Effect of glutaraldehyde cross-linking on the properties of chitosan/hydroxyapatite- gentamicin delayed materials

doi:10.3969/j.issn.2095-4344.2014.30.005

Abstract

Abstract:

BACKGROUND: Cross-linking is a common method for the modification of bone tissue engineering materials, but there are few studies about the effect of cross-linking on properties of drug-loaded artificial bone.
OBJECTIVE: To study the effect of glutaraldehyde cross-linking on mechanical strength, degradation rate and in vitro release behavior of chitosan/hydroxyapatite-gentamicin delayed materials.
METHODS: The chitosan/hydroxyapatite-gentamicin drug-loaded artificial bone and the cross-linked chitosan/hydroxyapatite-gentamicin drug-loaded artificial bone were prepared at 10%, 20% and 30% chitosan mass ratio. The mechanical strength, absorption rate, degradation rate and in vitro release behavior of materials in each group were determined.
RESULTS AND CONCLUSION: The compressive strength of 10%, 20%, 30% chitosan/hydroxyapatite- gentamicin drug-loaded artificial bone was (10.16±1.17) MPa, (28.4±0.64) MPa and (23.28±1.30) MPa, respectively. After cross-linking, the strength was increased to (36.3±1.20) MPa, (51.6±2.08) MPa and (36.9±3.22) MPa, respectively. The absorption rate and degeneration rate were reduced by cross-linking. In the first day of drug release in vitro, the rate of gentamicin released from non-cross-linked 30% chitosan/hydroxyapatite-gentamicin drug-loaded artificial bone was 42.2%, while the rate was decreased to 33.6% after cross-linking. At the following 9 days, the total release of the cross-linked material was lower than that of non-cross-linked material. Glutaraldehyde cross-linking could improve the stability, reduce the degradation rate, and significantly ameliorate the release of artificial bone.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: hydroxyapatites, chitosan, mechanics, biodegradation, environmental

CLC Number:

R318

Zhao Hong-xia. Effect of glutaraldehyde cross-linking on the properties of chitosan/hydroxyapatite- gentamicin delayed materials[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(30): 4783-4789.

Figures/Tables 2

2.1 材料的表面形貌图1是交联前后30%CS/HA-G缓释材料经冷等静压成型后的照片，从外观上观察，经戊二醛交联后材料的颜色由原来的白色变为淡黄色。从交联30% CS/HA-G的扫描电镜照片(图2)中可以看到，CS/HA-G粉体材料为球状和片状的混合体。与非交联材料相比，交联CS/HA-G其粉体粒径有所增大，说明戊二醛的交联作用使得材料颗粒发生了一定程度的团聚。 2.2 载药材料的体外药物释放载药材料的药物突释现象一直以来都是研究者急待解决的一个难题。制剂在进入体内的第1天前后会迅速、大量地释放药物，这种现象被称为突释。给药初期的突释有可能导致血药浓度接近或超过中毒水平，从而产生明显的不良反应[31]。从图3可看出，未经交联的CS/HA-G存在着较明显的突释现象，第1天时间内10%CS/HA-G、20%CS/HA-G和30% CS/HA-G的药物释放量分别达到29.9%，39.0%和42.2%；经戊二醛交联后各种材料第1天内庆大霉素的释放量分别下降至22.3%，30.1%和33.6%。药物缓释体系中药物突释的原因，一般从以下两个方面来解释，首先是因为药物分子和药物载体分子之间的相互作用太弱，导致药物很容易从载体进入释放介质中；其次，药物在药物缓释体系中的分布导致了突释，药物疏松地吸附于材料表层或被包埋在材料表层。在释放初期，药物从材料的孔洞和缝隙中释放出来[31]。 CS/HA-G中壳聚糖/羟基磷灰石载体对药物的控释主要包括：壳聚糖能对药物进行有效包埋，壳聚糖形成的三维网络结构对药物释放有较强的阻滞作用；壳聚糖分子链上的活性基团可与药物成键；纳米羟基磷灰石对药物的有效吸附。以戊二醛作为交联剂对壳聚糖/羟基磷灰石进行交联，主要是对材料中壳聚糖的化学作用。经戊二醛交联后，壳聚糖分子内及分子间的交联度增大，形成的多孔结构变得更加致密，对药物释放能起到更大的阻滞作用，从而降低了药物的突释。药物载体是药物释放体系的重要组成部分，其亲水性也影响了药物的缓释速度。一般多聚物的亲水性越强，突释越大，释放速度越快[32]。交联反应使得壳聚糖的亲水基团氨基参与了成键，减少了材料的亲水基团数目，使材料的吸水性降低，从而降低了药物的突释。体外药物释放实验结果表明交联作用能改善CS/HA-G缓释材料的突释现象，从而减小不良反应，延长释药时间，能达到更好的治疗目的。缓释体系中药物的释药原理主要有溶出、扩散、溶蚀、渗透压及离子交换作用，其中溶出和扩散释药机制远远超过了其他过程。在骨架型药物缓释体系中，药物分散在不溶性骨架材料中，药物释放是通过骨架中弯曲的孔道扩散进行的，药物释放速度取决于药物在骨架材料中的扩散速率[33-34]。缓释体系在长时间的浸泡过程中，材料会因吸水而出现膨胀，膨胀会使得材料的孔道变大，更利于药物的扩散溶出。在CS/HA-G缓释体系中，交联后材料的吸水率下降，因吸水而造成材料膨胀及药物扩散就会比未交联材料更迟缓。因此在长期浸泡过程中，非交联材料的膨胀表现为速率快而时间短地达到膨胀极值，而交联材料则表现为速率慢而时间长地达到膨胀极值。因此，在后期浸泡过程中，当非交联材料已无明显膨胀时，交联材料还会继续膨胀，此时交联材料因膨胀引起的药物扩散就能大于非交联材料。由于有机高分子壳聚糖在CS/HA-G材料的膨胀中占主导地位，因此壳聚糖含量越大，上述这种交联及非交联材料的膨胀行为差异就会越大。因此第9天时，出现了交联30%CS/HA-G的单次释放率高于非交联30%CS/HA-G的现象。而从总释放量来看，各种壳聚糖含量的交联CS/HA-G总释放量均低于相同壳聚糖含量的CS/HA-G，表明交联CS/HA-G将具备更长的有效药物浓度释放时间。 2.3 材料的亲水性能交联对CS/HA-G载药材料的吸水性能有较大影响，从图4可看出交联CS/HA-G的吸水率比未交联CS/HA-G低。这主要有两个原因：首先，交联反应减少了材料的亲水基团。材料中有机相壳聚糖分子侧链上有亲水基团氨基和羟基，戊二醛对材料的交联作用主要在于戊二醛与壳聚糖的氨基形成－C＝N－键，从而使材料的亲水基团数目减少，直接导致了材料的亲水性能减弱。其次，吸水率也与材料的孔隙率及孔径有关，材料的孔隙率和孔径越大，平衡时能容纳的水越多，从而吸水率也越大。交联反应使壳聚糖形成了更为致密的网络结构，材料的孔隙率下降，从而降低了材料的吸水率。从图4还可看出，3种壳聚糖含量材料的吸水率大小为：30%CS/HA-G> 20%CS/HA-G>10%CS/HA-G，交联30%CS/HA-G>交联20%CS/HA-G>交联10%CS/HA-G。这说明材料中无机相与有机相的含量配比也直接影响了材料的吸水率，CS/HA-G复合材料中无机相羟基磷灰石对水分子的吸附及容纳能力远远小于有机相壳聚糖，因此壳聚糖的含量越高，复合材料的吸水性越大。"

2.4 载药材料的机械性能良好的力学性能是组织工程人工骨材料的基本要求。从图5可看出，交联使CS/HA-G的机械强度得到显著提高。壳聚糖含量为10%，20%和30%的CS/HA-G抗压强度分别为(10.16±1.17)，(28.40±0.64)，(23.28±1.30) MPa，经戊二醛交联后材料的机械强度都得到了提高，其抗压强度分别达到(36.30± 1.20)，(51.60±2.08)，(36.90±3.22) MPa，这一结果表明，交联有利于改善CS/HA-G人工骨材料的力学性能。同时，从图5还可以看出，无论是否交联，20%CS/HA-G的机械强度均高于对应的10%和30%CS/HA-G。单纯的羟基磷灰石脆性较大、强度较低，研究者常采用有机高分子与羟基磷灰石复合来增加材料的弹性和韧性，从而提高材料的力学性能。壳聚糖与羟基磷灰石的复合有效改善了单纯羟基磷灰石的力学性能。随着壳聚糖含量的增大，复合材料的机械强度也随之增大，因此20%CS/HA-G的抗压强度高于10%CS/HA-G。但胶原、壳聚糖、丝素蛋白等这些天然高分子材料在提取制备过程中已经失去了其在生物组织中特有的三维结构，导致其在生物组织中特有的高机械性能也随之丧失了。所以，壳聚糖含量的进一步增大并不能带给复合材料更高的强度。相反，由于壳聚糖本身力学强度不高，复合物中壳聚糖含量过高反而会导致材料的机械强度降低，导致30%CS/HA-G的抗压强度反低于20%CS/HA-G。因此若单从CS/HA-G人工骨材料的机械强度这一角度考虑，复合材料以20%壳聚糖含量为最佳。 2.5 载药材料的体外降解图6为CS/HA-G在交联前后的体外降解情况，从图中可看到，交联反应降低了材料的降解速率。这主要有3个原因：首先，交联反应减少了材料的亲水基团数目，使材料的吸水性降低，这将减缓材料内部液态环境的物质交换，导致材料的累积失重率降低。其次，戊二醛使壳聚糖发生了分子间或分子内交联，使得材料在降解时需要断裂更多的化学键，从而使降解变得更困难。第三，交联反应使壳聚糖形成了更为致密的网络结构，使与壳聚糖复合的羟基磷灰石更难溶解或扩散到浸泡液中。在浸泡60 d时，未经交联10%，20%，30%CS/HA-G的降解失重率分别为24.2%，40.8%，48.0%，而交联CS/HA-G的失重率降低至20.5%，27.5%，30.8%，这一实验结果表明戊二醛交联使材料获得了更好的稳定性能。"

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