【作者】 申晓青；

【导师】 李少萍；

【作者基本信息】 第一军医大学 ， 口腔临床医学， 2005， 硕士

【摘要】 由于生物可降解吸收骨科材料不必二次手术取出,减少了病人的痛苦,同时又不会在人体内形成长期存在的异物,近年来发展迅猛。虽然目前国内外均已有以聚乳酸（PLA）及其衍生物为代表的成品可吸收骨折内固定系统用于临床,但是临床使用后追踪观察发现这些骨折内固定系统仍存在生物降解时间过长、降解速度不理想、与骨折愈合的过程不匹配、降解释放酸性产物引起不良的组织反应和延迟反应等缺点。 甲壳素（CHI）是一种碱性多糖,带正电荷,在体内可被溶菌酶完全降解为对人体无毒的N—乙酰氨基葡萄糖和氨基葡萄糖,具有优异的亲水性、优良的生物相容性和生物可降解性,无毒性、无刺激性,并具有抗菌、消炎、止血等作用,还有一定的骨传导作用,可促进骨愈合,以往主要用作骨缺损的填充材料。 本研究将左旋聚乳酸（PLLA）和甲壳素（CHI）共混,研制出左旋聚乳酸—甲壳素（PLLA/CHI）复合材料,期望通过两种材料特性的互补,从而使复合材料具有更优良的性能。理论上,PLLA/CHI复合材料有以下特点:①由于PLLA分子中含有大量的羟基（—OH）、羧基（—COOH）等活性基团,CHI分子中含有羟基（—OH）、氨基（—NH₂）,二者可通过氢键、酯键、胺键、离子键等多种形式结合,从而在材料分子量增加不多的情况下,使材料的机械强度增大;②PLLA具疏水性,CHI优异的亲水性可使复合材料的吸水率增加,从而加快PLLA的水解;③随着材料的降解,PLLA释放酸性产物,CHI释放氨基葡萄糖等碱性小分子,二者发生中和反应,可避免乳酸堆积引起的非炎性反应,减轻组织反应。更多还原

【Abstract】 Absorbable fracture fixation devices made of biodegradable synthetic polymers have become a focus of many researchers because of their potential advantages, such as free from a secondary operation to remove the devices, no or less foreign body reactions, etc. Although some absorbable fracture fixation systems have been used clinically around the world, most of them made of poly-lactic acid （PLA） and its derivatives, the follow-ups demonstrated not so ideally for many shortcomings of the material, including poor degradation rates and duration, mismatching between the material degradation and the bone healing, and development of severe tissue responses, and so on.Chitin, one of the most abundant polysaccharide in nature, can be degraded into N-acetylglucosamine and amino sugar by lysozyme. With excellent properties as hydrophilic, biocompatibility, biodegradation, non-toxicity, anti-inflammation, homeostasis, osteoconduction, and promotion of bone healing, it is always utilized as filling material for bone defects.In this study, a new kind of composite material, which is made of poly-L-lactic acid （PLLA） and CHI, was produced for the purpose of osteosynthesis. Theoretically, several improvements could be expected to the new material: firstly, high initial mechanical quality because of strong bonding formation between —OH, —COOH of PLLA and — OH, — NH₂ of CHI; secondly, rapid degradation since improvements of the water uptake rate of the composite material by CHI and acceleration of hydrolytic degradation of PLLA; and thirdly, relief of tissue response for the degradation products of PLLA （lactic acid） causing inflammation neutralized by the degradation products of CHI （amino sugar）.Objective:1. To investigate the degradation behavior and mechanism of PLLA/CHI incubated in PBS so as to study the effect of CHI on the degradation of PLLA in vitro.2. To investigate the degradation behavior and mechanism of PLLA/CHI implanted into animal’s body so as to study the effect of CHI on the degradation of PLLA in vivo.3. To observe the tissue response and the degradation outcome of PLLA/CHI implants in vivo.Methods:1. Fifteen PLLA/CHI rods and fifteen PLLA rods with the dimensions of 1.5 mm X3 mmX25 mm were incubated in PBS （pH 7.4） respectively and the test tubes were placed in water bath at 37 °C, 100 rounds per minute vibration. The incubating medium was changed every week after measuring pH value. Three rods of PLLA/CHI and PLLA respectively were selected randomly for measurement of bending strength, viscosity-average molecular weight （Mv）, and mass loss ratio and observation of macroscopic and ultramicroscopic changes at weeks 4, 8,12, 24, and 32.2. Fifteen PLLA/CHI rods and fifteen PLLA rods with the dimensions of 1.5 mm X 3 mm X 25 mm were implanted in the muscle tissues of the back of fifteen rabbits respectively. Three rabbits were sacrificed randomly and the rods of PLLA/CHI and PLLA were collected respectively for measurement of bending strength, Mv, and mass loss ratio and observation of macroscopic and ultramicroscopic changes at weeks 4, 8,12, 24, and 32 after implantation.3. Fifteen PLLA/CHI cubes and fifteen PLLA cubes with the dimensions of 1.5 mm X 3 mm X 3 mm were implanted in the muscle tissues of the back of fifteen rabbits respectively. Three rabbits were sacrificed randomly and the tissue specimens including cubes of implantation and the surrounding tissues were harvested respectively at weeks 4, 8, 12, 24, and 32 and destined for histological observation of the inflammatory reaction, which indicates the biocompatibility of the composites, and the biodegraded fragments of the material.Results:1. pH changes in vitro. The pH value of the degradation medium of PLLA/CHI stayed above 7.0 for the entire length of the study. It decreased sharply in the first 4 weeks, returned at the 5th week, and remained stable until the 14th week, since then it underwent a fluctuant period with slightly low value up to the 28th week. The pH value of the degradation medium of PLLA changed similarly to that of PLLA/CHI except for the first 4 weeks only with a slight decrease, which has statistically significant difference compared to that of PLLA/CHI.2. Degradation in vitro. The effect of CHI on the degradation of PLLA is reverse at different stages: speeding up the degradation at the early stage, and slowing down the degradation at the late stage.Bending strength changes. The bending strength of both PLLA/CHI and PLLA descended abruptly. The half-life of bending strength attenuation of PLLA/CHI was 4 weeks, longer than that of PLLA. The bending strength of PLLA/CHI remained stable from the 4th to the 8th weeks, and descended to 12.5 MPa in 12 weeks, when that of PLLA was less than one fifth of its initial strength.Mv changes. Both PLLA/CHI and PLLA had similar changes. The Mv of both materials declined rapidly in 12 weeks, especially that of PLLA/CHI, but there was an acceleration period from the 8th to the 12th weeks for Mv of PLLA. It was only 20%-30% of the initial Mv left at the 12th week, and around 10% left at the 32nd week, though Mv of PLLA/CHI was higher than that of PLLA since the 24th week.Mass loss ratio changes. Both PLLA/CHI and PLLA had similar changes. Little mass loss ratio change occurred in 12 weeks, but mass loss manifolded for PLLA/CHI since the 12th week and for PLLA since the 24th week. The mass loss ratio of PLLA/CHI was higher than that of PLLA in 24 weeks, and lower than that after 32 weeks.Macroscopic observation. The character of the rods of PLLA shifted from originally achromatic and transparent to chalky, brittle, and easy to be broken up, but no changes found on the surface. The rods of PLLA/CHI was bright brownish initially, then shaded and appeared pores on the surface. With the degradation of the material progressed, the number of the pores manifolded and showed spongy-liked appearance.Ultramicroscopic observation by scanning electron microscopy （SEM）. The rods of PLLA were evenly compact as regular layers, and little change occurred at 4 and 8 weeks. There appeared pores and channels at 12 weeks, then the pores and channels dilated but the margin of the material stayed intact until 32 weeks. The rods of PLLA/CHI appeared slightly irregular with a rigid bonding between particles of CHI and PLLA. Circular gaps around the CHI particles emerged at 4 weeks. With the CHI particles dissociated, many pores and channels presented by 12 weeks, and manifolded, dilated and extended to the margin of the material at 24 weeks. 3. Degradation in vivo. CHI delayed the degradation process of PLLA obviouslyso as to keep the initial stability of the material.Bending strength changes. The bending strength of PLLA/CHI was higher than that of PLLA for the entire length of the study. That of both materials dropped abruptly in 4 weeks. The half-life of bending strength attenuation of PLLA/CHI was 4 weeks, longer than that of PLLA. The bending strength of PLLA/CHI remained stable from the 4th to the 12th weeks, and maintained 30% of its initial strength （43.1 MPa） in 12 weeks, but that of PLLA was less than one fifth of its initial strength at 8 weeks.Mv changes. Both PLLA/CHI and PLLA had similar changes. The Mv of both materials declined rapidly, especially within the first 4 weeks by one half of its initial Mv. However, that of PLLA/CHI was higher than that of PLLA all along, with statistical difference at 4 and 24 weeks.Mass loss ratio changes. Both PLLA/CHI and PLLA had similar changes. The mass loss ratio of PLLA/CHI was lower significantly than that of PLLA all the while. Little mass loss change occurred in the first 12 weeks, but mass of both materials lost manifolded then, and ascended significantly at week 32.Macroscopic observation. With the degradation proceeded, the rods of PLLA appeared from achromatic and transparent to opaque gradually. The material became white and cracked at week 12, then chalkiness with fibrous tissues filling in the crack at week 32. The cortex seemed unchanged. The color of the rods of PLLA/CHI shifted from bright brownish to puce at week 4, then became shaded. Dense light color spots appeared on the surface at week 12, then pits, cracks, and broken. Gaps and fissures presented on the broken surface and developed then.Ultramicroscopic observation by SEM. The section of PLLA remainedunchanged within the first 8 weeks after implantation. There appeared fine channels by 12 weeks, and the channels dilated and deepened by 24 weeks. Meanwhile, fine channels presented at the margin of the material. The pores and channels bestrewed the material and the channels in the inner part of the material extended to the margin in 32 weeks. Circular gaps around a few CHI particles in the rods of PLLA/CHI emerged at week 4. With the CHI particles dissociated, the pores and channels presented, manifolded, dilated and the integrity of the cortex was destroyed at 12 weeks. The pores and channels with different sizes bestrewed the material and evacuation developed after 24 weeks. 4. Observation of the tissue response and the degraded fragments of materialsAll animals recovered well from their operations, and there were no signs of allergenic, toxic, or inflammatory reaction.Histological observation. A thin fibrous tissue layer, with collagenous fibers arranged along the surface of the implant regularly, was found surrounding the cubes of PLLA/CHI or PLLA 4 weeks after implantation. However, the fibrous tissue encapsulation surrounding PLLA/CHI was thicker than that of PLLA, with appearance of renascent capillaries and a few lymphocytes. The encapsulation around both of the materials became thickened then, and there appeared renascent capillaries and a few lymphocytes. More capillaries were found in the periphery of PLLA/CHI, accompanied by macrophage clumps and foam cells. The foreign body reaction to PLLA began to relieve up to 12 weeks, when many lymphocytes were detected in the encapsulation of PLLA/CHI and a layer of macrophages became thickened in the implant-tissue interfaces. There were a few lymphocytes, some piece of birefringent material and a thin layer of foam cells in the implant-tissue interfaces in the encapsulation of PLLA at 32nd week. However, though the layer of foam cells in the implant-tissue interfaces became thinner and birefringent material existed, there were still infiltration of lymphocytes in the encapsulation of PLLA/CHI, macrophages and giant cells.Conclusions:1. CHI played a role in the degradation of PLLA as speeding up the degradation at the early stage, and slowing down the degradation at the late stage in vitro.2. CHI delayed the degradation process of PLLA obviously so as to keep the initial stability of the material in vivo.3. Though the bending strength of PLLA/CHI has not been improved, it manifested resistance to attenuate initially and attenuated quickly after 12 weeks in vivo, which met for the clinical demands.4. There were degraded fragments of both PLLA/CHI and PLLA found until 32 weeks after implantation, and tissue response to both materials was mild.5. PLLA/CHI needs to be improved by application of smaller size of CHI particles and modified processing technique for osteosynthesis purpose.更多还原