本文選題：前交叉韌帶 + 聚對(duì)苯二甲酸乙二醇酯��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：前交叉韌帶(anterior cruciate ligament, ACL)損傷是臨床常見(jiàn)的運(yùn)動(dòng)損傷之一。目前臨床上多采用ACL重建術(shù)治療ACL損傷,從而避免內(nèi)側(cè)副韌帶損傷(MCL)、半月板損傷、膝關(guān)節(jié)軟骨損傷等一系列并發(fā)癥。重建材料的選擇大體上分為三種：自體肌腱、異體肌腱和人工韌帶。它們或多或少存在著取材受限、取材部位的并發(fā)癥、免疫排斥與疾病傳播等缺點(diǎn)。而人工韌帶則很大程度上規(guī)避了這些風(fēng)險(xiǎn),因此人工韌帶逐漸開(kāi)始運(yùn)用于臨床�，F(xiàn)在越來(lái)越多的臨床醫(yī)生開(kāi)始接受一種新型人工韌帶---[LARS(Ligament Advanced Reinforcement System)],因?yàn)樗^好的力學(xué)性能和生物相容性,能縮短ACL重建術(shù)后愈合時(shí)間,能盡早實(shí)現(xiàn)患者重返日常生活。 然而,越來(lái)越多的文章開(kāi)始關(guān)注LARS人工韌帶的失敗案例,他們發(fā)現(xiàn)在LARS人工韌帶與宿主骨之間存在阻礙愈合的纖維疤痕存在,這些很可能是由于LARS人工韌帶原材料-—PET材料的生物相容性不高導(dǎo)致的。已經(jīng)有很多文章開(kāi)始關(guān)注于改進(jìn)PET材料的生物相容性,作為首先接觸到宿主的材料表面很大程度上決定了移植材料的生物學(xué)性能,因此通過(guò)表面修飾對(duì)現(xiàn)有材料進(jìn)行改進(jìn)�，F(xiàn)有的材料學(xué)方面,如羥基化、磷酸化材料表面等得到了較為廣泛的運(yùn)用。 早在上世紀(jì)70年代,Larry Hench就發(fā)現(xiàn)了生物玻璃這種人造骨移植材料,它是一種硅酸鹽合成材料,其主要成分在于二氧化硅在混合物中的質(zhì)量比低于60%。由于它能在體內(nèi)形成羥基磷灰石層而廣泛應(yīng)用于被用作為骨性支架來(lái)研究Si02在混合物中的質(zhì)量比在45-52%下能快速的與宿主骨發(fā)生化學(xué)連接,從而發(fā)揮其生物學(xué)性能,雖然質(zhì)量比在55-60%下,較前反應(yīng)稍慢,但其能保持較長(zhǎng)的生物學(xué)活性。58S是生物玻璃家族中的一員,它比一般的生物玻璃有著更高的Ca/P比(CaO32.6%、SiO258.2%、P2059.2%)和緩釋型,因此較多的運(yùn)用在臨床。 本實(shí)驗(yàn)用生物玻璃58S涂層處理PET人工韌帶,希望處理過(guò)的PET人工韌帶能夠有更好的生物活性,能夠促進(jìn)PET韌帶與骨隧道之間的緊密連接,促進(jìn)移植物與骨的愈合,從而提高PET人工韌帶移植的成功率。我們用MC3T3-E1鼠成骨細(xì)胞和半關(guān)節(jié)模型來(lái)驗(yàn)證我們的假設(shè)。 第一部分以明膠為載體的生物玻璃涂層復(fù)合材料的構(gòu)建與測(cè)定 目的構(gòu)建并測(cè)定以明膠為載體的生物玻璃涂層復(fù)合材料。 方法將LARS韌帶骨道端(PET纖維)展平剪成24孔板大小(圖1),同時(shí)制備PET膜片成24孔板大小,將上述材料浸泡在75%的酒精中4h去漬,等離子水清洗,在37℃烘箱下烘干,等離子處理材料表面后,置于按質(zhì)量不同濃度配比(1:2,1:3,1：4,15)的58S與明膠的混合溶液中,磁力攪拌約5min,取出后烘干,隨機(jī)選取涂層前和涂層后各一個(gè)樣本,經(jīng)離子濺射儀表面噴金處理后,用掃描電鏡觀察材料表面特征,選取最佳濃度配比的混合溶液作為涂層溶液進(jìn)行涂層；同樣選取涂層和未涂層組樣本各一,運(yùn)用X射線能譜儀分析涂層前后材料的組成元素變化。余下樣本(n=48)經(jīng)環(huán)氧乙烷消毒后備用。 結(jié)果成功構(gòu)建58S-PET復(fù)合材料,確定質(zhì)量比為1：4的生物玻璃與明膠的混合溶液作為涂層溶液,掃描電鏡觀察材料表面特征能看見(jiàn)在原本光滑的PET材料表面附著了大量顆粒不等的物質(zhì),經(jīng)X射線能譜儀分析結(jié)果顯示,經(jīng)58S涂層處理的PET纖維,其Si、Ca、P元素明顯增高,符合58S的基本元素含量,佐證了在PET纖維上顆粒確為生物玻璃。余下樣本依次經(jīng)環(huán)氧乙烷消毒、DMEM培養(yǎng)液浸泡7天后用于成骨細(xì)胞培養(yǎng)。 結(jié)論成功構(gòu)建出58S-PET復(fù)合材料,有利于實(shí)驗(yàn)進(jìn)一步開(kāi)展。 第二部分58S-PET復(fù)合材料體外成骨細(xì)胞增殖及活性實(shí)驗(yàn) 目的觀察并測(cè)定58S-PET復(fù)合材料相較未經(jīng)處理的PET材料在體外成骨細(xì)胞中的效果。 方法經(jīng)上述處理好的PET纖維及膜片置于24孔培養(yǎng)板中,都加入MC3T3-E1細(xì)胞懸液(密度2×104/m1)。膜片組用倒置顯微鏡觀察72小時(shí)后的涂層組(58S-PET)與未經(jīng)涂層組(PET)之間的細(xì)胞形態(tài)變化,并在1,3,5天進(jìn)行細(xì)胞計(jì)數(shù)。纖維組在第1,3,5天分別進(jìn)行MTT、ALP測(cè)試其生物學(xué)活性及細(xì)胞增殖。 結(jié)果細(xì)胞在培養(yǎng)72小時(shí)后兩組細(xì)胞形態(tài)無(wú)明顯差別,且實(shí)驗(yàn)組(58S-PET)細(xì)胞密度明顯較未經(jīng)處理的對(duì)照組(PET)高；第1,3,5天的細(xì)胞計(jì)數(shù),細(xì)胞數(shù)量在實(shí)驗(yàn)組(58S-PET)和對(duì)照組(PET)均呈現(xiàn)增長(zhǎng)趨勢(shì),但是在第5天實(shí)驗(yàn)組明顯高于對(duì)照組(6.250±0.27,8.92±0.17;P0.05)；同樣,在四唑鹽比色法(MTT)和堿性磷酸酶(ALP)活性測(cè)定中,在涂層組在第3,5d均表現(xiàn)出明顯的優(yōu)勢(shì)(P0.05) 結(jié)論58S-PET復(fù)合材料未表現(xiàn)出對(duì)于成骨細(xì)胞的毒性,且還能明顯提高成骨細(xì)胞的數(shù)量和活性。 第三部分58S-PET復(fù)合材料體內(nèi)生物力學(xué)及組織學(xué)實(shí)驗(yàn) 目的了解58S-PET復(fù)合材料在體內(nèi)的生物力學(xué)及組織學(xué)效果。 方法將24只雄性新西蘭大白兔(體重2.7±0.3kg)并將其隨機(jī)分成A、B兩組：每組12只,造成雙側(cè)半關(guān)節(jié)移植物-骨愈合模型,其中A組植入經(jīng)生物玻璃(58S)涂層處理的PET材料組,作為實(shí)驗(yàn)組(58S-PET)；B組植入未經(jīng)處理組,作為對(duì)照組(PET)；為避免材料之間的相互影響,A、B兩組雙側(cè)均植入同一材料。術(shù)后6,12周分別取材行生物力學(xué)及組織學(xué)檢查。 結(jié)果生物力學(xué)顯示：兩組材料植入后均表現(xiàn)出隨時(shí)間推移最大拔出力均有顯著提高,術(shù)后6周、12周兩組間有顯著性差異(p0.05),實(shí)驗(yàn)組均高于對(duì)照組。實(shí)驗(yàn)組6周(實(shí)驗(yàn)組61.70±6.95N)高于對(duì)照組(45.21±9.78N,p=0.03),隨著材料植入術(shù)后時(shí)間的延長(zhǎng)兩組間最大拔出拉力呈現(xiàn)逐漸顯著的趨勢(shì),實(shí)驗(yàn)組12周(實(shí)驗(yàn)組89.25±9.50N)顯著高于對(duì)照組(71.38±6.26N,p=0.02)組織學(xué)觀察：材料植入后第6周,實(shí)驗(yàn)組及對(duì)照組材料與宿主骨與骨界面模糊,有新骨形成,與宿主骨組織結(jié)合緊密,以實(shí)驗(yàn)組明顯。第12周,兩組植入材料已與宿主骨逐漸融合,新生骨進(jìn)一步增多,移植物-骨愈合得到進(jìn)一步增強(qiáng)。 結(jié)論以58S作為涂層材料改進(jìn)現(xiàn)有PET材料能改善宿主與移植物的愈合能力,縮短愈合時(shí)間。
[Abstract]:Anterior cruciate ligament (ACL) injury is one of the most common sports injuries. At present, ACL reconstruction is used in the treatment of ACL injury, which avoids the complications of medial collateral ligament injury (MCL), meniscus injury, and knee cartilage injury. The selection of reconstruction materials is generally divided into three kinds: autologous tendon, Allogeneic tendons and artificial ligaments. They have more or less defects in material limitation, complications of taking parts, immune rejection and spread of disease. And artificial ligaments largely avoid these risks, so artificial ligaments are gradually starting to apply to the clinic. More and more clinicians are now beginning to accept a new type of artificial toughening. With ---[LARS (Ligament Advanced Reinforcement System)], because of its good mechanical properties and biocompatibility, it can shorten the healing time after ACL reconstruction and make the patient return to daily life as soon as possible.
However, more and more articles begin to pay attention to the failure cases of LARS artificial ligaments. They found that there is a fibrous scar that hinders healing between the LARS artificial ligaments and the host bone. These are probably due to the poor biocompatibility of the raw material of the LARS artificial ligament - the material of the PET. Many articles have begun to focus on the improvement. The biocompatibility of PET material, which is the first contact to the surface of the host material, largely determines the biological properties of the transplanted materials. Therefore, the existing materials are improved by surface modification. The existing materials, such as hydroxylation and phosphorylated materials, have been widely used.
As early as the 70s of last century, Larry Hench discovered the artificial bone graft of Bioglass. It is a silicate synthetic material. Its main component is that the mass ratio of silica in the mixture is less than 60%. because it can form a hydroxyapatite layer in the body, and it should be used as a bone scaffold to study Si02 in the mixture. The mass of the compound can be linked to the host bone quickly with the host bone, and thus exerts its biological performance. Although the mass is slower than that under 55-60%, the longer biological activity of.58S is a member of the Bioglass family, which has a higher Ca/P ratio than the normal Bioglass (CaO32.6%, SiO258.2%). P2059.2% and slow release, so it is more widely used in clinic.
This experiment treated the PET artificial ligament with the 58S coating of Bioglass. It is hoped that the treated PET artificial ligament can have better biological activity, can promote the close connection between the PET ligament and the bone tunnel, promote the healing of the graft and bone, and thus improve the success rate of the transplantation of the PET artificial ligament. We use the MC3T3-E1 mouse osteoblasts and the half joint model. Type to verify our hypothesis.
The first part is the construction and determination of the Bioglass coating composite with gelatin as carrier.
Objective to construct and determine the Bioglass coating composite with gelatin as carrier.
Methods the size of the LARS ligament end (PET fiber) was flattened into 24 orifice plates (Figure 1), and the size of the 24 hole plate was prepared by the PET diaphragm. The above material was soaked in 75% alcohol, 4H was soaked, plasma water was washed, dried at 37 centigrade oven, and after plasma treatment on the surface of the material, 58S and gelatin with different mass ratio (1:2,1:3,1:4,15) were placed on the surface of the material with different mass concentration (1:2,1:3,1:4,15). In the mixed solution, the magnetic force is stirred for about 5min, and then dried, and one sample is randomly selected before and after the coating. After spraying gold on the surface of the ion sputtering instrument, the surface characteristics of the material are observed by scanning electron microscope, and the mixed solution of the best concentration ratio is selected as the coating solution. The samples of the coating and the uncoated group are also selected. First, the changes in the composition of the material before and after coating were analyzed by X ray energy spectrometer. The remaining samples (n=48) were sterilized by ethylene oxide.
Results the 58S-PET composite was successfully constructed and the mixed solution of bio glass and gelatin with a mass ratio of 1:4 was determined as a coating solution. The surface characteristics of the material could be observed on the surface of the original smooth PET material by scanning electron microscope. The results of PET fiber treated by 58S coating showed that the PET fiber was treated by 58S coating. The Si, Ca, and P elements were significantly higher in dimension, which conformed to the basic elements content of 58S. It was confirmed that the particles on PET fiber were truly Bioglass. The remaining samples were sterilized by ethylene oxide in turn and DMEM culture solution was soaked for 7 days to be used for osteoblast culture.
Conclusion 58S-PET composites were successfully constructed, which is conducive to further development of the experiment.
The second part is about the proliferation and activity of 58S-PET composite osteoblasts in vitro.
Objective To observe and determine the effect of 58S-PET composite material on osteoblasts in vitro compared with untreated PET material.
Methods the treated PET fiber and film were placed in the 24 hole culture plate and added MC3T3-E1 cell suspension (density 2 x 104/m1). The cell morphology changes between the coating group (58S-PET) and the uncoated group (PET) were observed by inverted microscope for 72 hours after 72 hours, and the cell count was carried out on 1,3,5 days. The fiber group was respectively entered on the day of 1,3,5. MTT, ALP test its biological activity and cell proliferation.
Results there was no significant difference in cell morphology between the two groups after 72 hours of culture, and the cell density of the experimental group (58S-PET) was significantly higher than that of the untreated control group (PET). The cell count on day 1,3,5, the number of cells in the experimental group (58S-PET) and the control group (PET) showed a growing trend, but in the fifth day group, the experimental group was significantly higher than the control group (6.250 + 0.2). 7,8.92 + 0.17; P0.05); similarly, in four zolium salt Colorimetry (MTT) and alkaline phosphatase (ALP) activity determination, the coating group showed obvious advantages in 3,5d (P0.05).
Conclusion 58S-PET composite does not show toxicity to osteoblasts, and can significantly increase the number and activity of osteoblasts.
The third part is the biomechanics and histology experiment of 58S-PET composites.
Objective to understand the biomechanical and histological effects of 58S-PET composites in vivo.
Methods 24 male New Zealand white rabbits (2.7 0.3KG) were randomly divided into A, B two groups, 12 rats in each group, resulting in bilateral half joint graft bone healing model, and group A was implanted into the PET material group treated with biological glass (58S) coating, as the experimental group (58S-PET); B group was implanted in the untreated group as the control group (PET); to avoid the control group (PET). A and B two groups were implanted with the same material on both sides. Biomechanical and histological examinations were taken at 6,12 weeks after operation.
The results of biomechanics showed that the maximum pulling force of the two groups were significantly improved after the implantation of the two groups, and there were significant differences between the two groups after 6 weeks and 12 weeks after the operation. The experimental group was higher than the control group. The experimental group was 6 weeks (61.70 + 6.95N) higher than the control group (45.21 + 9.78N, p=0.03), with the delay of the time after the material implantation. The maximum pulling pull between the two groups showed a gradual and significant trend. The experimental group was significantly higher than that of the control group (71.38 + 6.26N, p=0.02) in the experimental group (71.38 + 6.26N, p=0.02). The material and the host bone and bone interface were blurred in the experimental group and the control group after sixth weeks of implantation. Twelfth weeks later, the two groups of implant materials were gradually fused with the host bone, and the new bone was further increased, and the graft bone healing was further enhanced.
Conclusion using 58S as the coating material to improve the existing PET materials can improve the healing ability of host and graft and shorten the healing time.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R873

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