CN108276487B - 一种可促进成骨并抑制破骨的活性多肽及其应用 - Google Patents
一种可促进成骨并抑制破骨的活性多肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种可促进成骨并抑制破骨的活性多肽及其应用。是在PTH1‑34的基础上将N端丝氨酸磷酸化，且在C端引入三个重复序列的谷氨酸或天冬氨酸，所述多肽序列为SEQ ID NO:1或SEQ ID NO:2所示；其具有类似BMP2的骨诱导活性，及类似甲状旁腺素PTH抑制破骨细胞的作用。该多肽C端重复序列结合于钙磷材料或表面钙磷涂层材料表面上，避免多肽的无规卷曲，不需要有机试剂的参入，有效地保护多肽的活性，并通过肽键的裂解实现缓慢控制释放，可避免PTH1‑34长期间断注射给药，减轻患者的注射痛，且达到促进成骨细胞、抑制骨质疏松的效果。本发明改变了以往PTH1‑34不能局部给药的观点。
Description
技术领域
本发明涉及骨生物材料与组织工程领域，具体涉及一种可促进成骨并抑制破骨的活性多肽及其应用。
背景技术
现有资料显示，全世界约2亿人患有骨质疏松症，其发病率在慢性疾病中目前已上升至第7位。在我国，60岁以上的骨质疏松症患者已突破8000万，而老年人骨质疏松性骨折的发生率也已达到6.3％-24.4％。骨质疏松性骨折愈合较慢，愈合过程中发生并发症的风险高，同时骨质疏松性骨折的致残率和致死率较高，严重影响患者生活质量，加重卫生系统负担。世界卫生组织(WHO)已将骨质疏松症列为影响老年人身体健康的三大疾病之一。
目前市场上骨质疏松的临床治疗用药以抑制骨吸收的药物为主，如雌激素、双膦酸盐类和降钙素，而其他一些药物则是骨形成促进剂，如氟化物、甲状旁腺激素(PTH)。PTH类药物是目前最有前途的骨形成促进剂，将开发用于原发性骨质疏松症的防治。大规模临床试验表明，甲状旁腺激素(PTH)能够刺激骨形成，明显提高(每年7％到10％)骨密度，组织形态测定研究表明，PTH能够改善骨质疏松中的微结构，并能逆转以前曾经认为不能逆转的骨退化过程。
人体天然PTH含有84个氨基酸，是骨骼、肾脏钙和磷酸盐代谢的主要调节激素，其生理作用包括调节骨代谢、肾小管对钙和磷酸盐的再吸收和肠内钙吸收，小剂量PTH有明显的成骨作用。然而，内源性PTH水平过高对皮质骨和松质骨的成骨作用有不同的影响，如甲状旁腺机能亢进者仅出现整体骨密度(BMD)降低，而松质骨BMD则无明显改变。PTH的N端与C端在骨代谢中发挥的作用不尽相同，N端主要与经典PTH-I型受体结合，影响成骨作用；而C端则通过与另一类受体结合，发挥其他生物学作用，甚至促进骨细胞凋亡。重组人甲状旁腺激素(rhPTH 1-34)特立帕肽(SVSEI-QLMHN-LGKHL-NSMER-VEWLR–KKL QD-VHNF)正是基于上述机制所研发的人PTH类似物，该产品具有与天然PTH N端34个氨基酸序列完全相同的结构，其生物学作用亦是通过与特异性高亲和性PTH-I受体结合介导的。与天然PTH相比，它对骨和肾脏具有相同的生理作用，但克服了C端对骨代谢的不利影响。特立帕肽是目前唯一获美国FDA批准的骨形成促进药物，它不仅可以增加骨量、抑制骨吸收进而逆转骨质疏松，而且可能促进骨折愈合，被认为是治疗严重骨质疏松症的"突破性"进展，但是其存在的问题是价格昂贵，只能注射给药，可引发高钙血症，甚至骨肉瘤风险，而且不能长期使用(2年)。为此，针对骨质疏松性骨折患者骨形成/愈合能力减弱，更需要研发出一种既能促进骨形成、逆转骨质疏松，又能促进骨折愈合和骨缺损修复的新型药物。
重组人甲状旁腺激素(rhPTH 1-34)特立帕肽的生物学活性尚不够稳定,其主要原因是由于该多肽序列较长(含34个氨基酸)，且存在一定的柔曲性，可能发生卷曲，空间结构仍不太稳定，因而与受体结合的优势构象不突出，影响到与受体结合的选择性及活性强度。因此迫切需要进一步优化，筛选出稳定其空间结构，以充分体现小分子活性药物的理想优势。同时，这种PTH活性多肽需要通过适当载体材料缓慢间断控制释放，才能最大程度发挥其骨诱导活性与逆转骨质疏松的能力。
发明内容
为解决以上问题，本发明提供了一种可促进成骨并抑制破骨的活性多肽，该多肽在PTH1-34的基础上将N端丝氨酸磷酸化，同时在C端引入三个重复序列的天冬氨酸(D)或谷氨酸(E)，所得到的多肽既具有类似BMP2的骨诱导活性，又具有类似甲状旁腺素PTH抑制破骨细胞的作用。将所述多肽与钙磷材料复合后，通过肽键的裂解实现缓慢控制释放。
本发明采用的技术方案如下：
本发明第一方面，提供一种可促进成骨并抑制破骨的活性多肽PTHrP-1或 PTHrP-2，该多肽是在PTH1-34的基础上将N端丝氨酸磷酸化，同时在C端引入三个重复序列的天冬氨酸(D)或谷氨酸(E)，其氨基酸序列如SEQ ID NO:1 或SEQ ID NO:2所示。
本发明第二方面，提供一种缓释复合材料，由上述可促进成骨细胞活性同时抑制破骨细胞活性的多肽PTHrP-1或PTHrP-2搭载钙磷支架材料或钙磷涂层支架材料得到。
优选地，所述复合材料中钙磷支架材料或钙磷涂层支架材料为介孔生物活性玻璃。
优选地，所述复合材料中钙磷支架材料或钙磷涂层支架材料为壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC)。
本发明第三方面，提供上述缓释复合材料的制备方法，包括以下步骤：
S1：将所述可促进成骨并抑制破骨的活性多肽PTHrP-1或PTHrP-2的水溶液加入钙磷载体支架材料或钙磷涂层载体支架材料中；
S2：将所述含多肽载体支架材料－55℃冻干，即得到所述载促进成骨细胞并抑制破骨细胞活性的缓释复合材料。
优选地，S1中所述复合材料中材料大小为直径5mm、厚度2mm或直径4mm、长度15mm或长度20mm、宽度5mm、厚度2mm中的一种。
优选地，S1中所述复合材料中上述多肽PTHrP-1或PTHrP-2的含量为0.1 -0.5mg。
本发明第四方面提供上述多肽PTHrP-1或PTHrP-2在制备促进成骨细胞并抑制破骨细胞活性药物中的应用。
优选地，该药物是多肽PTHrP-1或PTHrP-2搭载钙磷支架材料或钙磷涂层支架材料得到的复合材料。
优选地，该药物可以局部施用，而非只能全身给药。
在本发明的具体实施例中通过局部皮肤刺激试验、急性毒性试验、细胞毒性实验证实本发明提供的多肽安全性好。
在本发明的另一个具体实施例中将PTH相关多肽PTHrP-1或PTHrP-2和 PTH1-34分别与介孔生物活性玻璃(MBG)复合，分为两组，MBG+1000ngPTH 组和MBG+1000ngPTHrP-1或PTHrP-2组。分别将密度为1×105/ml的成骨细胞 MC3T3-E1和破骨细胞RAW加入材料中共培养1d、3d、5d、7d。通过激光共聚焦显微镜或扫描电镜观察发现，PTH1-34组第一天由于PTH1-34的爆释，细胞生长旺盛，第3d和第5d成骨细胞出现凋亡，数量减少，第7d开始破骨细胞增多，以破骨细胞为主。PTHrP-1或PTHrP-2多肽组释放稳定，成骨细胞呈稳定趋势增长，第5d和第7d以成骨细胞为主，少有破骨细胞。证实PTHrP-1或 PTHrP-2多肽具有促进成骨同时抑制破骨的作用。
在本发明的具体实施例中多肽PTHrP-1或PTHrP-2负载介孔生物活性玻璃 (MBG)促进大鼠腰椎横突间融合和大鼠颅骨缺损的修复以及多肽PTHrP-1负载壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC)支架材料修复兔桡骨缺损，证实多肽PTHrP-1或PTHrP-2搭载钙磷支架材料或钙磷涂层支架材料得到的复合材料可以局部施用。
本发明具有如下优点：
①所述多肽N端磷酸化丝氨酸可促进钙盐的沉积与矿化，骨诱导活性更佳。
②对PTH1-34进行优化修饰后，结构更加稳定，C端重复天冬氨酸或谷氨酸序列(3个D或3个E)能与钙磷材料表面的天然位点结合，从而可以在一定程度上避免多肽的无规卷曲，充分暴露多肽活性位点，骨诱导活性更佳。
③所述多肽通过重复的天冬氨酸或谷氨酸序列(3个D或3个E)结合于钙磷支架材料上，不需要有机试剂的参入，可以有效地保护多肽的活性，并通过肽键的裂解实现达到间断缓慢控制释放。
④所述多肽与钙磷材料结合植入或与可注射含钙磷材料复合直接注射骨折端和骨缺损处，能在局部募集成骨细胞或促进向成骨细胞分化，同时通过调节剂量，可以最大限度的减少破骨细胞及骨质疏松的发生。避免了PTH1-34需全身给药和长期给药带来的风险和副作用。
⑤降解产物为氨基酸，不产生免疫反应和炎症反应。
⑥制备工艺简单，便于规模化生产。
附图说明
图1为PTHrP-1多肽(A)和PTHrP-2多肽(B)高效液相色谱法HPLC检测图；
图2为PTHrP-1多肽(A)和PTHrP-2多肽(B)质谱法MS检测图；
图3为MC3T3-E1细胞在PTHrP-1多肽诱导作用下生长正常，无毒性；
图4为不同浓度PTHrP-1多肽诱导MC3T3-E1细胞ALP染色图，图中100ng/ml 组和200ng/ml组ALP阳性细胞明显多于50ng/ml组，100ng/ml组和200ng/ml 组无明显差别；
图5为不同浓度PTHrP-1多肽诱导MC3T3-E1细胞ALP活性检测图，图中三种含PTHrP-1多肽的不同浓度的培养基中MC3T3-E1细胞的ALP活性随着成骨诱导时间的延长均持续增高，但100ng/ml组和200ng/ml组中MC3T3-E1细胞的 ALP活性明显高于50ng/ml组，100ng/ml组和200ng/ml组无明显差别；
图6为不同浓度PTHrP-1多肽诱导MC3T3-E1细胞骨钙素OCN含量检测结果图，100ng/ml组和200ng/ml组中的OCN含量明显高于50ng/ml组和0ng/ml组， 100ng/ml组和200ng/ml组无明显差别；
图7为PTHrP-1多肽负载介孔生物活性玻璃促进成骨细胞生长并抑制破骨细胞生长激光共聚焦图；
图8为PTHrP-2多肽负载介孔生物活性玻璃促进成骨细胞生长并抑制破骨细胞生长扫描电镜图；
图9为不同浓度PTHrP-1多肽负载介孔生物活性玻璃缓释曲线图；
图10为PTH相关多肽PTHrP-1负载介孔生物活性玻璃(MBG)修复SD大鼠腰椎横突间融合Micro CT检测图，图中0.5mg PTHrP-1/MBG复合支架组(A)骨修复效果明显优于0.1mgPTHrP-1/MBG复合支架组(B)，明显优于单纯支架组 (C)；
图11为PTH相关多肽PTHrP-1负载介孔生物活性玻璃(MBG)修复SD大鼠腰椎横突间融合Van Gieson染色图，图中0.5mg PTHrP-1/MBG复合支架组(A) 骨修复效果好于0.1mgPTHrP-1/MBG复合支架组(B)，明显好于单纯支架组(C)；
图12为PTH相关多肽PTHrP-2负载介孔生物活性玻璃(MBG)修复SD大鼠颅骨缺损Micro CT检测图，图中0.5mg PTHrP-2/MBG复合支架组(A1、A2)骨修复效果明显优于0.1mgPTHrP-2/MBG复合支架组(B1、B2)，明显优于单纯支架组(C1、C2)；
图13为PTH相关多肽PTHrP-2负载介孔生物活性玻璃(MBG)修复SD大鼠颅骨缺损Van Gieson染色图，图中0.5mg PTHrP-2/MBG复合支架组(A)骨修复效果好于0.1mg PTHrP-2/MBG复合支架组(B)，明显好于单纯支架组(C)；
图14为PTH相关多肽PTHrP-1负载壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC) 支架材料修复兔桡骨缺损第6周和第12周CT图，0.1mg PTHrP-1/CH/TBC复合支架组修复效果明显优于其它三组；
图15为PTH相关多肽PTHrP-1负载壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC) 支架材料修复兔桡骨缺损第6周和第12周HE染色图，0.1mg PTHrP-1/CH/TBC 复合支架组修复效果明显优于其它三组。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
发明人在实验过程中试验了以PTH1-34为主体进行剪切的多种多肽，结果发现多肽促进成骨细胞活性均不及PTH1-34。发明人尝试将N端丝氨酸磷酸化，同时在C端引入三个重复序列的谷氨酸(D)或天冬氨酸(E)，结果出乎意料地发现，所得多肽不仅能促进成骨细胞和抑制破骨细胞，而且能局部给药，与钙磷支架材料复合后，能有效地修复骨缺损及抑制骨质疏松。
【实施例1】多肽PTHrP-1和PTHrP-2的合成
实验中所需的多肽分子采用自动多肽合成仪合成，并通过高效液相色谱纯化，用质谱仪、氨基酸及多肽分析仪检测其纯度和序列。PTHrP-1和PTHrP-2的序列分别为
和
合成得到的纯度大于95％，分子量为4543.07Da，PTHrP-2分子量为4585.15Da。HPLC和MS如图1、图2所示。
【实施例2】多肽PTHrP-1毒性实验
1、局部皮肤刺激试验
取健康新西兰大白兔3只，雌雄不限，体重2.0-2.5kg，剪去背部兔毛，观察2d，皮肤无伤痕刺激，将浓度为100ng/mlPTHrP-1多肽溶液和生理盐水各0.2ml 分别注射于脊柱两侧皮下，每侧取l0个点，注射后注射部位观察1d,3d,7d三个时间点。通过1d,3d,7d三个时间点观察，PTHrP-1多肽溶液和生理盐水注射部位均未见红肿，也未见红斑出现，提示PTHrP-1多肽对动物机体没有刺激性。
2、急性毒性试验
选健康小白鼠10只，体重20-25g，雌雄各半，分笼饲养。每只小白鼠腹腔内注射100ng/ml多肽溶液(50ml/kg)，注射后观察1d，3d，7d三个时间点，一周后处死小白鼠，组织学HE切片观察心、肝、脾、肺、肾等器官有无异常。所有小白鼠在试验期间均未出现毒性反应表现，进食正常，活动如常。各个时间点处死动物后各脏器大体观未见明显异常，各器官组织学HE染色切片未见异常。
3、细胞毒性实验
第3代小鼠MC3T3-E1细胞，密度为1×105/ml，加入6孔培养板中，每孔加入0.5ml细胞悬液，在37℃、体积分数为5％CO2培养箱中培养。将浓度为100ng/ml多肽溶液加入培养基中，每2-3d更换培养基。观察细胞生长情况，发现细胞生长正常，如图3所示。
【实施例3】多肽PTHrP-1促进MC3T3-E1细胞成骨分化最佳浓度
将四种含不同浓度的PTHrP-1多肽培养基(0ng/ml、50ng/ml、100ng/ml、 200ng/ml)分别加入24孔培养板中，每种5孔。取第3代小鼠MC3T3-E1细胞，密度为1×105/ml，每孔加入0.5ml细胞悬液，在37℃、体积分数为5％CO2培养箱中培养，每2d更换培养基。培养14d，采用ALP试剂盒进行ALP染色。结果发现含PTHrP-1多肽三组培养孔中，MC3T3-E1细胞聚集，呈复层生长，细胞形态由圆形或类圆形逐渐变为多角形或立方形，细胞外基质分泌明显增多， ALP染色可见细胞胞浆中出现红棕色颗粒或棕褐色或咖啡色颗粒，是为ALP阳性细胞。其中100ng/ml组和200ng/ml组ALP阳性细胞明显多于50ng/ml组， 100ng/ml组和200ng/ml组无明显差别，如图4所示。
同样方法，小鼠MC3T3-E1细胞培养5、10、15及20d，将24孔板中的细胞用PBS冲洗3次，用0.25％胰蛋白酶消化5～8min，每孔加入培养基1ml终止消化，4℃条件下10000rpm离心5min，再用PBS冲洗3次，加细胞裂解液破膜。按试剂盒说明进行ALP活性检测。通过紫外分光光度计在520nm波长处测定吸光度值(OD520)。ALP活性检测结果显示：三种含PTHrP-1多肽的不同浓度的培养基中MC3T3-E1细胞的ALP活性随着成骨诱导时间的延长均持续增高，但100ng/ml组和200ng/ml组中MC3T3-E1细胞的ALP活性明显高于50ng/ml 组，100ng/ml组和200ng/ml组无明显差别，如图5所示。
同样方法，细胞诱导培养1、2、3及4w时，收集陈旧培养液及洗涤液，采用ELISA试剂盒检测培养液中OCN的含量。结果显示：100ng/ml组和200ng/ml 组中的OCN含量明显高于50ng/ml组和0ng/ml组，100ng/ml组和200ng/ml组无明显差别，如图6所示。
ALP定性和定量实验以及OCN定量实验提示PTHrP-1多肽的体外诱导成骨最佳浓度为100ng/ml。
【实施例4】多肽PTHrP-1溶液与成骨细胞MC3T3-E1和破骨细胞RAW共培养
将多肽PTHrP-1和PTH1-34分别与介孔生物活性玻璃(MBG)复合，材料大小为10mm*10mm。分为两组，MBG+1000ngPTH组和MBG+1000ngPTHrP-1 组。分别将密度为1×105/ml的成骨细胞MC3T3-E1和破骨细胞RAW加入材料中共培养1d、3d、5d、7d。通过激光共聚焦显微镜观察发现，PTH1-34组第一天由于PTH1-34的爆释，细胞生长旺盛，第3d和第5d成骨细胞出现凋亡，数量减少，第7d开始破骨细胞增多，以破骨细胞为主。PTHrP-1多肽组释放稳定，成骨细胞呈稳定趋势增长，第5d和第7d以成骨细胞为主，少有破骨细胞。如图7所示，其中成骨细胞呈拉丝状，破骨细胞呈多核团状。证实PTHrP-1多肽具有促进成骨同时抑制破骨的作用。
【实施例5】多肽PTHrP-2溶液与成骨细胞MC3T3-E1和破骨细胞RAW共培养
将PTH相关多肽PTHrP-2和PTH1-34分别与介孔生物活性玻璃(MBG)复合，材料大小为10mm*10mm。分为两组，MBG+1000ngPTH组和 MBG+1000ngPTHrP-2组。分别将密度为1×105/ml的成骨细胞MC3T3-E1和破骨细胞RAW加入材料中共培养1d、5d。分别将在两个时间点加入固定液固定细胞，通过扫描电镜观察成骨和破骨细胞共培养过程。结果发现，PTH1-34组第一天成骨细胞生长旺盛，第5d成骨细胞部分凋亡，数量减少，以破骨细胞为主。 PTHrP-2多肽组成骨细胞呈稳定趋势增长，第5d仍以成骨细胞为主，少有破骨细胞。如图8所示，其中成骨细胞呈拉丝状，破骨细胞呈多核团状。证实PTHrP-2 多肽具有促进成骨同时抑制破骨的作用。
【实施例6】多肽PTHrP-1负载介孔生物活性玻璃(MBG)的缓释性
将制备好的0.1mg PTHrP-1/MBG材料和0.5mg PTHrP-1/MBG材料分别浸入 5ml配制好的PBS溶液中，在37℃条件下浸泡7d。在分别浸泡2h，6h,12h,24h, 2d,3d,4d,5d,6d和7d后，检测洗出液中PTHrP-1多肽的量。在每个时间间隔中，洗出液均被完全移除，然后重新倒入PBS溶液。所收集的洗出液中PTHrP-1 多肽的量用PTH ELISA试剂盒进行检测。如图9所示，不同浓度的 PTHrP-1/MBG材料均具有缓慢释放的特性。
【实施例7】多肽PTHrP-1负载介孔生物活性玻璃(MBG)促进大鼠腰椎横突间融合(骨修复)
将18只健康雄性SD大鼠随机分为3组，每组10只，其中A组植入0.5mg PTHrP-1/MBG材料，B组植入0.1mg PTHrP-1/MBG材料，C组植入单纯MBG 材料。10％水合氯醛腹腔注射麻醉，沿腰椎棘突作后中线切口，然后在距中线4mm 处做两个单独的筋膜切口。钝性分离，显露L4和L5横突，充分暴露后，用庆大霉素/生理盐水溶液冲洗融合床，并使用高速钻头去除横突皮质骨，然后在 L4-L5横突处分别植入三组材料，缝合伤口。术后12周行放射学和组织学检测。如图10、图11所示，PTH相关多肽PTHrP-1负载介孔生物活性玻璃(MBG) 能明显促进大鼠腰椎横突间融合。
【实施例8】多肽PTHrP-2负载介孔生物活性玻璃(MBG)促进大鼠颅骨缺损的修复
将30只健康雄性SD大鼠随机分为3组，每组10只，其中A组植入0.5mg PTHrP-2/MBG材料，B组植入0.1mg PTHrP-2/MBG材料，C组植入单纯MBG 材料。10％水合氯醛腹腔注射麻醉，取颅顶正中纵行切口，于颅骨正中缝和人字缝交叉的外下象限空白处两侧，分别作一直径为5mm的圆形骨缺损区，分别同时植入上述三组材料，缝合。分别于术后6周和12周每组各分别处死4只和6 只大鼠，取颅骨做放射学和组织学检测。如图12、图13所示，PTH相关多肽PTHrP-2负载介孔生物活性玻璃(MBG)能明显促进大鼠颅骨缺损的修复。
【实施例9】多肽PTHrP-1负载壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC)支架材料修复兔桡骨缺损
取4-6月龄新西兰大白兔48只，雄性，体重2.0-2.5kg，每只大白兔术前均用戊巴比妥钠(25mg/kg)和甲苯噻嗪(8mg/kg)肌肉注射麻醉。麻醉满意后，用弯剪剔除左前肢兔毛，然后用1％活力碘消毒灭菌。常规铺巾。以尺桡骨中点为中心作纵行切口，长约18mm，逐层切开皮肤、筋膜及肌层，显露左侧尺桡骨中段。用自制小刀锯造15mm长度桡骨中段缺损。分别将四组材料植入缺损处 (空白组、CH/TBC组、0.01mgPTHrP-1/CH/TBC组、0.1mgPTHrP-1/CH/TBC 组)。术后6周和12周行放射学和组织学检查。如图14、图15所示，0.1mgPTHrP-1 负载壳聚糖/纳米羟基磷灰石/煅烧骨(CH/TBC)支架材料能明显促进兔桡骨缺损修复。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<110> 武汉大学
<120> 一种可促进成骨并抑制破骨的活性多肽及其应用
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Claims (5)
1.一种可促进成骨细胞活性并抑制破骨细胞活性的多肽，其特征在于，所述多肽是在PTH1-34的基础上将N端的第一位丝氨酸磷酸化，同时在C端引入三个谷氨酸，其氨基酸序列如SEQ ID NO:2所示。
2.一种缓释复合材料，其特征在于，由权利要求1所述的可促进成骨细胞活性并抑制破骨细胞活性的多肽搭载钙磷支架材料得到。
3.根据权利要求2所述的缓释复合材料，其特征在于，所述的钙磷支架材料为介孔生物活性玻璃。
4.权利要求2或3所述的缓释复合材料的制备方法，其特征在于，包括以下步骤：
S1：将权利要求1所述的可促进成骨细胞活性并抑制破骨细胞活性的多肽的水溶液加入钙磷载体支架材料中；
S2：将S1获得的含多肽载体支架材料－55℃冻干，即得到载有可促进成骨细胞活性并抑制破骨细胞活性的多肽的缓释复合材料。
5.权利要求1所述的多肽或权利要求2或3所述的缓释复合材料在制备可促进成骨细胞活性并抑制破骨细胞活性的药物中的应用，其特征在于，所述药物通过局部施用。
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