以颗粒软骨构建定型移植体的实验研究

发布时间：2018-05-07 07:12

本文选题：肋软骨 + 实验动物模型　；参考：《第二军医大学》2015年博士论文

【摘要】：【研究背景】软骨组织在整形外科是一种重要移植材料,尤其在鼻整形、耳再造等手术中更是一种不可或缺的重要材料。传统的软骨移植是取整块状软骨,根据需要雕刻和拼接成一定形状后再移植体内。整块状软骨存在翘曲的问题,严重的翘曲有时不得不再次修整,因而影响手术的最终效果。为了克服整块软骨容易翘曲的问题,颗粒软骨应运而生,所谓颗粒软骨是由整块状软骨切成的0.5-2m m之间的颗粒。早在60多年前就曾出现过颗粒软骨应用于临床的报道,它被用于颅骨缺损修复和颌面凹陷的填充,还可以金属模具预构颗粒软骨支架用于耳再造及股骨头再造,均取得较好效果,但随后因造价及感染等原因被硅胶、聚乙烯、聚四氟乙烯等人工材料所取代。人工材料因可随意塑形、可大量制造、避免供区损伤等优势曾经大量使用,但随后暴露出感染、排异、外露等问题,这让作为自体材料的颗粒软骨再次获得外科医生的亲睐。近10多年来颗粒软骨再次得到广泛应用,应用形式除单纯软骨颗粒注射填充外,更常见的是以氧化纤维素膜、自体筋膜或脱细胞真皮支架(Allo Derm)包裹成“沙袋”状结构后用于移植,也有以纤维蛋白胶粘结后使用。颗粒软骨是一种接近理想的移植材料,跟整块状软骨相比,颗粒软骨具有来源丰富、容易制备、易于塑形、不易翘曲等优点;相对人工材料,颗粒软骨完全来源于自体,没有排异反应,而且感染和外露均较少。颗粒软骨虽然具有诸多优点,但若想得到更广泛应用,还存在以下问题:1、颗粒软骨移植物的力学强度能否满足临床需要?在植入初期颗粒软骨是没有力学强度的,而植入后期颗粒软骨移植物力学强度的变化至今尚不明了。2、早期以金属模具塑造颗粒软骨支架的方法因造价高昂、操作复杂和感染率高等问题被弃用,而人工材料的发展却为模具材料提供更多的选择,这些人工材料能否作为模具来构建特定形状的颗粒软骨移植体?3、现有取肋软骨手术的创伤仍然较大,并且软骨颗粒的制备过程也较繁琐。【目的】建立兔肋软骨解剖及生物力学的实验模型。以高分子材料作为模具,构建特定形状的颗粒软骨移植体。检测所构建颗粒软骨移植体的力学性能,观察颗粒软骨移植体力学性能在不同移植时间、不同模具材料的差异及与正常肋软骨的比较。另外,为解决现有取肋软骨手术会在胸部留下较大疤痕的问题,探讨经微创切口切取颗粒软骨的可行性。【方法】(1)对新西兰大白兔进行解剖,观察胸廓及肋骨、肋软骨的结构和排列,测量剥离软骨膜后单根肋软骨的长度及横径;对第4至第7肋软骨进行压缩测试和拉伸测试,得到正常新西兰大白兔的肋软骨的压缩和拉伸破坏强度、应力-应变曲线及弹性模量等参数。(2)以电纺丝技术制备聚乳酸-聚羟基乙酸(PLGA)可吸收模具,以冷冻粉碎热压技术制备高密度聚乙烯(HDPE)不可吸收模具。压缩实验组选取新西兰大白兔12只,手术方法相同,分别切取双侧第6、7、8肋软骨,其中双侧7肋切为4段,3段回植,1段送力学测试;剩余肋软骨全部切成颗粒,分别填充于可吸收及不可吸收模具后回植体内。拉伸实验组的动物数量及手术方法与压缩实验组相同。分别于术后1月、3月、6月取材,对不同样本分别做压缩及拉伸实验。所取样本送组织学检查。(3)通过对现有旋切器械的观察和比对,以计算机辅助设计软件Auto CAD构建数字化旋切刀头模型,通过金属3D打印得到旋切刀头,配合管状刀身、套管、动力装置组装成电动肋软骨旋切器,在离体猪软骨、尸体及家猪上验证旋切器的工作方式,比较不同旋切刀头的切削效率。将切取的软骨碎屑及刀片切取的颗粒软骨以II型胶原酶消化分离出软骨细胞,对软骨细胞作台盼蓝染色计数。【结果】(1)兔拥有与人类类似的胸腔结构及肋软骨分布,多数兔与人类一样拥有12对肋骨,12.5%有13对肋骨;其中第1-7肋通过软骨直接与胸骨相连,称为实肋,第8-10肿软骨不直接与胸骨相连,而是通过韧带在内侧相连并指向剑突,称为虚肋,第11-13肋游离在腹壁中,称为浮肋。肋软骨长度从第1肋至第7肋逐渐增加,而从第8肋至第13肋逐渐减少。实肋的横径明显比虚肋及浮肋的大,实肋中第1肋较其肋软骨均粗,其余实肋粗细基本相同。正常兔肋软骨抗压强度平均12.73±1.06MPa,弹性模量平均27.64±1.88Mpa,极限应变平均63.77%±2.67%;抗拉强度平均3.78±0.48Mpa,弹性模量平均25.94±4.09Mpa,极限应变19.79%±2.38%。(2)以可吸收模具及不可吸收模具成功构建颗粒软骨移植物,软骨颗粒可在模具内结合成一个整体,其中可吸收模具在术后3月时已完全吸收。正常软骨段及颗粒软骨移植物的重量均有所增加,其中颗粒软骨移植物的增加幅度更大;PL GA模具术后1月时有明显溶胀,重量增加超过1倍。在压缩实验组,术后1月、3月、6月可吸收模具颗粒软骨(ADC)的压缩弹性模量为6.63±1.20MPa、8.00±1.94MPa、10.50±1.82MPa,同期不可吸收模具颗粒软骨(NDC)压缩弹性模量为5.07±1.00MPa、10.61±1.80MPa、13.11±2.59MPa,同期ADC与NDC的压缩弹性模量相比无统计学差异(p0.05),但均明显小于术前正常软骨段的压缩弹性模量28.14±1.96MPa。正常软骨段术后压缩模量随时间延长呈增加趋势,1月、3月、6月时压缩弹性模量分别为29.11±3.00MPa、32.45±2.79MPa、32.70±2.56MPa。在拉伸实验组,术后1月、3月、6月ADC抗拉强度分别为0.41±0.06Mpa、0.91±0.25MPa、1.24±0.11MPa,同期NDC的抗拉强度为0.15±0.05MPa、0.85±0.18MPa、1.09±0.10MP。6月ADC的抗拉强度大于NDC,这种差别具有统计学意义(p0.05)。组织学检查显示软骨颗粒被大量纤维组织包裹粘结,其胶原含量随移植时间延长而减少,并且跟整块状软骨相比炎性反应更强。颗粒软骨边缘可见少量的新生软骨。(3)在五种旋切刀头中优选出切削效率最高的椭圆面双直刃侧面开刃的刀头,成功研制肋软骨旋切器。经小切口验证微创旋切软骨技术可行,在手柄或电机带动下,可在肋软骨表面旋转切取螺旋片状软骨碎片。该技术装置已申请国家发明专利,申请号:201510023304.4,已通过初审,待实质性审查。通过该旋切刀切取的软骨碎片,其细胞活率与以刀片切取的颗粒软骨细胞活力相比没有统计学差异。【结论】本研究结果首次构建兔肋软骨解剖及生物力学模型,并以可吸收和不可吸模具构建颗粒软骨移植体,同时测试其力学强度。初期颗粒软骨移植体的强度较弱,随移植时间延长会逐渐增加,并且其抗压性能要明显优于抗拉性能,表明颗粒软骨目前仅可用于强度要求不高的移植部位,在组织缺损部位作为有一定强度的填充材料。借助Auto CAD及快速成形技术,试制成旋切刀头,以此为基础组装成旋切器,分别在尸体及家猪体内成功切取肋软骨碎片,验证了经微创切口直接切取碎软骨技术的可行性。
[Abstract]:[background] cartilage tissue is an important transplant material in plastic surgery, especially in the surgery of rhinoplasty and ear reconstruction. The traditional cartilage transplantation is a piece of cartilage, which is carved and spliced into a certain shape and replanted in a certain shape. The problem of the warpage is the problem of the whole cartilage. Heavy warp sometimes has to be repaired again, thus affecting the final effect of the operation. In order to overcome the problem that the whole cartilage is easy to warp, the granular cartilage comes into being, the so-called granular cartilage is a particle between the 0.5-2m m cut by the whole cartilage. It has been reported that the granular cartilage has been applied to the clinic more than 60 years ago. The repair of skull defects and the filling of the maxillofacial depression can also make the metal die prefabricated granular cartilage scaffold for the ear reconstruction and the femoral head reconstruction, which have achieved good results, but they are then replaced by artificial materials such as silica gel, polyethylene and polytetrafluoroethylene because of the cost and infection. The advantages of injury and other advantages have been used extensively, but then infection, rejection and exposure have been exposed. This makes the granular cartilage of the autogenous material get the attention of the surgeon again. In the past 10 years, the granular cartilage has been widely used. The fascia or acellular dermal scaffold (Allo Derm) is used as a "sandbag" structure and is used for transplantation. It is also used with fibrin glue. Granular cartilage is an ideal material for transplantation. Compared with the bulk cartilage, granular cartilage has the advantages of rich source, easy preparation, easy to shape and not warp easily; relative artificial material, Granular cartilage is completely derived from autologous, no rejection, and less infection and exposure. Although granular cartilage has many advantages, but if it is to be more widely used, there are still some problems: 1, the mechanical strength of granular cartilage grafts can meet the clinical needs? In the early stage of implantation, the granular cartilage is not mechanical strength, and implantation The changes in the mechanical strength of the late granular cartilage grafts are still unknown.2. The early method of molding the granular cartilage scaffold with metal molds was abandoned because of its high cost, complex operation and high infection rate, while the development of artificial materials provided more choices for mould materials. The shape of granular cartilage graft? 3, the trauma of the existing costal cartilage surgery is still large, and the preparation of cartilage granules is more complicated. [Objective] to establish an experimental model of the anatomy and biomechanics of the rabbit costal cartilage. The mechanical properties of the implant were observed, and the physical properties of the grafted cartilage were observed at different time of transplantation, the difference of the different mold materials and the comparison with the normal costal cartilage. In addition, to solve the problem that the existing costal cartilage surgery would leave a large scar on the chest, the feasibility of cutting the granular cartilage through the minimally invasive incision was discussed. [method] (1) new West The Landa rabbit was dissected to observe the structure and arrangement of the chest and ribs, the costal cartilage, and measure the length and transverse diameter of the single rib cartilage after the exfoliation of the cartilage membrane. The compression and tensile strength of the costal cartilage of the normal New Zealand white rabbits, the stress strain curve and the modulus of elasticity were obtained by compression test and tensile test of the fourth to seventh rib cartilage. (2) the preparation of poly (lactic acid polyglycolic acid) (PLGA) absorbable mould with electrospinning technology, and the preparation of high density polyethylene (HDPE) non absorbable mould with freezing crushing hot pressing technology. The experimental group selected 12 New Zealand white rabbits with the same operation methods, respectively, and cut bilateral 6,7,8 costal cartilage respectively, of which the bilateral 7 ribs were cut into 4 segments, 3 segments replanted, 1 The residual costal cartilage was completely cut into particles and filled in the absorbable and non absorbable moulds. The number of animals in the experimental group and the method of operation were the same as those in the compression experimental group. The samples were collected in January, March and June respectively. The compression and tensile tests were performed on the different samples respectively. (3 ) through the observation and comparison of the existing rotary cutting instruments, the computer aided design software Auto CAD is used to construct the digital rotary cutting tool head model, the rotary cutting tool head is obtained through the metal 3D printing, and the tube body, the casing and the power device are assembled into the electric rib cutter, and the working mode of the rotary cutter is verified in the pig cartilage, the corpse and the domestic pig. The cutting efficiency of different rotary cutting knives was compared. The cartilage cells were separated by II collagenase digestion and the cartilage cells were digested by II collagenase, and the cartilage cells were counted by trypan blue staining. [results] (results) rabbits had similar thoracic cavity structure and rib cartilage distribution, most rabbits had 12 pairs of ribs like humans, 12.5% There are 13 pairs of ribs; of which the 1-7 rib is connected directly with the sternum through the cartilage, which is called the real rib. The 8-10 swollen cartilage is not connected directly with the sternum, but is connected by the ligament on the inside and pointing to the sword process. It is called the virtual ribs. The 11-13 rib is free in the abdominal wall and is called the floating ribs. The length of the rib cartilage increases from the first ribs to the seventh ribs, and from the eighth ribs to the thirteenth ribs. The transverse diameter of the real rib is obviously larger than that of the virtual rib and the floating ribs. The first ribs in the real rib are thicker than the costal cartilage, and the rest of the rib are basically the same. The average compressive strength of the normal rabbit costal cartilage is 12.73 + 1.06MPa, the modulus of elasticity is 27.64 + 1.88Mpa, the average of the ultimate strain is 63.77% + 2.67%, the average tensile strength is 3.78 0.48Mpa, and the modulus of elasticity is 25.94 + 4. .09Mpa, the ultimate strain 19.79% + 2.38%. (2) can be successfully constructed with the absorbable die and the non absorbable die. The cartilage particles can be synthesized in the mold. The absorbable die is fully absorbed in March. The weight of the normal cartilage and the granular cartilage graft is increased, among which the grained cartilage is transplanted. The increase of the substance was greater; the PL GA mold was swelling in January, and the weight increased by more than 1 times. In the compression experimental group, the compression modulus of the absorbable die granular cartilage (ADC) was 6.63 + 1.20MPa, 8 + 1.94MPa and 10.50 + 1.82MPa in June, March and June. The compression modulus of the non absorbable die granular cartilage (NDC) was 5.07 + 1.00M at the same time. Pa, 10.61 + 1.80MPa, 13.11 + 2.59MPa, the compression modulus of ADC and NDC had no statistical difference at the same time (P0.05), but it was significantly less than the compression modulus of normal cartilage segment before operation 28.14 + 1.96MPa. normal cartilage segments increased with time. In January, March, and June, the compression modulus was 29.11 + 3.00MPa, respectively. The tensile strength of 32.45 + 2.79MPa and 32.70 + 2.56MPa. in the tensile test group was 0.41 + 0.06Mpa, 0.91 + 0.25MPa and 1.24 + 0.11MPa in June, March and June. The tensile strength of NDC was 0.15 + 0.05MPa, 0.85 + 0.18MPa, and 1.09 + 0.10MP.6 month ADC was greater than NDC, and the difference was statistically significant. Histological examination showed that The cartilage particles were wrapped and bonded by a large number of fibrous tissue, and the collagen content decreased with the prolongation of the transplantation time, and the inflammatory reaction was stronger compared with the bulk cartilage. A small amount of new cartilage was seen on the edge of the granular cartilage. (3) a knife with the highest cutting efficiency with the highest cutting efficiency on the side of the ellipsoid and two straight edges was selected, and the ribs were successfully developed. The technique of minimally invasive circumflex cartilage is proved to be feasible by a small incision and can be rotated on the surface of the rib cartilage by the handle or motor. The technical device has applied for a national patent for invention. The application number: 201510023304.4, it has been examined in the first instance, to be examined in a real quality. There was no statistical difference between the viability of the cells and the vitality of the granular cartilaginous cells taken by the blade. [Conclusion] the results of this study were the first to construct the anatomical and biomechanical models of the rabbit costal cartilage, and to construct the grafted body with absorbable and non absorbable molds and test their mechanical strength at the same time. The strength of the initial granular cartilage graft is weak. The prolongation of the transplantation time will increase gradually, and its compression performance is obviously superior to the tensile property. It shows that the granular cartilage can only be used in the grafts with low strength and is used as a filling material in the tissue defect. With the help of Auto CAD and rapid forming technology, a spin cutting tool is made, which is based on the rotation cutting. The rib cartilage fragments were successfully cut out in cadavers and pigs, which verified the feasibility of directly cutting the cartilage by minimally invasive incision.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R622

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