yuan.
针对关节软骨再生和修复方面的研究目前是骨关节炎治疗研究的热点。目前的尝试有如下几类：Research on the regeneration and repair of articular cartilage is currently a hot spot in the treatment of osteoarthritis. Current attempts fall into the following categories:
1. 微骨折法：为关节镜微创手术，清除已钙化软骨，在缺损部位转孔，令骨髓和血液渗出，形成凝血块，诱导软骨细胞生长，缺点：非真正软骨，恢复期长需要半年到一年以上。1. Microfracture method: It is a minimally invasive arthroscopic surgery. It removes the calcified cartilage, and drills a hole in the defect site to make the bone marrow and blood seep out, form a blood clot, and induce the growth of chondrocytes. Disadvantages: It is not real cartilage, and the recovery period is long. Six months to more than a year.
2. 自体骨软骨移植：将自身非承重软骨移植到缺损区，缺点：手术操作繁琐，人体创伤大，软骨供区有限。2. Autologous osteochondral transplantation: Transplant your own non-load-bearing cartilage to the defect area. Disadvantages: cumbersome operation, large human trauma, and limited cartilage donor area.
3. 自体软骨细胞移植（ACI）：取自身关节软骨细胞在体外培养，再植入缺损区，缺点：需二次手术、培养条件苛刻，需要相应认证的实验室进行。3. Autologous chondrocyte transplantation (ACI): take autologous articular chondrocytes and culture them in vitro, and then implant them into the defect area. Disadvantages: secondary surgery is required, the culture conditions are harsh, and correspondingly certified laboratories are required.
4．富血小板血浆凝胶修复：为自体血浓缩的含血小板的血浆，加入牛凝血酶和氯化钙成凝胶状，植入缺损区进行修复，缺点：需要无菌操作，环境、人员要求较高，操作过程尚无标准化程序，牛凝血酶加入有免疫原性风险。4. Platelet-rich plasma gel repair: platelet-containing plasma concentrated from autologous blood, added with bovine thrombin and calcium chloride to form a gel, and implanted into the defect area for repair. Disadvantages: aseptic operation is required, and the environment and personnel requirements are high , there is no standardized procedure in the operation process, and the addition of bovine thrombin has the risk of immunogenicity.
因此，临床上急需一种操作方便，再生效果显著并且稳定的产品用于软骨修复。Therefore, there is an urgent need for a product that is easy to operate, has a significant regenerative effect and is stable for cartilage repair.
由于关节软骨没有血液供应，自身修复能力极差，创伤、骨性关节炎所致的关节软骨损伤和缺损是临床治疗的难题。近年来，应用基因技术和组织工程方法来治疗关节软骨损伤是研究的两大热点。组织工程通过三维培养，模拟体内生物和力学环境来构建功能性组织工程软骨。在构建组织工程化软骨的过程中，细胞支架为细胞提供一个稳定的三维空间结构，并保持足够的孔隙率，供其进行物质交换、生长代谢，从而引导软骨形成。可注射性的生物材料具有可以与细胞均匀混合，与修复组织之间嵌合连接好，塑型容易的特点而且能通过关节镜操作，治疗简单、安全、有效，符合现代微创手术的要求，因此，可注射性的软骨组织工程产品应该是临床应用的发展方向。Since articular cartilage has no blood supply and its self-repair ability is extremely poor, articular cartilage damage and defects caused by trauma and osteoarthritis are difficult problems in clinical treatment. In recent years, the application of gene technology and tissue engineering methods to treat articular cartilage damage are two hot spots of research. Tissue engineering constructs functional tissue engineered cartilage by simulating the biological and mechanical environment in vivo through three-dimensional culture. In the process of constructing tissue-engineered cartilage, the cell scaffold provides cells with a stable three-dimensional space structure and maintains sufficient porosity for material exchange, growth and metabolism, thereby guiding cartilage formation. Injectable biomaterials have the characteristics of uniform mixing with cells, good fit and connection with repaired tissues, easy shaping, and can be operated through arthroscopy. The treatment is simple, safe and effective, and meets the requirements of modern minimally invasive surgery. Therefore, injectable cartilage tissue engineering products should be the development direction of clinical application.
可注射性壳聚/β甘油磷酸二钠凝胶复合由种异休一软骨细胞修复兔膝关节软骨缺损及威灵仙的干预效应，公开了选用壳聚糖、β-甘油磷酸二钠作为合成支架的材料，用于可注射型C/β-GP复合软骨细胞凝胶的制备。其具体步骤是，将1.2克壳聚糖溶解于60ml0.1mol/l稀盐酸溶液中，室温下充分搅拌混匀，高压蒸汽灭菌消毒10分钟，4℃冰箱保存。2.8克β-甘油磷酸二钠充分溶解于5ml去离子水中，0.22μm滤器过滤除菌，4℃冰箱保存。Injectable chitosan/β-glycerol phosphate disodium gel combined with heterochondrocytes to repair rabbit knee articular cartilage defects and the intervention effect of clematis, disclosed that chitosan and β-glycerophosphate disodium were used as synthetic Scaffold materials for the preparation of injectable C/β-GP composite chondrocyte gels. The specific steps are as follows: dissolving 1.2 grams of chitosan in 60 ml of 0.1 mol/l dilute hydrochloric acid solution, fully stirring and mixing at room temperature, sterilizing with high-pressure steam for 10 minutes, and storing in a refrigerator at 4°C. Dissolve 2.8 g of β-glycerophosphate disodium in 5 ml of deionized water, sterilize with a 0.22 μm filter, and store in a refrigerator at 4°C.
可注射型C/β-GP复合软骨细胞凝胶的制备：将已经制备好的20g/L壳聚糖溶液和560g/Lβ-甘油磷酸二钠溶液在冰浴条件下以7：1混匀，pH计测pH值为7.2，后与软骨细胞混匀备用，细胞浓度为5×1010L-1。该方法的缺点是：（1）需二次手术，第一次手术取自身关节软骨细胞在体外培养，第二次手术再将软骨和支架混合植入缺损区；（2）培养条件苛刻，需要相应认证的实验室进行；（3）感染和传染病毒的风险大，在培养时需要用到完全培养基，完全培养基内含有动物血清，感染和传染病毒的风险增大。Preparation of injectable C/β-GP composite chondrocyte gel: mix the prepared 20g/L chitosan solution and 560g/L β-glycerol phosphate disodium solution at 7:1 under ice bath conditions, The pH value measured by the pH meter was 7.2, and then mixed with chondrocytes for later use, and the cell concentration was 5×1010L-1. The disadvantages of this method are: (1) Second surgery is required, the first surgery takes its own articular chondrocytes to culture in vitro, and the second surgery mixes cartilage and scaffolds into the defect area; (2) The culture conditions are harsh and require Correspondingly certified laboratories; (3) The risk of infection and transmission of viruses is high, and complete medium is required for cultivation. The complete medium contains animal serum, and the risk of infection and transmission of viruses increases.
发明内容Contents of the invention
为了解决上述技术问题，本发明提供了一种可吸收软骨修复系统，该系统包括可吸收软骨修复支架和自体血，可吸收软骨修复支架的主要成分为壳聚糖、甘油磷酸盐、pH值调节剂、渗透压调节剂和水。该系统在人体生理温度（37℃）下成凝胶状，修补于软骨缺损部位，可实现软骨组织的修复。In order to solve the above-mentioned technical problems, the present invention provides an absorbable cartilage repair system, which includes an absorbable cartilage repair scaffold and autologous blood. The main components of the absorbable cartilage repair scaffold are chitosan, glycerol phosphate, pH value adjustment agent, osmotic pressure regulator and water. The system becomes gelatinous at the physiological temperature of the human body (37°C), and repairs the cartilage defect to realize the repair of cartilage tissue.
可吸收软骨修复支架的主要成分中，壳聚糖0.1%～5%，甘油磷酸盐0.1%～10%，pH值调节剂0.01%～5%，渗透压调节剂0.001%～2%，余量为水（以上的比例为质量浓度百分比）。Among the main components of the absorbable cartilage repair scaffold, chitosan is 0.1% to 5%, glycerol phosphate is 0.1% to 10%, pH regulator is 0.01% to 5%, osmotic pressure regulator is 0.001% to 2%, and the balance is For water (the ratio above is the mass concentration percentage).
上述的原料中，壳聚糖其脱乙酰度应为不小于70%。壳聚糖(Chitosan)又名甲壳胺； 脱乙酰甲壳质；几丁聚糖； 脱乙酰几丁质；聚氨基葡糖，为虾壳、蟹壳提取的甲壳素经脱乙酰的产物。壳聚糖生物相容性好、无毒、无免疫原性，在医疗、制药、化妆品和食品领域有诸多应用。在医疗领域中，壳聚糖具有止血、促进伤口愈合、防止病理性粘连、抗菌、抗真菌和抗肿瘤性能。Among the above raw materials, the degree of deacetylation of chitosan should be not less than 70%. Chitosan (Chitosan), also known as chitosan; deacetylated chitin; chitosan; deacetylated chitin; Chitosan has good biocompatibility, non-toxicity and non-immunogenicity, and has many applications in medical, pharmaceutical, cosmetic and food fields. In the medical field, chitosan has hemostasis, promotes wound healing, prevents pathological adhesions, antibacterial, antifungal and antitumor properties.
壳聚糖可被诸如溶菌酶的某些特定酶降解成氨基葡萄糖和乙酰氨基葡萄糖，最终进入人体的糖代谢循环中，是一种良好的可吸收生物大分子。Chitosan can be degraded into glucosamine and acetylglucosamine by certain specific enzymes such as lysozyme, and finally enters the carbohydrate metabolism cycle of the human body, and is a good absorbable biomacromolecule.
壳聚糖可用于止血，壳聚糖为带正电荷的大分子多糖，和红细胞膜表面的阴离子结合使红细胞凝集，同时激活血小板聚集，活化凝血酶，从而达到快速止血的目的。Chitosan can be used for hemostasis. Chitosan is a positively charged macromolecular polysaccharide that combines with anions on the surface of red blood cell membranes to agglutinate red blood cells, activate platelet aggregation, and activate thrombin to achieve rapid hemostasis.
本发明所述组合物的甘油磷酸盐为甘油磷酸钠、甘油磷酸钾、甘油磷酸钙、甘油磷酸铁的一种或几种组合。甘油磷酸盐是细胞壁磷壁酸的主要成分之一，可以作为人体磷补充剂，用以满足人体每天对磷的需要。甘油磷酸参与骨质的形成，以磷脂形式参与细胞膜的组成，同时磷与许多代谢中的酶活性有关，在能量代谢中的作用至关重要。The glycerophosphate in the composition of the present invention is one or more combinations of sodium glycerophosphate, potassium glycerophosphate, calcium glycerophosphate and iron glycerophosphate. Glycerophosphate is one of the main components of cell wall teichoic acid, which can be used as a phosphorus supplement for the human body to meet the daily phosphorus needs of the human body. Glycerophosphate is involved in the formation of bone, and participates in the composition of cell membranes in the form of phospholipids. At the same time, phosphorus is related to the enzyme activities in many metabolisms, and plays an important role in energy metabolism.
本发明所述的组合物pH值应为6.0～8.0之间，pH值调节剂为盐酸、硫酸、磷酸、硝酸、醋酸、柠檬酸、琥珀酸、酒石酸的一种或几种组合。The pH of the composition of the present invention should be between 6.0 and 8.0, and the pH regulator is one or more combinations of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, citric acid, succinic acid, and tartaric acid.
本发明所述的组合物渗透压应为160mOsm/Kg～360mOsm/Kg，渗透压调节剂为氯化钠、磷酸氢二钠、磷酸二氢钠、氯化钙、氯化钾、乳酸钠、醋酸钠的一种或几种组合。The osmotic pressure of the composition of the present invention should be 160mOsm/Kg～360mOsm/Kg, and the osmotic pressure regulator is sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, calcium chloride, potassium chloride, sodium lactate, sodium acetate one or a combination of several.
本发明所述的组合物经制备后可以形成一种稳定的水凝胶，水凝胶是高度水合的大分子网络，该组合物的大分子网络为壳聚糖。该制备方法包括以下步骤：将壳聚糖溶解于酸溶液中，将甘油磷酸盐溶解于水中，将二者混合形成复合溶液，将复合溶液的pH调节至6.0～8.0，再加入渗透压调节剂将所述复合溶液的渗透压调节至160mOsm/Kg～360mOsm/Kg。如此形成的复合溶液即为可吸收软骨修复支架，其和自体血混合后，在升温到37℃可形成稳定的水凝胶，该水凝胶即为本发明所述的可吸收软骨修复系统。The composition of the invention can form a stable hydrogel after being prepared, and the hydrogel is a highly hydrated macromolecular network, and the macromolecular network of the composition is chitosan. The preparation method comprises the following steps: dissolving chitosan in acid solution, dissolving glycerol phosphate in water, mixing the two to form a composite solution, adjusting the pH of the composite solution to 6.0-8.0, and adding an osmotic pressure regulator Adjust the osmotic pressure of the composite solution to 160mOsm/Kg-360mOsm/Kg. The composite solution formed in this way is the absorbable cartilage repair scaffold. After it is mixed with autologous blood, it can form a stable hydrogel when the temperature is raised to 37°C. The hydrogel is the absorbable cartilage repair system of the present invention.
修复支架具体的配制方法是，配制质量百分比为0.1%～5%壳聚糖溶液，将上述的壳聚糖溶液置于0.1M～10M酸性溶液中，得混合溶液；The specific preparation method of the repair bracket is to prepare a chitosan solution with a mass percentage of 0.1% to 5%, and place the above chitosan solution in a 0.1M to 10M acidic solution to obtain a mixed solution;
配制质量百分比为0.1%～5%0.1%～20%甘油磷酸盐水溶液；Prepare a mass percentage of 0.1% to 5% and 0.1% to 20% glycerol phosphate aqueous solution;
将混合溶液与甘油磷酸盐水溶液按0.1～10:1的体积比例混合，调节pH至6.0～8.0，再加入渗透压调节剂调整渗透压为160mOsm/Kg～360mOsm/Kg，得可吸收软骨支架。Mix the mixed solution with glycerophosphate aqueous solution at a volume ratio of 0.1 to 10:1, adjust the pH to 6.0 to 8.0, and then add an osmotic pressure regulator to adjust the osmotic pressure to 160mOsm/Kg to 360mOsm/Kg to obtain an absorbable cartilage scaffold.
修复系统中可吸收软骨支架与自体血的体积比为1-20：1，它们相结合后，在人体生理温度37℃下成凝胶状，修补于软骨缺损部位，实现软骨组织的修复。The volume ratio of the absorbable cartilage scaffold to autologous blood in the repair system is 1-20:1. After they are combined, they will form a gel at the physiological temperature of the human body at 37°C, and repair the cartilage defect to realize the repair of cartilage tissue.
本发明的可吸收软骨修复系统植入人关节损伤部位，在人体生理温度条件下形成稳定的水凝胶，具有促进软骨组织修复的作用。The absorbable cartilage repairing system of the present invention is implanted in human joint injury sites, forms stable hydrogel under physiological temperature conditions of human body, and has the function of promoting cartilage tissue repairing.
本发明所述的可吸收软骨修复系统利用壳聚糖可吸收软骨修复支架和自体血混合后形成支架凝胶，直接植入软骨损伤区域，利用自体血的各种生长因子和壳聚糖诱导软骨细胞及粘附软骨细胞的作用，加快软骨组织的修复和再生，一次微创手术即可完成。解决了关节软骨没有血液供应、为软骨细胞生长提供稳定三维空间和微创手术完成软骨修复等诸多临床难题。The absorbable cartilage repair system of the present invention utilizes the chitosan absorbable cartilage repair scaffold and autologous blood to form a scaffold gel after being mixed with autologous blood, which is directly implanted in the damaged cartilage area, and utilizes various growth factors of autologous blood and chitosan to induce cartilage The role of cells and adherent chondrocytes can speed up the repair and regeneration of cartilage tissue, which can be completed in one minimally invasive operation. It solves many clinical problems such as lack of blood supply to articular cartilage, providing a stable three-dimensional space for chondrocyte growth, and minimally invasive surgery to complete cartilage repair.
可吸收软骨修复支架用于软骨修复具有以下优点：Absorbable cartilage repair scaffolds for cartilage repair have the following advantages:
1. 无菌、无热原、无免疫原性，生物相容性良好，可降解吸收；1. Sterile, non-pyrogenic, non-immunogenic, good biocompatibility, degradable and absorbable;
2. 利用壳聚糖作为支架可诱导软骨细胞并粘附其表面；2. Using chitosan as a scaffold can induce chondrocytes and adhere to their surface;
3. 壳聚糖和自体血混合可形成稳定的支架凝胶，植入骨缺损区域稳定可靠，自体血内的生长因子可促进软骨细胞的生长，缩短软骨组织的形成时间；3. The mixture of chitosan and autologous blood can form a stable scaffold gel, which is stable and reliable for implantation in bone defect areas. Growth factors in autologous blood can promote the growth of chondrocytes and shorten the formation time of cartilage tissue;
4. 一次微创手术即可完成软骨修复，节省医生时间，减少病人的痛苦；4. Cartilage repair can be completed in one minimally invasive operation, which saves time for doctors and reduces pain for patients;
5. 操作过程简单，微生物感染几率小；5. The operation process is simple, and the probability of microbial infection is small;
6. 相对于背景技术中提供的方法，本发明为可吸收软骨修复支架和自体血混合，仅需要一次手术，无需体外培养，不存在免疫原性及感染和传染动物病毒的风险。6. Compared with the method provided in the background technology, the present invention mixes the absorbable cartilage repair scaffold with autologous blood, requires only one operation, does not require in vitro culture, and does not have the risk of immunogenicity and infection and infection of animal viruses.
具体实施方式detailed description
下面通过具体实施例对本发明进行进一步阐述，以便本领域技术人员更了解本发明，但并不因此限制本发明。The present invention will be further described below through specific examples, so that those skilled in the art can better understand the present invention, but the present invention is not limited thereby.
实施例1Example 1
可吸收软骨修复支架制备方法具体如下步骤：The preparation method of the absorbable cartilage repair scaffold is as follows:
配制1%的壳聚糖于0.2mol/L 醋酸溶液中，配制10%的磷酸甘油钾水溶液，称取1%壳聚糖溶液20g，滴加10%β-磷酸甘油钾12.0g，边加边搅拌，滴加完毕后用醋酸调节pH为7.54，然后加入醋酸钠适量调节渗透压为300 mOsm/Kg，继续搅拌30min。该混合液2ml，加入1ml大鼠血液，混合均匀后，置于37℃的水浴箱中，即可成凝胶状的可吸收软骨修复支架。Prepare 1% chitosan in 0.2mol/L acetic acid solution, prepare 10% aqueous solution of potassium glycerophosphate, weigh 20g of 1% chitosan solution, add 12.0g of 10% β-potassium glycerophosphate dropwise, add as you go Stir, after the dropwise addition, adjust the pH to 7.54 with acetic acid, then add an appropriate amount of sodium acetate to adjust the osmotic pressure to 300 mOsm/Kg, and continue stirring for 30 minutes. Add 1ml of rat blood to 2ml of the mixture, mix evenly, and place in a water bath at 37°C to form a gel-like absorbable cartilage repair scaffold.
实施例2Example 2
配制2%的壳聚糖于0.1mol/L 盐酸溶液中，配制7.5%的磷酸甘油钠水溶液，称取2%壳聚糖溶液40g，滴加7.5%β-磷酸甘油钠36g，边加边搅拌，滴加完毕后用盐酸调节pH为6.56，然后加入氯化钠调节渗透压为310 mOsm/Kg，继续搅拌30min。该混合液5ml，加入1ml兔血液，混合均匀后，置于37℃的水浴箱中，即可成凝胶状可吸收软骨修复支架。Prepare 2% chitosan in 0.1mol/L hydrochloric acid solution, prepare 7.5% sodium glycerophosphate aqueous solution, weigh 40g of 2% chitosan solution, add 36g of 7.5% β-sodium glycerophosphate dropwise, and stir while adding After the dropwise addition, adjust the pH to 6.56 with hydrochloric acid, then add sodium chloride to adjust the osmotic pressure to 310 mOsm/Kg, and continue stirring for 30 minutes. Add 1ml of rabbit blood to 5ml of the mixed solution, mix evenly, and place in a water bath at 37°C to form a gel-like absorbable cartilage repair scaffold.
实施例3Example 3
配制磷酸缓冲液：取磷酸氢二钠71.64g，加水溶解成1000ml，作为甲液。另取磷酸二氢钠27.60g，加水溶解成1000ml，作为乙液。取甲液81ml与乙液19ml，混匀，调节pH值至7.3。Prepare phosphate buffer solution: Take 71.64g of disodium hydrogen phosphate, add water and dissolve it into 1000ml, and use it as solution A. Take another 27.60 g of sodium dihydrogen phosphate, add water and dissolve it to 1000 ml, and use it as liquid B. Take 81ml of solution A and 19ml of solution B, mix well, and adjust the pH value to 7.3.
配制4%的壳聚糖于0.1mol/L 磷酸溶液中，配制15%的磷酸甘油钠水溶液，称取4%壳聚糖溶液30g，滴加15%β-磷酸甘油钠25g，边加边搅拌，滴加完毕后用磷酸调节pH为6.56，再加入以上磷酸缓冲液调节渗透压为302 mOsm/Kg继续搅拌30min。该混合液10ml，加入1ml人血液，混合均匀后，置于37℃的水浴箱中，即可成凝胶状可吸收软骨修复支架。Prepare 4% chitosan in 0.1mol/L phosphoric acid solution, prepare 15% sodium glycerophosphate aqueous solution, weigh 30g of 4% chitosan solution, add 25g of 15% β-sodium glycerophosphate dropwise, and stir while adding After the dropwise addition, adjust the pH to 6.56 with phosphoric acid, then add the above phosphate buffer to adjust the osmotic pressure to 302 mOsm/Kg and continue stirring for 30 min. Add 1ml of human blood to 10ml of the mixed solution, mix evenly, and place in a water bath at 37°C to form a gel-like absorbable cartilage repair scaffold.
实施例4Example 4
大鼠软骨细胞制备：取4～5周龄，体重185～200的SD大鼠，断颈处死， 无菌条件下切取双侧髋膝关节软骨，放入培养皿，浸入L-DMEM培养基中漂洗数次，剪碎，0.25%的胰蛋白酶消化，经80目筛网过滤后，加入L-DMEM完全培养基培养。待细胞融合为单层时，用胰蛋白酶消化并进行细胞传代。Preparation of rat chondrocytes: SD rats aged 4 to 5 weeks and weighing 185 to 200 were killed by dislocation of the neck, and bilateral hip and knee articular cartilages were excised under aseptic conditions, put into a petri dish, and immersed in L-DMEM medium Rinse several times, cut into pieces, digest with 0.25% trypsin, filter through 80-mesh sieve, add L-DMEM complete medium for culture. When the cells were confluent into a monolayer, they were digested with trypsin and the cells were passaged.
取实施例1混合溶液和新鲜大鼠血清按1:1的比例混合均匀，将上述大鼠软骨细胞混悬其中，加入6孔培养板中的3孔，每孔1ml，放于37℃孵箱15min形成凝胶后，每孔加入4mlL-DMEM完全培养基进行培养，其余3孔直接加入大鼠软骨细胞液1ml和4mlL-DMEM完全培养基进行培养。结果表明，加入实施例1混合溶液和新鲜大鼠血清的样品软骨细胞倍增时间为5天，而未加的样品软骨细胞倍增时间为7天，说明本发明组合物具有促进软骨生长的作用。Take the mixed solution of Example 1 and fresh rat serum and mix evenly at a ratio of 1:1, suspend the above-mentioned rat chondrocytes in it, add 1ml to 3 wells of a 6-well culture plate, and place in a 37°C incubator After 15 minutes of gel formation, add 4ml L-DMEM complete medium to each well for culture, and directly add 1ml of rat chondrocyte liquid and 4ml L-DMEM complete medium for culture in the remaining 3 wells. The results showed that the chondrocyte doubling time of the sample added with the mixed solution of Example 1 and fresh rat serum was 5 days, while the chondrocyte doubling time of the sample without adding was 7 days, indicating that the composition of the present invention has the effect of promoting cartilage growth.
实施例5Example 5
将成年兔膝关节内侧纵向切口，逐层切开皮肤、皮下组织及关节囊，牵拉髌骨使向外侧脱位，显露股骨滑车关节面，用直径4mm的钻头在右股骨内外髁，左股骨内髁造出直径0.6cm，深度0.2cm全层厚软骨缺损模型。Make a longitudinal incision on the medial side of the knee joint of adult rabbits, cut the skin, subcutaneous tissue and joint capsule layer by layer, pull the patella to dislocate laterally, and expose the articular surface of the femoral trochlear. A full-thickness cartilage defect model with a diameter of 0.6 cm and a depth of 0.2 cm was created.
取实施例1混合溶液0.5ml，加入新鲜兔血2.5ml，混合均匀，通过外科手术植入兔后腿软骨缺损模型区，等待15min即形成完全凝胶，缝合伤口。6周后再次观察软骨缺损区，表明平整光滑，与周围正常软骨难以区分，色泽为瓷白色。采用Wakitani组织学评分方法对软骨缺损区进行评分，结果6周后兔后腿软骨缺损模型区评分为4.1±0.8。Take 0.5ml of the mixed solution of Example 1, add 2.5ml of fresh rabbit blood, mix evenly, implant into the rabbit hind leg cartilage defect model area through surgery, wait for 15 minutes to form a complete gel, and suture the wound. After 6 weeks, the cartilage defect area was observed again, which showed that it was smooth and indistinguishable from the surrounding normal cartilage, and its color was porcelain white. Wakitani histological scoring method was used to score the cartilage defect area. After 6 weeks, the score of the cartilage defect model area in the rabbit hind legs was 4.1±0.8.
实施例6Example 6
将成年羊膝关节内侧纵向切口，逐层切开皮肤、皮下组织及关节囊，牵拉髌骨使向外侧脱位，显露股骨滑车关节面，用直径10mm的钻头在右股骨内外髁，左股骨内髁造出直径0.8cm，深度0.4cm全层厚软骨缺损模型。Make a longitudinal incision on the medial side of the knee joint of adult sheep, cut the skin, subcutaneous tissue and joint capsule layer by layer, pull the patella to dislocate laterally, and expose the articular surface of the femoral trochlear. A full-thickness cartilage defect model with a diameter of 0.8 cm and a depth of 0.4 cm was created.
取实施例3混合溶液0.5ml，加入新鲜羊血3.5ml，混合均匀，通过外科手术植入羊膝关节软骨缺损模型区，等待15min即形成完全凝胶，缝合伤口。6个月再次观察软骨缺损区，表明平整光滑，与周围正常软骨难以区分，色泽为瓷白色。组织学观察发现，6个月后软骨细胞体积较大，数量较多，可见柱状排列，与周围正常软骨组织相近，修复组织和周围软骨整合良好。Take 0.5ml of the mixed solution of Example 3, add 3.5ml of fresh sheep blood, mix well, and implant it into the sheep knee articular cartilage defect model area through surgery, wait for 15 minutes to form a complete gel, and suture the wound. The cartilage defect area was observed again at 6 months, which showed that it was smooth and indistinguishable from the surrounding normal cartilage, and its color was porcelain white. Histological observation found that after 6 months, the chondrocytes were larger in size and more in number, arranged in columns, which were similar to the surrounding normal cartilage tissue, and the repair tissue was well integrated with the surrounding cartilage.
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| CN201610410453.0ACN105944149A (en) | 2016-06-13 | 2016-06-13 | Absorbable cartilage repair system and preparation method thereof |
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| CN201610410453.0ACN105944149A (en) | 2016-06-13 | 2016-06-13 | Absorbable cartilage repair system and preparation method thereof |
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| CN107349478A (en)* | 2017-06-08 | 2017-11-17 | 西安交通大学 | Sodium glycero-phosphate/chitosan/polyethylene glycol sustained-release gel and its production and use |
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