CN101829354B - 一种微囊缓释中药复合磷酸钙骨水泥的制备方法 - Google Patents
一种微囊缓释中药复合磷酸钙骨水泥的制备方法 Download PDFInfo
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Abstract
本发明提供一种微囊缓释中药复合磷酸钙骨水泥的制备方法,微囊是以***胶和β-环状糊精混合物作为壁材,采用羟基磷灰石和磷酸三钙的CPC固相,蒸馏水为液相,固-液比为2.5g∶1.6ml,将微囊包裹的杜仲提取物与固-液相混合,搅拌溶解,在复合物即将硬化前填入磨具,固化成型。该方法有效地提高了CPC的成骨性能,而微囊化能提高杜仲稳定性,缓释内容物释放,使液体药物固化并使药物浓度集中于靶区。因此,杜仲叶提取物的载入对CPC理化性能无明显影响及CPC/杜仲叶提取物复合材料具有显著促进成骨细胞增殖的作用,可在制备人工关节材料中应用。
Description
技术领域
本发明属于材料科学、生物医学和中医药学的交叉领域,具体涉及一种以微囊缓释中药复合磷酸钙骨水泥的制备方法,经缓释微囊包裹的杜仲提取物复合磷酸钙骨水泥可提高磷酸钙骨水泥的成骨功效,较好地充填、修复、替换骨组织的缺损。
技术背景
磷酸钙骨水泥(Calcium Phosphate Cement CPC)自80年代中期问世以来因其具有良好的生物相容性、可注射性、骨传导性并可降解,以及可即时随意塑形性已成为骨缺损临床修复的常用材料。但因磷酸钙骨水泥本身不具有骨诱导性,且其降解速度慢于新骨的爬行替代,可导致材料强度的降低,故作为一种良好的载体近年来将它复合其他材料或一些生物因子进行改性的研究层出不穷,而与中药复合共同发挥作用的研究报道稀少。杜仲具有补肝肾、强筋骨的功效,研究表明其提取物对成骨细胞的增殖具有明显的促进作用。但单纯以中药提取物复合骨修复材料极易使药物作用较快流失而不易持久作用于靶细胞,且理化性能不稳定,易受外界影响而分解,因此本研究小组采用缓释微囊包裹杜仲提取物以不同比例复合磷酸钙骨水泥修复兔股骨髁贯穿性缺损,并与普通CPC相比较,确定了复合材料成骨性能及适合比例。
发明内容
本发明的目的是提供一种微囊缓释中药复合磷酸钙骨水泥的制备方法,该方法有效地提高了CPC的成骨性能。
本发明包括如下步骤:
(1)微囊的制备:将***胶和β-环状糊精混合物以质量比1∶1配成溶液,作为微囊壁材溶液;
(2)杜仲微囊粉末制备:称取适量杜仲提取物,用少量酒精溶解后,与微囊壁材溶液按照质量比1∶10混合,将混合溶液加热至60℃左右,经高压均质处理;控制进风温度150℃,出风温度90℃,流量25mL/min,对均质处理的溶液进行喷雾干燥,得到杜仲微囊粉末;
(3)复合磷酸钙骨水泥的制备:以羟基磷灰石和磷酸三钙按质量比1∶1为固相物,取该干粉2.5g,在液相固化液蒸馏水1.6ml中加入0.608mg/ml的杜仲微囊粉末混合,溶解后加入干粉充分搅拌,约2分钟后混合物开始固化成面团状,趁未硬前将其填入模具中,用小铲压平,五分钟后硬化倒出,收集后分别以聚乙烯袋独立包装,环氧灭菌。
本发明的另一个目的是提供所述的微囊缓释中药复合磷酸钙骨水泥在制备人工关节材料中的应用。
所述的中药为杜仲提取物,其有效成分对成骨细胞成骨性能具有促进作用。微囊是以***胶和β-环状糊精混合物作为壁材,所采用的CPC固相成分为质量比1∶1的羟基磷灰石和磷酸三钙,液相部分为蒸馏水,固-液比例为2.5g∶1.6ml,将微囊包裹的杜仲提取物溶解于CPC液相,再与CPC固相充分搅拌混合成复合物,在复合物即将硬化前填入事先所设磨具,固化成型。
磷酸钙骨水泥有骨传导性但不具有骨诱导性,降解时间较长,而自古来中草药对于骨修复有着卓越的成效。杜仲(Euconnia ulmoides Oliv)是我国特有的名贵中药材,始载于《神农本草经》,药用部位为干燥树皮,其味甘、微辛,性温,归肝肾经,补肝肾、强筋骨,为骨伤科常用药物之一。现代医学研究证实了杜仲叶中含有几十种药用有效成分,如绿原酸、京尼平苷、桃叶珊瑚甙、生物碱、黄酮类、内脂等,杜仲叶提取物的载入对CPC理化性能无明显影响及CPC/杜仲叶提取物复合材料具有显著促进成骨细胞增殖的作用。
但杜仲叶提取物有如下缺点:1.一些成分如绿原酸等的性质很不稳定,提取后易被氧化降解;2.其中的黄酮类物质大都是醇溶性物质,遇水难溶;3.杜仲提取物局部使用很快被稀释和代谢,故需反复大剂量使用。而微囊化能提高药物稳定性,缓释内容物释放,使液体药物固化并使药物浓度集中于靶区。因此,制备微囊应用其缓释功效使杜仲提取物在CPC中持续缓慢释放在本实验研究中具有关键意义。
附图说明
图1为术后8周、12周X线图。
图2为术后4周病理切片图。
图3为术后4周电镜图。
图4为术后4周B组BMP因子的免疫组化图。
图5为术后4周B组VEGF因子的免疫组化图。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1
1.1 材料:
1.1.1 磷酸钙骨水泥(CPC):羟基磷灰石和磷酸三钙按质量比1∶1为固相物干粉2.5g和固化液(蒸馏水1.6ml/支)组成;
1.1.2 杜仲提取物制备:按照药典颁布的方法,将100g杜仲叶和800g 50%浓度的乙醇(按1∶8比例)混合,置于烧瓶中加热2次,合并提取液,将提取液中溶剂回收,转溶于水将其过滤出水溶液和沉淀,再将水溶液用石油醚萃取得萃取液和水溶液,回收萃取液,得浸膏,干燥,粉碎成粉状干物质,即为杜仲提取物;
1.1.3 杜仲微囊制备:将壁材(***胶和β-环状糊精混合物配比1∶1)配成溶液,称取适量杜仲提取物,用少量酒精溶解后,与壁材溶液按照1/10混合,将溶液加热至60℃左右,经高压均质处理。控制进风温度150℃,出风温度90℃,流量25mL/min,对均质处理的溶液进行喷雾干燥,所得干燥的杜仲微胶囊为黄色固体粉末;
1.1.4 CPC/杜仲微囊复合物制备:CPC固化液中加入0.304mg/ml的杜仲微囊粉末,溶解后,与CPC干粉充分搅拌,约2分钟后混合物开始固化成面团状,在混合物即将硬化前将其填入直径为6mm,高度为4mm的模具中,用小铲压平,五分钟后硬化倒出,收集后分别以聚乙烯袋独立包装,环氧灭菌后作为CPC+杜仲1组(实验组A);而加入0.608mg/ml的杜仲微囊粉末同前步骤作为CPC+杜仲2组(实验组B)。
1.2 修复骨缺损
1.2.1 建立股骨髁缺损模型 健康雄性新西兰大白兔16只,体重2.5±0.5kg,年龄1周岁左右。标准饲料术前喂养2周。8%水合氯醛4ml/kg腹腔注射麻醉后,动物仰卧位,剪去膝内侧被毛,常规消毒铺巾。取膝内侧3cm纵行切口,逐层分离达股骨内髁,平髌骨下缘,垂直股骨髁钻一直径6mm、深约12mm的贯穿性骨洞。用生理盐水将骨屑和血块冲洗净,干纱布填塞保持创面干燥,两侧股骨均就此造模。
1.2.2 动物分组 采用平衡不完全区组实验设计方法,同一兔左右侧股骨缺损采用不同材料填充或不予填充(空白),分别为CPC+杜仲1组(实验组A);CPC+杜仲2组(实验组B);CPC组(对照组C);不予填充组为(空白组D)。将16只兔随机分为4组(n=4),每组4只,即左CPC/右空白组、左CPC+杜仲1组/右CPC组、左CPC+杜仲1组/右CPC+杜仲2组及左CPC+杜仲2/右空白组。
1.2.3 术后处理 为预防感染,所有动物术后伤口碘伏纱布湿覆并40万单位青霉素肌注3天,每日一次。浙大动物房统一饲料分笼喂养。于术后2、4、8、12周每组各处死1只兔子,每次4只,取出双侧完整股骨,做以下检测。
1.3 观察指标
1.3.1 术后观察 动物饮食、患肢活动及伤口变化。1.3.2 X线 分别在术后2、4、8、12周摄片观察四组植入材料吸收及新骨长成情况。
1.3.3 组织学观察 垂直股骨纵轴截取包括植入材料在内大小约为1.2mm×6mm×4mm的标本32枚,10%的中性甲醛固定,常规脱钙,石蜡包埋,沿骨纵轴切5~10um厚组织切片,HE染色,光镜下观察成骨及材料降解情况。
1.3.3 扫描电镜观察 于术后4周、8周截取标本2.5%的戊二醛4℃固定,磷酸缓冲液漂洗,1%锇酸固定,梯度酒精脱水,醋酸异戊酯处理,临界点干燥、喷金,在XL30ESEM型电镜中观察。
1.3.4 免疫组化 将术后2、4、8周每标本切取5um厚免疫组化染色切片各2张,在20×20视野,每张切片随机取10个视野,利用MIAS-300型真彩医用图像分析仪测量切片中BMP-2、VEGF 2种骨诱导因子染色平均深度(灰度值)
2 结果
2.1 术后观察
除有两只兔子一周内死亡及时得以补充外,余均饮食如常。各组动物术后患肢跛行,约二周后除空白组肢体仍蜷缩、拖地、不敢活动外,实验及对照组肢体均正常行动,活动自如,有一例骨缺损未予填充肢体发生骨折。各伤口无化脓感染,平均10d愈合。2周取材时伤口愈合好,皮下有肉芽肿形成,骨面无材料外露及排出。
2.2 X线
术后4周:A、B、C各组骨缺损区植入材料高密度状,清晰可见,骨皮质已开始愈合;8周:B组可见较淡缺损区轮廓影,其中CPC+杜仲2材料已大部分吸收,被新骨替代,A组和C组尚可见骨缺损区明显骨水泥高密度影;C组有骨吸收低密度影现象;12周:A组骨水泥还清晰可见,C组尚可见骨水泥轮廓,B组骨髓腔贯通,骨水泥几乎未见。空白D组12周时未完全愈合。参见图1A(a1、a 2、a 3)分别为术后8周ACB组,图1B(b1、b2、b3)分别为术后12周ACB组。
2.3 组织学检查
A(CPC+杜仲1)组:术后4周:原始骨小梁交织成片状,CPC均匀红染嵌入其间,水泥与骨交界处可见新生血管、成纤维细胞及多核巨噬细胞。边缘有幼稚小梁结构侵入,成骨细胞增生旺盛。原始骨小梁内成骨细胞小点状散在分布,少量钙盐沉积。8周:骨小梁增粗,纤维成分减少。CPC部分降解成团状,少数被骨小梁包绕,多数在小梁中呈小灶性分布。软骨成骨活跃,出现破骨细胞及脂肪滴。12周:大量新生骨组织中可见少量CPC颗粒,骨小梁融合,髓腔贯通,可见脂肪细胞大量出现。
B(CPC+杜仲2)组:术后4周:骨小梁排列较规则成条索状,完全侵入CPC,材料开始降解,可见多核巨噬细胞和丰富新生血管。CPC中大量梭形成骨细胞浸润。多处巢状软骨形成,新生骨小梁软骨钙化,大片蓝色钙盐沉积,小梁上成骨细胞胞体大,胞浆丰富椭圆形。8周:骨小梁明显增粗,出现成熟板层骨及哈佛氏***,管壁上多行排列成骨细胞密集,植入材料大部分降解成松散颗粒,面积减少。12周:新生骨组织中未见CPC颗粒,髓腔贯通,正常骨形成。
C(CPC)组:术后4周:骨小梁略微稀疏,部分CPC吸收呈空泡结构。8周:骨小梁增粗,成骨细胞密集。
12周:出现脂肪细胞,其间夹杂编织骨小梁和CPC颗粒,髓腔未完全贯通。
D(空白)组:术后4周:少量骨小梁出现,有大量血细胞分布;12周:骨小梁形态纤细,稀疏,出现脂肪细胞。(见图2ABCD)
2.4 电镜观察
术后四周电镜下观察材料中杜仲微囊清晰可辨,直径约50um(图3D),均破裂,杜仲得以释放。
术后4周:A组:蜂巢状小梁骨与CPC骨水泥交界;B组:骨小梁开始呈编织状,攀伸到骨水泥界面,骨水泥散裂成小块;C组:骨小梁类似A组,骨水泥仍大块连接。(见图3ABC) 8周:A组:小梁增粗,与骨水泥分界清楚;B组:可见骨水泥与小梁骨结合较紧密,无明显界限,中间无纤维组织样结构。骨小梁明显增粗伸人材料内部,材料减少;C组:材料较致密,偶见骨小梁伸人材料内部。12周:B组骨水泥大量降解,被骨组织替代,A、C组骨水泥仍明显存在。
实施例2
本研究选用CPC已在国内外临床使用,成骨性能确实可靠,将它作为对照更具有无干扰性和准确性。我们设计0.304,0.608mg/ml两种杜仲浓度,并采用兔股骨髁直径6mm的骨缺损模型从几方面评价不同比例下材料的成骨作用及降解速度。从X线表现来看(图1),CPC+杜仲2(0.608mg/ml)组材料降解速度与新骨长成速度相匹配,略优于CPC+杜仲1(0.304mg/ml)组,显著优于CPC组,12周时CPC+杜仲2(0.608mg/ml组)几乎看不到骨水泥影,髓腔贯通,材料基本降解,与新骨长成速度一致;CPC+杜仲1(0.304mg/ml)组可见骨水泥影,材料降解为小颗状存在,新骨长成良好;CPC组则明显骨水泥影尚未降解。从组织学观察4周时CPC+杜仲2组骨水泥中大量梭形成骨细胞浸润及丰富新生血管,CPC+杜仲1组同CPC组只在宿主骨表面有成骨细胞和新生血管,CPC组骨小梁明显稀疏。12周时实验组髓腔均贯通,但CPC+杜仲1组材料未完全降解。表明CPC+杜仲2组成骨性能更活跃且降解速度适宜。而从电镜下微囊清晰可辨,较制成时略有缩小,可能与其中内容物得以释放有关。
BMP是迄今为止所发现的作用最为肯定的骨生长因子之一,而BMP-2是其家族中成骨活性最强的生长因子之一,其主要作用是诱导未分化的间充质细胞分化形成软骨和骨,从术后四周CPC+杜仲2组免疫组化图像观在软骨细胞和成骨细胞周围BMP-2因子呈高表达(图4),说明0.608mg/ml杜仲加入后赋予CPC一定的骨诱导性质。而VEGF主要作用于血管内皮细胞,具有强的促内皮细胞有丝***的作用,增加血管通透性及血管维持功能,有利于新生血管的生成,为局部材料的降解及新骨的形成创造最佳微环境。骨折断端存在VEGFmRNA高表达,且高表达贯穿于骨折愈合的全过程,本研究4周时VEGF在血管壁和骨髓基质细胞中表达达一高峰(图5),CPC+杜仲2组亦呈优势反应,进一步证明了适量杜仲的加入可起到改善局部血供的作用,这与有报道杜仲可通过诱导血管内皮产生扩血管物质有一致性。
结果表明杜仲提取物给微囊包裹,以0.608mg/ml比例与磷酸钙骨水泥复合修复兔股骨髁缺损诱导新骨形成作用强,降解时间较快,优于磷酸钙骨水泥。作为祖国传统医药,能通过现代缓释技术进行改进,具有生长因子相似的促进骨组织生长的作用。
Claims (2)
1.一种微囊缓释中药复合磷酸钙骨水泥的制备方法,通过以下步骤实现:
(1)微囊的制备:将***胶和β-环状糊精混合物以质量比1∶1配成溶液,作为微囊壁材溶液;
(2)杜仲微囊粉末制备:称取杜仲提取物,用酒精溶解后,与微囊壁材溶液按照质量比1∶10混合,将混合溶液加热至60℃,经高压均质处理;控制进风温度150℃,出风温度90℃,流量25mL/min,对均质处理的溶液进行喷雾干燥,得到杜仲微囊粉末;
(3)复合磷酸钙骨水泥的制备:以羟基磷灰石和磷酸三钙按质量比1∶1为固相物,取该混合干粉2.5g,在液相固化液蒸馏水1.6mL中按0.608mg/mL加入杜仲微囊粉末混合,溶解后加入干粉充分搅拌,2分钟后混合物开始固化成面团状,在混合物即将硬化前将其填入模具中,用小铲压平,五分钟后硬化倒出,收集后分别以聚乙烯袋独立包装,环氧灭菌。
2.根据权利要求1所述的一种微囊缓释中药复合磷酸钙骨水泥在制备人工关节材料中的应用。
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