TW201900226A - Drug-containing composite scaffold - Google Patents

Abstract

The present invention relates to a drug-containing composite scaffold comprising a porous bone material and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous bone material.

Description

一種含藥物之複合性支架  Drug-containing composite stent  

本發明係關於一種含藥物之複合性支架，其特徵在於將一藥物釋放奈米載體置於一多孔性陶瓷骨材內，提供局部藥物長效緩釋的效果，以加速骨頭缺損處的骨再生能力。 The present invention relates to a drug-containing composite stent characterized in that a drug-release nanocarrier is placed in a porous ceramic aggregate to provide a sustained-release effect of a local drug to accelerate bone at the bone defect. Regeneration ability.

孔性雙磷酸鈣陶瓷，臨床上被認可的生醫材料，其多孔性與局部分解性也會有利於新生骨向內生長。亦有文獻指出由此類多孔性雙相磷酸鈣陶瓷支架在孔徑大小為80-160μm或500-1000μm時最能有效的使新生血管與新生骨組織向內生長。不過對於不癒合(non-union)與較大骨缺損的無法完全癒合的情況，則仍須骨誘導的因子來協助促進骨生長，以加強骨癒合。 Porous calcium biphosphate ceramics, clinically recognized biomedical materials, their porosity and local decomposition are also conducive to new bone ingrowth. It has also been pointed out that such porous biphasic calcium phosphate ceramic scaffolds are most effective for ingrowth of neovascularization and new bone tissue at a pore size of 80-160 μm or 500-1000 μm. However, for cases where non-union and large bone defects cannot heal completely, osteoinductive factors are still required to help promote bone growth to enhance bone healing.

辛伐他汀(Simvastatin；SIM)，是羥甲基戊二酸單醯輔酶A(hydroxy-3-methylglutaryl coenzyme A；HMG-CoA)還原酶之抑制劑，為臨床上常用的降血脂藥物。近年來，一些活體內與活體外研究指出辛伐他汀(SIM)具有促進骨生成的效果。而過去活體外研究發現辛伐他汀可以促進骨母細胞的增生，並誘導人類骨髓幹細胞走向骨化作用。但是，許多研究指出辛伐他汀以全身性地給予方式對於促進骨生成的效果仍無一致的結 果，原因是口服之辛伐他汀大多在肝臟被代謝，到達骨骼周圍的藥量無法達到有效促骨化濃度。市售含藥醫材，例如BMP2(INFUSE^®)及BMP7(OP-1^®)之骨誘導作用生長因子產品。但使用上仍有一些限制，如：(1)無藥物載體控制其釋放，容易造成短期間濃度太高及藥效不持久之虞；(2)蛋白質產品保存不易；(3)價格高昂；和(4)機械強度不足。 Simvastatin (SIM) is an inhibitor of hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which is a commonly used hypolipidemic drug. In recent years, some in vivo and in vitro studies have indicated that simvastatin (SIM) has an effect of promoting osteogenesis. In vitro studies in vitro have found that simvastatin can promote the proliferation of osteoblasts and induce the ossification of human bone marrow stem cells. However, many studies have shown that the effect of simvastatin on systemic administration has no consistent effect on promoting osteogenesis, because oral simvastatin is mostly metabolized in the liver, and the amount of drug reaching the bones cannot be effectively promoted. Concentration. Commercially available medicinal materials such as BMP2 (INFUSE ^® ) and BMP7 (OP-1 ^® ) osteoinductive growth factor products. However, there are still some restrictions on the use, such as: (1) no drug carrier to control its release, which is likely to cause too short a concentration and a long-lasting effect; (2) protein products are not easy to store; (3) high prices; (4) Insufficient mechanical strength.

傳統人工替代性骨材，僅具有骨傳導功能，故大多用來治療骨折，雖有骨癒合的效果，但往往時間甚長或是無法有效骨整合導致癒合成效不佳之情況發生；這時需要配合自體骨移植幫助癒合，但自體骨來源短缺以及取骨處會使患者疼痛。因此，目前骨缺損患者的治療上，除了使用替代性骨材外，會同時施予骨生長藥物以提高骨修復效果，但該骨生長藥物因沒採用藥物載體之形式，故會造成短時間藥物濃度過高以及長時間藥效越來越差的缺點。因此，另有研究將含有辛伐他汀(SIM)的微米級粉體載體與人工骨材的結合於臨床上合併使用，以含有辛伐他汀之藥物緩慢釋放微粒載體(例如SIM/PLGA-HAp或是SIM/rhBMP-2/PLGA-HAp)，其粒徑大小約為117,000±25μm，其可控制釋放辛伐他汀，故該藥物緩慢釋放微粒載體於壓確後以局部性的給予方式應用於骨折之修復，但是在臨床使用上，骨缺損患者透過使用人工骨材並搭配藥物緩慢釋放微粒載體來進行骨生長時，臨床醫師仍須根據經驗來判定，視多少骨頭缺損區域以及擺放的位置而評估給予多少量的藥物，目前並沒有此種客製化骨材，所以每次手術前，必須針對推估藥量放入人工骨材，這又涉及如何製備出具有此等藥物含量之人工骨材，又需精確釋放有效量藥物，在臨床上對臨床醫師 極度困擾。 Traditional artificial replacement bones have only bone conduction function, so most of them are used to treat fractures. Although there is bone healing effect, it often takes a long time or can not effectively achieve osseointegration, resulting in poor healing results. Body bone grafts help heal, but a shortage of autologous bone sources and bone removal can cause pain in the patient. Therefore, at present, in the treatment of patients with bone defects, in addition to the use of alternative bone materials, bone growth drugs are simultaneously administered to improve the bone repair effect, but the bone growth drug does not use the form of a drug carrier, so it will cause short-term drugs. The disadvantage of excessive concentration and prolonged drug efficacy. Therefore, another study used a combination of a micron-sized powder carrier containing simvastatin (SIM) and an artificial bone material in combination with a clinically slow-release microparticle carrier containing simvastatin (eg, SIM/PLGA-HAp or Is SIM/rhBMP-2/PLGA-HAp), its particle size is about 117,000±25μm, which can control the release of simvastatin, so the drug slowly releases the microparticle carrier and applies it to the fracture in a localized manner after compression. The repair, but in clinical use, bone defect patients through the use of artificial bone and with the drug to slowly release the particulate carrier for bone growth, the clinician still has to judge according to experience, depending on the number of bone defects and the location To assess how much drug is administered, there is currently no such customized bone material, so before each operation, artificial bone material must be placed for the estimated dose, which involves how to prepare artificial bone with such drug content. Materials, but also need to accurately release an effective amount of drugs, clinically extremely troubled by clinicians.

是以，人工骨材和含有促進骨生長藥物的釋放載體要如何搭配使用，以提升醫師於臨床上使用的方便度，是目前業界欲解決的問題。 Therefore, how to use the artificial bone material and the release carrier containing the drug for promoting bone growth to improve the convenience of the doctor in clinical use is a problem that the industry is currently trying to solve.

本發明藉由將生物陶瓷骨移植材料與含有辛伐他汀之奈米載體複合成一體以形成一含藥醫材，來克服醫師於臨床上要給予多少促進骨生長之藥物劑量的問題。本發明使用聚乳酸甘醇酸-聚乙二醇(PLGA-PEG)奈米粒子或微脂體來包裹油溶性促進骨生長藥物(如史他汀類(statin)藥物)，故具每日釋放藥物特性，且不具生物毒性。而含有藥物的奈米載體進一步與人工骨移植材料複合，以製作出具有骨傳導與骨誘導作用的含藥骨材。本發明亦證實具有辛伐他汀(simvastatin)的含藥骨材可持續釋放辛伐他汀並能提高D1細胞的鹼性磷酸酶(Alkaline phosphatase；ALP)活性，其代表可有效促進骨生成；同時在動物實驗中，於骨頭缺損處植入含藥骨材，有效修補該骨頭缺損處，顯示出該含藥骨材具有骨修復能力。因此，本發明所製作出的含藥骨材具有局部長效緩釋藥物的用途，且因為可調整最適合藥物釋放濃度，有利於臨床上醫師使用，並加速骨頭嚴重缺損部位的骨再生能力。 The present invention overcomes the problem of how much a physician can clinically give a drug dosage for promoting bone growth by combining a bioceramic bone graft material with a simvastatin-containing nanocarrier to form a medicated medical material. The present invention uses polylactic acid glycolic acid-polyethylene glycol (PLGA-PEG) nanoparticles or microlipids to encapsulate oil-soluble bone growth promoting drugs (such as statin drugs), so that the drug is released daily. Characteristics, and not biologically toxic. The drug-containing nanocarrier is further compounded with the artificial bone graft material to produce a drug-containing aggregate having bone conduction and osteoinduction. The present invention also demonstrates that simvastatin-containing medicinal aggregates can continuously release simvastatin and increase Alzine phosphatase (ALP) activity of D1 cells, which is representative of effective bone growth; In the animal experiment, the drug-containing bone material was implanted in the bone defect to effectively repair the defect of the bone, indicating that the drug-containing bone material has bone repairing ability. Therefore, the medicated aggregate produced by the present invention has the use of a local long-acting sustained-release drug, and because it can be adjusted to be most suitable for drug release concentration, it is advantageous for clinical use by a physician and accelerates the bone regeneration ability of a severely damaged bone site.

本文中的用語「一」或「一種」係用以敘述本發明之元件及成分。此術語僅為了敘述方便及給予本發明之基本觀念。此敘述應被理解為包括一種或至少一種，且除非明顯地另有所指，表示單數時亦包括複數。於申請專利範圍中和”包含”一詞一起使用時，該用語「一」可意謂一個或超 過一個。 The articles "a" or "an" are used herein to describe the elements and compositions of the invention. This terminology is only for convenience of description and the basic idea of the invention. This description is to be construed as inclusive of the singular When used in conjunction with the word "comprising", the term "a" may mean one or more than one.

本文中的用語「或」其意同「及/或」。 The term "or" in this document means "and/or".

本發明提供一種含藥物之複合性支架，其包含一多孔性骨材以及一藥物釋放奈米載體，其中該藥物釋放奈米載體包含一促進骨生長藥物，且分佈於該多孔性骨材中，其中該藥物釋放奈米載體為一兩性分子載體。 The present invention provides a drug-containing composite scaffold comprising a porous aggregate and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous aggregate Wherein the drug releases the nanocarrier as an amphoteric carrier.

本發明的多孔性骨材係屬於一種生醫材料，具有良好的生物相容性。於一具體實施例中，該多孔性骨材的組成材料包含一羥基磷灰石(hydroxyapatite；HAp)、一β-磷酸三鈣(β-tricalcium phosphate；β-TCP)、一羥基磷灰石-磷酸三鈣(hydroxyapatite tricalcium phosphate；HATCP)、一α-磷酸三鈣(α-tricalcium phosphate；α-TCP)、一生物活性玻璃陶瓷(bioactive glass ceramic)、一硫酸鈣、一骨水泥(bone cement)或其上述組合。於一較佳具體實施例中，該多孔性骨材為含有雙相磷酸鹽的多孔性骨材。於另一具體實施例中，該雙相磷酸鹽為一羥基磷灰石-磷酸三鈣(HATCP)。於一較佳具體實施例中，該雙相磷酸鹽為一羥基磷灰石-β-磷酸三鈣(HAp-β-TCP)。 The porous aggregate of the present invention belongs to a biomedical material and has good biocompatibility. In one embodiment, the constituent material of the porous aggregate comprises hydroxyapatite (HAp), a β-tricalcium phosphate (β-TCP), and a hydroxyapatite- Hydroxapatite tricalcium phosphate (HATCP), alpha-tricalcium phosphate (α-TCP), bioactive glass ceramic, calcium monosulfate, bone cement Or a combination thereof. In a preferred embodiment, the porous aggregate is a porous aggregate comprising a dual phase phosphate. In another embodiment, the dual phase phosphate is monohydroxyapatite-tricalcium phosphate (HATCP). In a preferred embodiment, the dual phase phosphate is monohydroxyapatite-[beta]-tricalcium phosphate (HAp-[beta]-TCP).

於另一具體實施例中，該多孔性骨材是一多孔性陶瓷骨材。於一較佳具體實施例中，該多孔性陶瓷骨材是一以溫感水膠為模板所製備的多孔性陶瓷骨材。於一更佳具體實施例中，該以溫感水膠為模板所製備的多孔性陶瓷骨材之製作方法包含以下步驟：(a)合成氮-異丙基丙烯醯胺-甲基丙烯酸聚合物(poly(N-isopropylacrylamide-co-methacrylic acid)；(b) 將羥基磷灰石或磷酸鈣鹽類與分散劑混合，其中該分散劑為聚丙烯酸鈉鹽(polyacrylic acid；PAA)、聚甲基丙烯酸鈉(polymethacrylic acid；PMA)或聚乙烯醇(poly-vinyl-alcohol；PVA)；(c)混合步驟(a)之氮-異丙基丙烯醯胺-甲基丙烯酸聚合物與水以得出膠體溶液；(d)混合步驟(c)之膠體溶液與步驟(b)之產物以得出混合物；(e)加入高分子顆粒至步驟(d)之混合物中並攪拌以得出漿體，其中該高分子係聚乙烯，其顆粒體積係步驟(d)之混合物總體積之5%到20%；(f)將步驟(e)之漿體填入模板槽中；及(g)將步驟(f)之填入漿體的模板槽裝載於陶瓷坩堝中，然後送入高溫爐內以高溫燒結，以得出含多孔性陶瓷組合物。該以溫感水膠為模板所製備的多孔性陶瓷骨材之製備方法可參照美國專利公告號之第8940203號的專利案、臺灣專利公告號之第I411595號的專利案或Yin-Chih Fu的文獻(Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers：In Vitro and In Vivo evaluation,Journal of the Mechanical Behavior of Biomedical Materials,27：64-76,2013)。上述專利案及文獻之內容納入本發明中。 In another embodiment, the porous aggregate is a porous ceramic aggregate. In a preferred embodiment, the porous ceramic aggregate is a porous ceramic aggregate prepared by using a warm water gel as a template. In a more preferred embodiment, the method for preparing a porous ceramic aggregate prepared by using a temperature sensitive water gel as a template comprises the following steps: (a) synthesizing nitrogen-isopropyl acrylamide-methacrylic acid polymer (poly(N-isopropylacrylamide-co-methacrylic acid); (b) mixing hydroxyapatite or calcium phosphate with a dispersing agent, wherein the dispersing agent is polyacrylic acid (PAA), polymethyl Polymethacrylic acid (PMA) or poly-vinyl-alcohol (PVA); (c) mixing the nitrogen-isopropyl acrylamide-methacrylic acid polymer of step (a) with water to give a colloidal solution; (d) mixing the colloidal solution of step (c) with the product of step (b) to obtain a mixture; (e) adding the polymer particles to the mixture of step (d) and stirring to obtain a slurry, wherein The polymer-based polyethylene has a particle volume of 5% to 20% of the total volume of the mixture of the step (d); (f) the slurry of the step (e) is filled into the template tank; and (g) the step ( f) The template tank filled with the slurry is placed in a ceramic crucible and then sent to a high temperature furnace for sintering at a high temperature to obtain a porous ceramic composite The preparation method of the porous ceramic aggregate prepared by using the temperature sensitive water gel as a template can be referred to the patent of No. 8940203 of the US Patent Publication No. No. I411595, or the patent of Yin-Chih of Taiwan Patent Publication No. I411595. (Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers: In Vitro and In Vivo evaluation, Journal of the Mechanical Behavior of Biomedical Materials, 27: 64-76, 2013). The contents of the literature and the contents of the literature are incorporated in the present invention.

於一具體實施例中，該多孔性骨材的孔隙率為15-85%。於一較佳具體實施例中，該多孔性骨材的孔隙率為20-75%。於一更佳具體實施例中，該多孔性骨材的孔隙率為40-75%。於另一具體實施例中，該多孔性骨材的孔洞大小為5-1000μm。於一較佳具體實施例中，該多孔性骨材的孔洞大小為50-800μm。於一更佳具體實施例中，該多孔性骨材的孔洞大小為100-600μm。 In one embodiment, the porous aggregate has a porosity of 15-85%. In a preferred embodiment, the porous aggregate has a porosity of 20-75%. In a more preferred embodiment, the porous aggregate has a porosity of 40-75%. In another embodiment, the porous aggregate has a pore size of from 5 to 1000 μm. In a preferred embodiment, the porous aggregate has a pore size of 50-800 μm. In a more preferred embodiment, the porous aggregate has a pore size of from 100 to 600 μm.

本發明的多孔性骨材具有骨傳導的功效。本文中使用之「骨 傳導」係指一植入基質(如骨材)被容許或加強於其表面或其孔洞、通道或其他內部空隙之新骨生長。當移植材料或移植基質(如骨材)可作為用於新骨生長之支架時，其被視為具有”骨傳導性”。本發明的多孔性骨材可作為骨架而使修補中主骨缺損位置之骨母細胞(成骨細胞)擴散及產生新骨。此外，本發明的多孔性骨材可依被修補骨骼區域或特定骨缺損所需形狀而設計成特定形狀。根據一具體實施例中，該多孔性骨材係一多孔塊狀組成。該多孔性骨材之適當形狀包括球形、片狀、立方形、楔形、楕圓形、圓柱形，或其組合。 The porous aggregate of the present invention has the effect of bone conduction. As used herein, "bone conduction" refers to the growth of new bone that is implanted into a substrate (e.g., an aggregate) that is tolerated or strengthened on its surface or its pores, channels, or other internal voids. When a graft material or a graft matrix (such as an aggregate) can be used as a scaffold for new bone growth, it is considered to have "osteoconductivity." The porous aggregate of the present invention can be used as a skeleton to diffuse osteoblasts (osteoblasts) at the position of the main bone defect in the repair and to generate new bone. Further, the porous aggregate of the present invention can be designed into a specific shape depending on the shape of the bone to be repaired or the desired shape of the specific bone defect. According to a specific embodiment, the porous aggregate is composed of a porous block. Suitable shapes for the porous aggregate include a sphere, a sheet, a cuboid, a wedge, a dome, a cylinder, or a combination thereof.

本發明的包含該促進骨生長藥物的藥物釋放奈米載體需要置入於該多孔性骨材內，即包含該促進骨生長藥物的藥物釋放奈米載體分佈於該多孔性骨材中。因此，該藥物釋放奈米載體藉由局部長效緩釋促進骨生長藥物，加強該多孔性骨材應用於骨頭癒合之功效。本文中「促進骨生長藥物」包含但不限於具有加速或促進骨折、截骨手術、骨延長手術及骨移植手術等中損傷之骨組織的修復過程之藥物。藉由該促進骨生長藥物之作用縮短骨組織修復為止之期間，或者藉由使修復後之骨強度恢復骨缺損骨折前水準。故該促進骨生長藥物係一種促進骨組織生長與維持骨質量之藥物。如本文使用用語「促進」一詞是指活性、反應或其他生物參數的增進。 The drug-releasing nanocarrier comprising the drug for promoting bone growth of the present invention needs to be placed in the porous aggregate, that is, the drug-releasing nanocarrier containing the drug for promoting bone growth is distributed in the porous aggregate. Therefore, the drug-releasing nanocarrier promotes the bone growth drug by local long-acting sustained release, and enhances the effect of the porous aggregate on bone healing. The "promoting bone growth drug" herein includes, but is not limited to, a drug having a repair process for accelerating or promoting fracture, bone osteotomy, bone lengthening surgery, and bone graft surgery. The period of bone tissue repair is shortened by the action of promoting the bone growth drug, or the bone defect is restored to the pre-fracture level of the bone defect. Therefore, the drug promoting bone growth is a drug that promotes bone growth and maintains bone quality. As used herein, the term "promoting" refers to an increase in activity, response, or other biological parameter.

於一具體實施例中，該促進骨生長藥物具有骨誘導作用。本文中所使用之「骨誘導作用」一詞係指刺激骨祖細胞(osteoprogenitor cells)分化成骨母細胞、其接著開始形成新骨之過程。當一化學或生物組成物能 刺激原始、未分化和多功能細胞變成成骨細胞系時被稱為具有”骨誘導性”。因此，該促進骨生長藥物能誘發或刺激骨誘導生長因子的產生。於另一具體實施例，該骨誘導生長因子包含但不限於骨成型蛋白(bone morphogenetic protein)、骨鈣素(ostercalcin)、成骨素(osteogenin)或鹼性磷酸酶(alkaline phosphatase；ALP)等。該骨誘導生長因子能主動促進或誘導骨細胞分化生長，以有效縮短骨缺陷癒合。 In a specific embodiment, the bone growth promoting drug has an osteoinductive effect. As used herein, the term "osteoinductive action" refers to the process of stimulating osteoprogenitor cells to differentiate into osteoblasts, which in turn begin to form new bone. A chemical or biological composition is said to have "osteoinductive" when it stimulates primitive, undifferentiated, and multifunctional cells to become osteoblastic lines. Therefore, the drug for promoting bone growth can induce or stimulate the production of osteoinductive growth factors. In another embodiment, the osteoinductive growth factor comprises, but is not limited to, bone morphogenetic protein, osteocalcin, osteogenin or alkaline phosphatase (ALP), and the like. . The osteoinductive growth factor can actively promote or induce the differentiation and growth of bone cells to effectively shorten the healing of bone defects.

此外，於另一具體實施例中，該促進骨生長藥物誘發血管生成。於一較佳具體實施例中，該誘發血管生成係透過增加一血管新生蛋白的生成。於一更佳具體實施例中，該血管新生蛋白包含一溫偉伯氏因子(von Willebrand factor；vWF)。 Moreover, in another embodiment, the bone promoting growth promoting drug induces angiogenesis. In a preferred embodiment, the induced angiogenic system increases the production of an angiogenic protein. In a more preferred embodiment, the angiogenic protein comprises a von Willebrand factor (vWF).

於另一具體實施例中，該促進骨生長藥物為一油溶性促進骨生長藥物。於一較佳具體實施例中，該油溶性促進骨生長藥物包含一史他汀類(statin)藥物。於一更佳具體實施例中，該史他汀類藥物包含辛伐他汀(simvastatin)。 In another embodiment, the bone growth promoting drug is an oil soluble drug that promotes bone growth. In a preferred embodiment, the oil soluble bone growth promoting drug comprises a statin drug. In a more preferred embodiment, the statin comprises simvastatin.

該藥物釋放奈米載體即一藥物載體，其具有減低副作用、改善藥物的耐受性、提高服藥的便利性、維持較長的療效時程。理想的藥物載體必須是生物可分解性(biodegradable)、小尺寸、穩定性佳、高藥物負載率(high loading capacity)、在體內有長循環時間(prolonged circulation)及病灶的大量積聚。本文所述之「兩性分子載體」意指以兩性分子所組成之載體。於一具體實施例中，該兩性分子載體為一微脂體(liposome)、一高分子微胞(polymeric micelle)或一樹枝狀高分子聚合物(dendrimer)。其 中，微脂體係由兩性分子形成，微脂體是利用磷脂雙分子層膜所形成的囊泡，其中最具有代表性的是卵磷脂與磷脂醯膽鹼，此外膽固醇也是脂質體另一個重要組成成分。而高分子微胞為一具有核-殼型(Core-shell)形態的奈米球，而高分子微胞是由兩性高分子(aliphatic polyester)鏈段所組成，一端帶有親水性鏈段，另一端則為親油性鏈段，兩性高分子鏈段中的親油性鏈段在水相中會透過凡德瓦力結合成一核心疏水區，該區域可作為脂溶性藥物的儲存槽，親水性鏈段則位於疏水核心的外部以增進高分子微胞於水相中的結構穩定性；因此，高分子會自組裝(selfassembly)形成奈米尺寸的微胞。故藉由奈米微胞結構的獨特性及尺寸效應，奈米微胞在做為藥物載體(drug Carrier)控制藥物釋放的材料上，於藥物傳送及基因治療等領域具備極大的應用潛力。目前含有藥物的高分子微胞的製備方法是將藥物溶解、陷入、附著於基材，或是利用基材將藥物包覆，形成奈米微粒、奈米球、奈米膠囊等。 The drug releases a nano carrier, that is, a drug carrier, which has the advantages of reducing side effects, improving drug tolerance, improving the convenience of taking the drug, and maintaining a long therapeutic time course. The ideal drug carrier must be biodegradable, small size, good stability, high loading capacity, prolonged circulation in the body, and substantial accumulation of lesions. As used herein, "amphoteric carrier" means a carrier composed of amphiphilic molecules. In one embodiment, the amphiphilic carrier is a liposome, a polymeric micelle or a dendrimer. Among them, the microlipid system is formed by amphiphilic molecules, and the liposome is a vesicle formed by the phospholipid bilayer membrane. The most representative one is lecithin and phospholipid choline. In addition, cholesterol is another important component of liposome. ingredient. The polymer microcell is a nanosphere having a core-shell shape, and the polymer microcell is composed of an amphoteric polyester segment with a hydrophilic segment at one end. The other end is a lipophilic segment, and the lipophilic segment in the amphoteric polymer segment is combined in the aqueous phase by van der Waals to form a core hydrophobic region, which can serve as a storage tank for the fat-soluble drug, a hydrophilic chain. The segment is located outside of the hydrophobic core to enhance the structural stability of the polymer microcells in the aqueous phase; therefore, the polymer will self-assemble to form nano-sized micelles. Therefore, by virtue of the uniqueness and size effect of the nano-cell structure, nano-cells have great potential for application in drug delivery and gene therapy as materials for controlling drug release by drug carriers. At present, a polymer microcapsule containing a drug is prepared by dissolving, immersing, or adhering to a substrate, or coating a drug with a substrate to form a nanoparticle, a nanosphere, a nanocapsule, or the like.

於一具體實施例中，該藥物釋放奈米載體為一高分子微胞。於一較佳具體實施例中，該高分子微胞係由兩性高分子所構成。於一更佳具體實施例中，該兩性高分子係選自於由聚酯、聚酸酐及聚醚所組成之群組。本發明所述之聚酯、聚酸酐、及聚醚沒有特別限制，只要是具有生物相容性與生物降解性者，皆可使用於本發明。特別是已獲准可實施於人體或動物體使用的生物材料，由於已通過生物安全性等的測試，為本發明較佳選用的材料。 In one embodiment, the drug-releasing nanocarrier is a polymeric microcell. In a preferred embodiment, the polymeric microcells are composed of an amphoteric polymer. In a more preferred embodiment, the amphoteric polymer is selected from the group consisting of polyesters, polyanhydrides, and polyethers. The polyester, polyanhydride, and polyether of the present invention are not particularly limited, and any of those having biocompatibility and biodegradability can be used in the present invention. In particular, biological materials that have been approved for use in human or animal body are preferred materials for the present invention because they have been tested by biosafety and the like.

此處所用之「聚酯」一詞包含聚己內酯(polycaprolactone； PCL)、聚戊內酯(polyvalerolactone；PVL)、聚丙內酯(polypropiolactone；PPL)、聚丁內酯(polybutyrolactone；PBL)、聚乳酸甘醇酸(poly(lactide-co-glycolide)；PLGA)、聚乳酸(polylactic acid；PLA)、聚乙交酯(polyglycolide；PGA)、聚異丁基氰基丙烯酸酯(poly(isobutylcyanoacrylate)；PIBCA)、聚間苯二甲酸(polyisophthalic acid；PIPA)、聚1,4-苯二丙酸(poly-1,4-phenylene dipropionic acid；PPDA)、聚杏仁酸(poly(mandelic acid)；PMDA)、聚富馬酸丙二酯(poly(propylene fumarate)；PPF)、聚原酸酯(poly(ortho ester)；POE)、或上述之組合。 The term "polyester" as used herein includes polycaprolactone (PCL), polyvalerolactone (PVL), polypropiolactone (PPL), polybutyrolactone (PBL), Poly(lactide-co-glycolide; PLGA), polylactic acid (PLA), polyglycolide (PGA), poly(isobutylcyanoacrylate) ; PIBCA), polyisophthalic acid (PIPA), poly-1,4-phenylene dipropionic acid (PPDA), poly(mandelic acid); PMDA ), poly(propylene fumarate; PPF), poly(ortho ester); POE), or a combination thereof.

此處所用之「聚酸酐」一詞包含聚癸二酸酐(poly(sebacic anhydride)；PSA)、聚雙羧基對苯丙醇酸酐(poly-(bis(p-carboxyphenoxy)propane anhydride)；PCPPA)、聚雙對羧基甲烷酸酐(poly-(bis(p-carboxy)methane anhydride)；PCMA)、聚雙羧基對苯丙醇與癸二酸的聚合物(poly-carboxyphenoxypropane-co-sebacic acid；p(CPP-SA))、聚雙羧基對苯丙醇與間苯二甲酸的聚合物(poly-carboxyphenoxypropane-co-isophthalic acid；p(CPP-IPA))、聚脂肪酸二元體與癸二酸的聚合物(poly(fatty acid dimmer-co-sebacic acid)；p(FAD-SA))、或上述之組合。 The term "polyanhydride" as used herein includes poly(sebacic anhydride; PSA), poly-(bis(p-carboxyphenoxy)propane anhydride; PCPPA), Poly-carboxyphenoxypropane-co-sebacic acid (PCP), poly-carboxyphenoxypropane-co-sebacic acid (pPP) -SA)), poly-carboxyphenoxypropane-co-isophthalic acid (p(CPP-IPA)), polymer of poly-fatty acid binary and sebacic acid (poly(fatty acid dimmer-co-sebacic acid); p(FAD-SA)), or a combination thereof.

此處所用之「聚醚」一詞包含聚乙二醇(poly(ethylene glycol)；PEG)、聚丙二醇(poly(propylene glycol)；PPG)、聚丁二醇(poly(butylene glycol)；PBG)、或上述之組合。 The term "polyether" as used herein includes poly(ethylene glycol); PEG, poly(propylene glycol; PPG), poly(butylene glycol); PBG. Or a combination of the above.

於一具體實施例中，該兩性高分子為一聚乳酸甘醇酸-聚乙 二醇(PLGA-PEG)。 In one embodiment, the amphoteric polymer is a polylactic acid glycol-polyethylene glycol (PLGA-PEG).

在本發明所指的「載體」意指可攜帶活性物質(如藥物)的攜帶物質。於一具體實施例中，該藥物釋放奈米載體為一緩釋型的藥物釋放奈米載體。故該藥物釋放奈米載體會控制緩慢釋放藥物，以有效促進骨生長。在本發明所指的「緩慢釋放」或「緩釋」意指在一定時間內，以穩定速率逐漸釋放活性物質(如藥物)。緩慢釋放或緩釋狀態係可讓活性物質在個體(如動物體、人類)的體內或血液內維持一定濃度持續一段時間。 The term "carrier" as used in the present invention means a carrier substance which can carry an active substance such as a drug. In one embodiment, the drug release nanocarrier is a sustained release drug release nanocarrier. Therefore, the release of the drug carrier by the drug controls the slow release of the drug to effectively promote bone growth. The term "slow release" or "slow release" as used in the present invention means that the active substance (e.g., drug) is gradually released at a steady rate for a certain period of time. The slow release or sustained release state allows the active substance to maintain a certain concentration in the body or blood of an individual (such as an animal, a human) for a period of time.

本發明亦提供一種治療骨缺損的方法，其包含給一種含藥物之複合性支架予一具有一骨缺損的一個體，其中該含藥物之複合性支架植入進該骨缺損的部位，其中該含藥物之複合性支架包含一多孔性骨材以及一藥物釋放奈米載體，其中該藥物釋放奈米載體包含一促進骨生長藥物，且分布於該多孔性骨材中。 The present invention also provides a method for treating a bone defect, comprising: administering a drug-containing composite stent to a body having a bone defect, wherein the drug-containing composite stent is implanted into a site of the bone defect, wherein the The drug-containing composite scaffold comprises a porous aggregate and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous aggregate.

人體內骨頭的體積及重量是佔最大的部份，最主要的功能即是協助身體行動以及支撐身體結構。臨床的治療中常出現骨頭缺損情形，造成的原因主要是因為嚴重骨折所造成的缺損，骨折未癒合造成缺損，骨髓炎後造成骨缺損，骨腫瘤術後缺損，脊椎體塌陷，人工關節再重建時髖臼骨缺損等。目前臨床上，仍以骨移植術為常見的處理方式。因此，本發明是以含藥物之複合性支架進行骨移植以治療骨缺損。 The volume and weight of bones in the human body are the largest part. The most important function is to assist the body in exercising and supporting the body structure. Bone defects often occur in clinical treatment, mainly due to defects caused by severe fractures, defects caused by unhealed fractures, bone defects after osteomyelitis, postoperative defects of bone tumors, collapse of vertebral bodies, and reconstruction of artificial joints Acetabular defects and so on. At present, bone grafting is still a common treatment in clinical practice. Accordingly, the present invention is a bone graft for treating bone defects with a drug-containing composite stent.

本文中「骨缺損」一詞包含需恢復骨原狀的骨任何部位的缺損，不管其缺損原因為何，例如，由外科手術引起的，瘤割除引起的，潰瘍引起的，植入引起的，或骨折引起的，都是骨缺損或骨組織缺損。於一 具體實施例中，該骨缺損為骨折、或骨骼移植部位或植入部位。本發明的含藥物之複合性支架應用於一個體上各處骨缺損的部位的治療，若為頭蓋骨等僅由膜內骨化形成之骨骼以外之骨組織受損患者，無特別限定，若為蓋骨等僅由膜內骨化形成之骨骼以外之骨骼，則任意部位之損傷皆可適用，宜為頭部(眼眶骨、顴骨、下顎骨)、體幹部(肋骨、髖骨、頸椎、胸椎、腰椎、骶骨、尾骨)、上肢(肩胛骨、鎖骨、肱骨、肘、橈骨、尺骨、舟骨、鉤骨、掌骨、指骨)、下肢(股關節、大腿骨、脛骨、腓骨、足關節、踵骨、舟骨、蹠骨)。又，骨組織之損傷形態亦無特別限定，例如截骨術或骨延長手術所切斷之骨的癒合、骨折(骨斷裂、龜裂、粉碎性骨折、複雜性骨折等)之治療等。 In this context, the term "bone defect" includes a defect in any part of the bone that needs to be restored to the original bone, regardless of the cause of the defect, for example, caused by surgery, caused by a tumor, caused by an ulcer, caused by an implant, or fractured. All caused by bone defects or bone tissue defects. In a specific embodiment, the bone defect is a fracture, or a bone graft site or an implant site. The drug-containing composite stent of the present invention is applied to a site of a bone defect in a body, and is not particularly limited as long as it is a bone tissue damaged by a bone formed by ossification in the membrane, such as a skull bone. For bones other than the bone formed by ossification in the membrane, any part of the injury can be applied. It should be the head (eye, humerus, mandible) and body trunk (rib, hip, cervical, Thoracic vertebrae, lumbar vertebrae, humerus, coccyx), upper limbs (scapula, clavicle, humerus, elbow, humerus, ulna, scaphoid, hookbone, metacarpal, phalanx), lower extremity (femoral joint, femur, tibia, tibia, tibia, foot joint, iliac crest) Bone, scaphoid, tibia). Further, the damage form of the bone tissue is not particularly limited, and for example, the healing of the bone cut by the osteotomy or the bone extension surgery, and the treatment of the fracture (bone fracture, crack, comminuted fracture, complex fracture, etc.).

本文中用語「個體(subject)」一詞係指動物。於一較佳具體實施例中，該個體係指哺乳動物。於一更佳具體實施例中，該個體係指人類。 The term "subject" as used herein refers to an animal. In a preferred embodiment, the system refers to a mammal. In a more preferred embodiment, the system refers to a human.

於一具體實施例中，該多孔性骨材的組成材料包含一羥基磷灰石(hydroxyapatite；HAp)、一β-磷酸三鈣(β-tricalcium phosphate；β-TCP)、一羥基磷灰石-磷酸三鈣(hydroxyapatite tricalcium phosphate；HATCP)、一α-磷酸三鈣(α-tricalcium phosphate；α-TCP)、一生物活性玻璃陶瓷(bioactive glass ceramic)、一硫酸鈣、一骨水泥(bone cement)或其上述組合。於一較佳具體實施例中，該多孔性骨材為含有雙相磷酸鹽的多孔性骨材。於另一具體實施例中，該雙相磷酸鹽為一羥基磷灰石-磷酸三鈣(HATCP)。於一較佳具體實施例中，該雙相磷酸鹽為一羥基磷灰石-β-磷酸三鈣 (HAp-β-TCP)。 In one embodiment, the constituent material of the porous aggregate comprises hydroxyapatite (HAp), a β-tricalcium phosphate (β-TCP), and a hydroxyapatite- Hydroxapatite tricalcium phosphate (HATCP), alpha-tricalcium phosphate (α-TCP), bioactive glass ceramic, calcium monosulfate, bone cement Or a combination thereof. In a preferred embodiment, the porous aggregate is a porous aggregate comprising a dual phase phosphate. In another embodiment, the dual phase phosphate is monohydroxyapatite-tricalcium phosphate (HATCP). In a preferred embodiment, the biphasic phosphate is monohydroxyapatite-β-tricalcium phosphate (HAp-β-TCP).

於另一具體實施例中，該多孔性骨材是一多孔性陶瓷骨材。於一較佳具體實施例中，該多孔性陶瓷骨材是一以溫感水膠為模板所製備的多孔性陶瓷骨材。 In another embodiment, the porous aggregate is a porous ceramic aggregate. In a preferred embodiment, the porous ceramic aggregate is a porous ceramic aggregate prepared by using a warm water gel as a template.

於一具體實施例中，該多孔性骨材的孔隙率為15-85%。於一較佳具體實施例中，該多孔性骨材的孔隙率為20-75%。於一更佳具體實施例中，該多孔性骨材的孔隙率為40-75%。於另一具體實施例中，該多孔性骨材的孔洞大小為5-1000μm。於一較佳具體實施例中，該多孔性骨材的孔洞大小為50-800μm。於一更佳具體實施例中，該多孔性骨材的孔洞大小為100-600μm。 In one embodiment, the porous aggregate has a porosity of 15-85%. In a preferred embodiment, the porous aggregate has a porosity of 20-75%. In a more preferred embodiment, the porous aggregate has a porosity of 40-75%. In another embodiment, the porous aggregate has a pore size of from 5 to 1000 μm. In a preferred embodiment, the porous aggregate has a pore size of 50-800 μm. In a more preferred embodiment, the porous aggregate has a pore size of from 100 to 600 μm.

於一具體實施例中，該促進骨生長藥物具有骨誘導作用。因此，本發明的含藥物之複合性支架可於需治療的骨缺損的位置促進形成形態正常的成熟骨。成熟骨為任何型的骨，不論是皮質骨還是小梁骨，較新生模型的不成熟骨或軟骨性骨都礦物化。形態上正常的骨即是組織學上檢查正常的骨(即由軟骨內骨或膜樣片狀骨構成的骨，包括有成骨細胞和破骨細胞的骨髓腔)。此與骨折修復第一階段所見的有纖維基質的骨痂形成相反。是以，骨誘導」一詞不僅意謂骨折時所見的骨再生的加速，也意謂刺激骨的形成至其正常的形態。於一較佳具體實施例中，該促進骨生長藥物誘發或刺激骨誘導生長因子的產生。於一更佳具體實施例，該骨誘導生長因子包含骨成型蛋白(bone morphogenetic protein)、骨鈣素(ostercalcin)、成骨素(osteogenin)或鹼性磷酸酶(alkaline phosphatase；ALP)。 In a specific embodiment, the bone growth promoting drug has an osteoinductive effect. Thus, the drug-containing composite scaffold of the present invention promotes the formation of mature bone of normal morphology at the site of the bone defect to be treated. Mature bone is any type of bone, whether cortical or trabecular, mineralized than the immature or cartilage bone of the new model. A morphologically normal bone is a histologically examined normal bone (ie, a bone composed of endochondral bone or membrane-like bone, including the medullary cavity with osteoblasts and osteoclasts). This is in contrast to the formation of osteophytes with fibrous stroma seen in the first stage of fracture repair. Therefore, the term "osseoinduction" means not only the acceleration of bone regeneration seen during fracture, but also the stimulation of bone formation to its normal form. In a preferred embodiment, the bone growth promoting drug induces or stimulates the production of osteoinductive growth factors. In a more preferred embodiment, the osteoinductive growth factor comprises a bone morphogenetic protein, ostercalcin, osteogenin or alkaline phosphatase (ALP).

此外，於另一具體實施例中，該促進骨生長藥物誘發血管生成。於一較佳具體實施例中，該誘發血管生成係透過增加一血管新生蛋白的生成。於一更佳具體實施例中，該血管新生蛋白包含一溫偉伯氏因子(von Willebrand factor；vWF)。 Moreover, in another embodiment, the bone promoting growth promoting drug induces angiogenesis. In a preferred embodiment, the induced angiogenic system increases the production of an angiogenic protein. In a more preferred embodiment, the angiogenic protein comprises a von Willebrand factor (vWF).

於一具體實施例中，該促進骨生長藥物為一油溶性促進骨生長藥物。於一較佳具體實施例中，該油溶性促進骨生長藥物包含一史他汀類(statin)藥物。於一更佳具體實施例中，該史他汀類藥物包含辛伐他汀(simvastatin)。 In one embodiment, the bone growth promoting drug is an oil soluble drug that promotes bone growth. In a preferred embodiment, the oil soluble bone growth promoting drug comprises a statin drug. In a more preferred embodiment, the statin comprises simvastatin.

於另一具體實施例中，該藥物釋放奈米載體為一微脂體(liposome)、一高分子微胞(polymeric micelle)、或一樹枝狀高分子聚合物(dendrimer)。於一較佳具體實施例中，該藥物釋放奈米載體為一高分子微胞。於一更佳具體實施例中，該高分子微胞係選自於由聚酯、聚酸酐及聚醚所構成之群組所組成。 In another embodiment, the drug-releasing nanocarrier is a liposome, a polymeric micelle, or a dendrimer. In a preferred embodiment, the drug-releasing nanocarrier is a polymeric microcell. In a more preferred embodiment, the polymeric microcells are selected from the group consisting of polyesters, polyanhydrides, and polyethers.

於另一具體實施例中，該高分子微胞係由兩性高分子所構成。於一較佳具體實施例中，該兩性高分子為一聚乳酸甘醇酸-聚乙二醇(PLGA-PEG)。 In another embodiment, the polymeric microcells are comprised of an amphoteric polymer. In a preferred embodiment, the amphoteric polymer is a polylactic acid glycolic acid (PLGA-PEG).

因此，本發明的含藥物之複合性支架可在或接近骨折、骨頭損傷或骨頭缺損的位置處形成一控制藥物釋放型植入物。於一具體實施例中，該藥物釋放奈米載體為一緩釋型的藥物釋放奈米載體。藉由該藥物釋放奈米載體控制緩慢釋放出一有效劑量的促進骨生長藥物，以有效穩定地促進骨缺損處的骨生長。本文中「有效劑量」一詞為一治療劑量可在特定條 件下可預防、降低、阻止或逆轉一個體的一症狀的發展，或部分、完全舒緩該個體開始接受治療時於特別情況下已存在的症狀。 Thus, the drug-containing composite stent of the present invention can form a controlled drug release implant at or near the location of a fracture, bone injury or bone defect. In one embodiment, the drug release nanocarrier is a sustained release drug release nanocarrier. The release of the nanocarrier by the drug controls the slow release of an effective dose of the drug for promoting bone growth to effectively and stably promote bone growth at the bone defect. As used herein, the term "effective dose" is a therapeutic dose that prevents, reduces, prevents, or reverses the development of a symptom of a body under certain conditions, or partially, completely relieves the individual's existence in a particular condition when it begins treatment. Symptoms.

以本發明的紐西蘭白兔的骨缺損模式(φ：3.5~3.6mm、L：10mm)來看，其骨缺損約為0.384立方公分(cc)，而使用160μl SIM/PP(含10μg simvastatin)，可換算出個體內之每立方公分(cc)骨缺損大小應給予416.7μl SIM/PP(含26μg simvastatin)奈米藥物載體。因此，依據該個體之骨缺損大小的情況，每立方公分(cc)骨缺損相對應所給予之該藥物釋放奈米載體所包裹之該促進骨生長藥物的有效劑量大於20μg，較佳為大於26μg。 According to the bone defect model of the New Zealand white rabbit of the present invention (φ: 3.5-3.6 mm, L: 10 mm), the bone defect is about 0.384 cubic centimeters (cc), and 160 μl of SIM/PP (containing 10 μg of simvastatin) is used. ), 416.7 μl of SIM/PP (containing 26 μg simvastatin) nano drug carrier should be administered for each cubic centimeter (cc) of bone defect size in the individual. Therefore, depending on the size of the bone defect of the individual, the effective dose of the bone-promoting drug coated per cubic centimeter (cc) of the bone defect corresponding to the drug-released nanocarrier is greater than 20 μg, preferably greater than 26 μg. .

於本發明中，該藥物釋放奈米載體所釋放出的該促進骨生長藥物之有效劑量超過0.5μM以上。於一具體實施例中，該藥物釋放奈米載體的藥物持續釋放天數為10天以上。該藥物釋放奈米載體的藥物持續釋放天數為7天以上。於一更佳具體實施例中，該藥物釋放奈米載體的藥物持續釋放天數為3天以上。此外，該藥物釋放奈米載體於給予後第1及2天所釋放出的該促進骨生長藥物之有效劑量超過0.5μM以上。 In the present invention, the effective amount of the drug for promoting bone growth released by the drug release nanocarrier exceeds 0.5 μM or more. In one embodiment, the drug releases the nanocarrier for a sustained release of the drug for more than 10 days. The drug releases the nanocarrier for a sustained release of the drug for more than 7 days. In a more preferred embodiment, the drug releases the nanocarrier for a sustained release of the drug for more than 3 days. Further, the drug-releasing nanocarrier has an effective dose of the bone-promoting drug released on the first and second days after administration of more than 0.5 μM.

本發明所提供之含藥物之複合性支架，與其他習用技術相互比較時，更具有下列之優點： The drug-containing composite stent provided by the invention has the following advantages when compared with other conventional techniques:

傳統的人工骨材於骨缺損的治療上，會有不癒合或是碰上較大骨缺損無法完全癒合的情況，此時需要另藉助骨誘導的因子來促進骨生長以填補無法癒合之骨缺損處；但臨床醫師又須根據經驗來判定多少骨缺損區域給予多少促進骨生長藥物，因此，臨床上治療會缺乏效率。故本發 明將促進骨生長藥物以奈米載體包覆，使其能直接融入且分佈於人工骨材中；而奈米載體緩慢釋放促進骨生長藥物之特性，故該人工骨材於植入故缺損處後，能準確地釋放在促進骨生長藥物治療範圍且控制釋放速率，提高骨缺損治療的效果，能達成有效藥物的減量，且無需臨床醫師另外評估要額外給予多少藥物劑量，僅需如先前於骨缺損處進行人工骨材植入即可。 In the treatment of bone defects, traditional artificial bones may not heal or encounter large bone defects and cannot completely heal. In this case, bone-inducing factors are needed to promote bone growth to fill the bone defect that cannot be healed. However, clinicians must rely on experience to determine how much bone-promoting drug is administered to the bone defect area. Therefore, clinical treatment may be inefficient. Therefore, the invention promotes the bone growth drug coated with the nano carrier, so that it can be directly integrated into and distributed in the artificial bone material; and the nano carrier slowly releases the characteristics of the drug for promoting bone growth, so the artificial bone material is implanted. After the defect, it can accurately release the therapeutic range of bone growth and control the release rate, improve the effect of bone defect treatment, achieve effective drug reduction, and do not need additional evaluation by the clinician to add additional drug dose, just as Artificial bone implants were previously performed at the bone defect.

故本發明的含藥物之複合性支架具有局部長效緩釋藥物的用途，且因為能調整最適合藥物釋放濃度，有利於臨床上使用。是以，本發明的含藥物之複合性支架不但具有促進骨骼生成的功能，且無蛋白質類藥物不易保存之缺點。 Therefore, the drug-containing composite stent of the present invention has the use of a local long-acting sustained-release drug, and is advantageous for clinical use because it can adjust the most suitable drug release concentration. Therefore, the drug-containing composite stent of the present invention not only has a function of promoting bone formation, but also has no disadvantage that proteinaceous drugs are difficult to store.

圖1為含辛伐他汀之聚乳酸甘醇酸-聚乙二醇奈米藥物載體之結構圖。SIM：辛伐他汀(Simvastatin)；以及PP：聚乳酸甘醇酸-聚乙二醇(PLGA-PEG)。 Figure 1 is a structural diagram of a polylactic acid glycolic acid-polyethylene glycol nano drug carrier containing simvastatin. SIM: Simvastatin; and PP: polylactic acid glycolic acid-polyethylene glycol (PLGA-PEG).

圖2為包裹辛伐他汀之微脂體奈米粒子的結構圖。SIM：辛伐他汀(Simvastatin)。 Figure 2 is a structural diagram of the liposome nanoparticles coated with simvastatin. SIM: Simvastatin.

圖3A為含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷之每日的辛伐他汀釋放量；及圖3B為含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷之辛伐他汀之累積釋放率。80μL Sim-PP/BC：含有80μL的包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷；160μL Sim-PP/BC：含有160μL的包覆辛伐他汀之PLGA-PEG 奈米粒子之多孔性生物陶瓷。 3A is the daily simvastatin release amount of the porous bioceramic containing the simvastatin-coated PLGA-PEG nanoparticle; and FIG. 3B is the porous containing the simvastatin-coated PLGA-PEG nanoparticle. Cumulative release rate of simvastatin in bioceramics. 80 μL Sim-PP/BC: Porous bioceramics containing 80 μL of simvastatin-coated PLGA-PEG nanoparticles; 160 μL Sim-PP/BC: 160 μL of simvastatin-coated PLGA-PEG nanoparticles Porous bioceramics.

圖4為含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷對D1細胞株的促骨生成影響。ALP：鹼性磷酸酶。控制組：僅使用培養基；辛伐他汀組：加入0.5μM辛伐他汀(SIM)於培養基中；以及辛伐他汀釋放組：加入第一天含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷之辛伐他汀(SIM)釋放液於培養基中。以各組別的第5天之ALP活性進行比較，**：p<0.01；***：p<0.001。 Figure 4 is a graph showing the effect of porous bioceramics containing PLVA-PEG nanoparticles coated with simvastatin on the osteogenic effect of D1 cell lines. ALP: alkaline phosphatase. Control group: medium only; simvastatin group: 0.5 μM simvastatin (SIM) was added to the medium; and simvastatin release group: PLGA-PEG nanoparticles containing simvastatin were added on the first day. The simvastatin (SIM) release solution of the porous bioceramic is in the medium. The ALP activity on day 5 of each group was compared, **: p < 0.01; ***: p < 0.001.

圖5A為紐西蘭白兔缺損模式之X光追蹤之結果；圖5Bi為紐西蘭白兔缺損模式之H&E染色之結果；圖5Bii為紐西蘭白兔缺損模式之H&E染色之定量結果，藉由影像軟體定量圖5Bi中H&E染色結果，以計算出新生骨形成率(new bone formation)；以及圖5C為紐西蘭白兔缺損模式之vWF之免疫組織染色的結果，其中vWF所呈顏色為褐色，100X的影像為10X的影像中紅框部分放大10倍的結果。BC組(數量為3)：以生物陶瓷(Bioceramics；BC)治療；BC+80μL SIM/PP NPs組(數量為3)：以含有80μL的包覆辛伐他汀之PLGA-PEG奈米粒子(5μg的辛伐他汀)之生物陶瓷治療；及BC+160μL SIM/PP NPs組(數量為3)：以含有160μL的包覆辛伐他汀之PLGA-PEG奈米粒子(10μg的辛伐他汀)之生物陶瓷治療。比例尺：10000μm。 Figure 5A shows the X-ray tracking results of the New Zealand white rabbit defect pattern; Figure 5Bi shows the H&E staining results of the New Zealand white rabbit defect pattern; Figure 5Bii shows the quantitative results of H&E staining of the New Zealand white rabbit defect pattern. The results of H&E staining in Figure 5Bi were quantified by image software to calculate the new bone formation; and Figure 5C is the result of immunohistochemical staining of vWF in the New Zealand white rabbit defect pattern, in which the color of vWF was In brown, the 100X image is a 10-fold magnified result of the red frame in the 10X image. BC group (quantity 3): treated with bioceramics (BC); BC + 80 μL SIM/PP NPs group (3 in number): PLGA-PEG nanoparticles containing 80 μL of coated simvastatin (5 μg Bioceramic treatment with simvastatin; and BC+160 μL SIM/PP NPs group (quantity 3): organisms containing 160 μL of simvastatin-coated PLGA-PEG nanoparticles (10 μg of simvastatin) Ceramic treatment. Scale bar: 10000 μm.

圖6A為大鼠頭蓋骨缺損之Micro-CT追蹤之影像圖。圖6B為大鼠頭蓋骨缺損之缺損處骨質新生之分析結果。控制組(數量為3)：不用任何生物陶瓷；BC組(數量為3)：僅使用生物陶瓷；BC+2.5 SIM-PP 組(數量為3)：使用含2.5μg的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷；以及BC+5SIM-PP組(數量為3)：使用含5μg的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷。 Figure 6A is an image of a Micro-CT trace of a rat skull defect. Fig. 6B is an analysis result of bone regeneration in a defect of a rat skull defect. Control group (quantity 3): no bioceramics; BC group (quantity 3): only bioceramics; BC+2.5 SIM-PP group (quantity 3): using 2.5 μg of coated simvastatin PLGA-PEG (simvastatin/PLGA-PEG) nanoparticle bioceramic; and BC+5SIM-PP group (quantity 3): PLGA-PEG (simvastatin/PLGA-PEG) containing 5 μg of coated simvastatin Bioceramics of nanoparticles.

圖7A為含有辛伐他汀之微脂體(simvastatin/Liposome)奈米粒子的多孔性陶瓷之每日辛伐他汀釋放量；以及圖7B為含有辛伐他汀之微脂體奈米粒子的多孔性陶瓷之辛伐他汀累積釋放濃度。Lipo 40μL：含有40μL的辛伐他汀之微脂體(simvastatin/Liposome)奈米粒子的多孔性陶瓷；Lipo 80μL：含有80μL的辛伐他汀之微脂體(simvastatin/Liposome)奈米粒子的多孔性陶瓷。 Figure 7A shows the daily release of simvastatin in a porous ceramic containing simvastatin/Liposome nanoparticles; and Figure 7B shows the porosity of simvastatin-containing liposome nanoparticles. The cumulative release concentration of simvastatin from ceramics. Lipo 40 μL: Porous ceramic containing 40 μL of simvastatin/Liposome nanoparticles; Lipo 80 μL: Porosity of 80 μL of simvastatin/Liposome nanoparticles ceramics.

本發明包括但不限於上述與下開之說明。實施方式則如下範例所示。 The invention includes, but is not limited to, the description above and below. The embodiment is shown in the following example.

1.方法與材料 1. Methods and materials

A.製備含辛伐他汀之聚乳酸甘醇酸-聚乙二醇奈米藥物載體 A. Preparation of simvastatin-containing polylactic acid glycolic acid-polyethylene glycol nano drug carrier

將聚乳酸甘醇酸(poly(lactic-co-glycolic acid)；PLGA)與聚乙二醇(polyethylene glycol；PEG)以1：10或2：10之比例聚合以形成直鏈聚乳酸甘醇酸-聚乙二醇(PLGA-PEG；PP)共聚物，該PP之化學結構式如下： Poly(lactic-co-glycolic acid; PLGA) and polyethylene glycol (PEG) are polymerized at a ratio of 1:10 or 2:10 to form a linear polylactic acid glycolic acid. - Polyethylene glycol (PLGA-PEG; PP) copolymer, the chemical structure of the PP is as follows:

Me：甲基 Me: methyl

將直鏈聚乳酸甘醇酸-聚乙二醇共聚物(PP)對辛伐他汀(Simvastatin；SIM)藥物以水包油法(O/W)進行包覆以形成奈米粒子(nanoparticles；NPs)，其結構如圖1所示。所透析出的包覆辛伐他汀之PLGA-PEG奈米粒子(Simvastatin/PLGA-PEG nanoparticles；SIM/PP NPs)再以高性能液相層析(HPLC)進行藥物包覆率(Entrapment efficiency；EE%)與藥物承載率(Loading content；LC%)之分析，其中EE%與LC%之計算公式分別下列之式(1)及式(2)所示： The linear polylactic acid glycolic acid-polyethylene glycol copolymer (PP) is coated with simvastatin (SIM) by oil-in-water (O/W) to form nanoparticles (NPs). ), its structure is shown in Figure 1. The dialyzed simvastatin-coated PLGA-PEG nanoparticles (Simvastatin/PLGA-PEG nanoparticles; SIM/PP NPs) were further subjected to high performance liquid chromatography (HPLC) for drug coverage (Entrapment efficiency; EE). %) and the analysis of the loading rate (LC%), wherein the calculation formulas of EE% and LC% are as shown in the following formulas (1) and (2):

PP：聚乳酸甘醇酸-聚乙二醇(poly(lactic-co-glycolic acid)-polyethylene glycol) PP: poly(lactic-co-glycolic acid-polyethylene glycol)

透過上述EE%與LC%之公式以決定聚乳酸甘醇酸-聚乙二醇(PP)的最佳包覆辛伐他汀藥物的條件。本發明是採取固定材料重量(25 mg/批)之方式，以考量取最適或最大值之包覆率與承載率的條件。於本發明中，包覆藥物之奈米粒子透析純化後以HPLC檢測，其包覆率達35%。 The conditions for optimally coating simvastatin of polylactic acid glycolic acid-polyethylene glycol (PP) are determined by the above formulas of EE% and LC%. The invention adopts the method of fixing the material weight (25 mg/batch) to consider the conditions of the optimum or maximum coverage ratio and the bearing ratio. In the present invention, the nanoparticle coated with the drug is purified by dialysis and detected by HPLC, and the coating ratio is 35%.

B.多孔性兩相生物陶瓷支架製備 B. Preparation of porous two-phase bioceramic scaffold

B-1.溫感膠體製備 B-1. Temperature sensitive colloid preparation

將25g之N-異丙基丙烯醯胺(N-isopropylacrylamide)與200μl的甲基丙烯酸(Methacrylic Acid)相繼加入裝有125ml水之500ml圓底燒瓶中至完全溶解；並且依序添加0.25g起始劑過硫酸銨(ammoniumpersulfate；APS)以及催化劑四甲基乙二胺(N,N,N’,N’-tetramethylethylenedia-mine；TEMED)入500ml圓底燒瓶內，持續攪拌24小時後，抽取其液體進行透析以利將N-異丙基丙烯醯胺與甲基丙烯酸單體排出，達成純化效果。將純化後的液體冷凍至液態氮送至冷凍乾燥機，利用昇華的方式將水除去，得到溫感膠體之產物，該溫感膠體之化學式為：poly(N-isopropylacrylamide-co-Metharylic acid(p(NIPAAM-co-MAA))，其結構式如下： 25 g of N-isopropylacrylamide and 200 μl of Methacrylic Acid were successively added to a 500 ml round bottom flask containing 125 ml of water to completely dissolve; and 0.25 g of starting was sequentially added. Ammonium persulfate (APS) and catalyst tetramethylethylenediamine (TEM, TEMED) were placed in a 500 ml round bottom flask and stirred for 24 hours. Dialysis was carried out to facilitate the removal of N-isopropylacrylamide and methacrylic acid monomers to achieve a purification effect. The purified liquid is frozen to liquid nitrogen and sent to a freeze dryer, and the water is removed by sublimation to obtain a product of a temperature-sensitive colloid having a chemical formula of poly(N-isopropylacrylamide-co-Metharylic acid (p) (NIPAAM-co-MAA)), its structural formula is as follows:

B-2.製備多孔性生物陶瓷 B-2. Preparation of porous bioceramics

將氫氧基磷灰石(hydroxyap-atide；HAp)與β-三鈣磷酸鹽 (β-Tricalcium phosphate；β-TCP)混合生物陶瓷粉體與上述溫感水膠置入離心式攪拌機進行多孔性生物陶瓷漿體(HAp/β-TCP/p(NIPAAM-co-MAA)slurry)的混練，以達均勻穩定製程的要求，可依要求製備出HAp/β-TCP相結構比在3/7至7/3的範圍、孔隙度可達20至75%的範圍、孔洞尺寸在5至500μm的範圍。此外，這多孔性生物陶瓷的抗壓樣本以ASTM F451-99a規範製作尺寸為Φ6mm×h12mm，以得抗壓強度達>5MPa以上的多孔錠狀支架材料。 Mixing hydroxyap-atide (HAp) and β-tricalcium phosphate (β-TCP) mixed bioceramic powder with the above-mentioned warm water gel into a centrifugal mixer for porosity The mixing of bioceramic slurry (HAp/β-TCP/p(NIPAAM-co-MAA)slurry) to achieve uniform and stable process requirements, HAp/β-TCP phase structure ratio can be prepared according to requirements to 3/7 to The range of 7/3, the porosity can be in the range of 20 to 75%, and the pore size is in the range of 5 to 500 μm. Further, the pressure-resistant sample of the porous bioceramic was made into a porous ingot-shaped scaffold material having a size of Φ6 mm × h12 mm in accordance with ASTM F451-99a specification to have a compressive strength of > 5 MPa or more.

以上步驟可以參考Yin-Chih Fu的文獻(Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers：In Vitro and In Vivo evaluation,Journal of the Mechanical Behavior of Biomedical Materials,27：64-76,2013)。 For the above steps, refer to the literature of Yin-Chih Fu (Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2 carriers: In Vitro and In Vivo evaluation, Journal of the Mechanical Behavior of Biomedical Materials, 27: 64-76 , 2013).

C.包覆辛伐他汀之PLGA-PEG奈米粒子結合多孔性陶瓷之藥物釋放計算 C. Drug release calculation of PLGA-PEG nanoparticles coated with simvastatin combined with porous ceramics

本發明將包覆辛伐他汀之PLGA-PEG奈米粒子(SIM/PP NPs)以每次20μL滴入多孔性生物陶瓷後置入抽風櫃中風乾，即可製作出含藥陶瓷。因此，本發明的實驗組別係分別將80和160μL的包覆辛伐他汀之PLGA-PEG奈米粒子滴入多孔性生物陶瓷中，各獲取5顆含藥陶瓷，再放入3mL磷酸鹽緩衝生理鹽水(PBS)中，於37℃下以60rpm進行釋放24小時，每次收取2mL並補2mL PBS，連續收取7天後，以高性能液相層析法(HPLC)檢測辛伐他汀(SIM)的釋放量。 In the present invention, PLVA-PEG nanoparticles (SIM/PP NPs) coated with simvastatin are dropped into a porous bioceramic at 20 μL each time, and then placed in a draft cabinet to be air-dried to prepare a medicated ceramic. Therefore, the experimental group of the present invention separately drops 80 and 160 μL of simvastatin-coated PLGA-PEG nanoparticles into a porous bioceramic, each of which obtains 5 medicinal ceramics, and then puts 3 mL of phosphate buffer. In physiological saline (PBS), release at 60 ° C for 24 hours at 37 ° C, each time 2 mL was taken and 2 mL of PBS was added for 7 days, and simvastatin was detected by high performance liquid chromatography (HPLC). The amount of release.

D.紐西蘭白兔橈骨缺損模式試驗 D. New Zealand white rabbit tibia defect model test

於紐西蘭白兔橈骨缺損實驗中，本發明分成三組實驗組：(1)BC組：以生物陶瓷(Bioceramics；BC)治療；(2)BC+80μL SIM/PP NPs組：以含80μL的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷治療；及(3)BC+160μL SIM/PP NPs組：以含160μL的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷治療。每組以3隻紐西蘭白兔進行測試。將紐西蘭白兔右前肢橈骨以鑽頭截斷1公分，以生物陶瓷放置缺損處治療，每2週以X光追蹤連續10週，犧牲後取下橈骨進行脫鈣、石蠟包埋與組織切片，並進行H&E染色和免疫組織染色檢測新生骨與血管新生因子vWF(Von Willebrand factor)。此外，新骨形成率是以缺損區域內新生骨(紅色染色部分)/缺損區域所算出。 In the New Zealand white rabbit tibia defect test, the present invention was divided into three experimental groups: (1) BC group: treated with bioceramics (BC); (2) BC + 80 μL SIM/PP NPs group: containing 80 μL Bioceramic treatment of simvastatin-coated PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles; and (3) BC+160 μL SIM/PP NPs group: PLGA-containing 160 μL of coated simvastatin Bioceramic treatment of PEG (simvastatin/PLGA-PEG) nanoparticles. Each group was tested with 3 New Zealand white rabbits. The New Zealand white rabbit's right forelimb tibia was cut off by 1 cm with a drill bit, treated with a bioceramic placement defect, followed by X-rays for 10 weeks every 2 weeks. After sacrifice, the tibia was removed for decalcification, paraffin embedding and tissue sectioning. H&E staining and immunohistochemical staining were used to detect the new bone and angiogenesis factor vWF (Von Willebrand factor). In addition, the new bone formation rate is calculated from the new bone (red stained portion)/deficient area in the defect area.

E.大鼠頭蓋骨缺損模式試驗 E. Rat skull bone defect model test

大鼠頭蓋骨之嚴重骨缺損模式係將SD大鼠(Sprague-Dawley rats；滿九週大)以Ketamine^®(150mg/kg體重)進行腹腔注射以進行全身麻醉。以牙科電動鑽進行外科手術，於大鼠的頭蓋骨產生直徑5mm之嚴重骨缺損(critical size defect)。再以含辛伐他汀(SIM)之奈米藥物載體複合多孔性生物陶瓷支架置入大鼠頭蓋骨的骨缺損處後，進行縫合。本發明將大鼠分為四組(每組各3隻)，分別是(1)控制組：不用任何生物陶瓷；(2)BC組：僅使用生物陶瓷；(3)BC+2.5 SIM-PP組：使用含有2.5μg的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷；以及(4)BC+5 SIM-PP：使用含有5μg的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子之生物陶瓷。大鼠頭骨 縫合後，每4週以微計算機斷層掃描技術(micro computed tomography；Micro-CT)進行追蹤持續8週，並分別於植入生物陶瓷後第0、4、8週將動物進行安樂死，將頭蓋骨取出，進行脫鈣、包埋和切片以及H&E組織染色以評估骨質新生能力。 A severe bone defect model of the rat skull was performed by intraperitoneal injection of SD rats (Sprague-Dawley rats; nine weeks old) with Ketamine ^® (150 mg/kg body weight) for general anesthesia. Surgical operation with a dental electric drill produced a critical size defect of 5 mm in diameter in the skull of the rat. Then, a bone biodegradation containing a simvastatin (SIM)-containing porous bioceramic stent was placed in the bone defect of the rat skull and sutured. The invention divides the rats into four groups (3 in each group), which are (1) control group: no bioceramics are used; (2) BC group: only bioceramics are used; (3) BC+2.5 SIM-PP Group: Bioceramics containing 2.5 μg of simvastatin-coated PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles; and (4) BC+5 SIM-PP: using 5 μg of coated simvastatin Bioceramics of PLGA-PEG (simvastatin/PLGA-PEG) nanoparticle. After suture of the rat skull, it was followed by micro computed tomography (Micro-CT) for 4 weeks every 4 weeks, and the animals were euthanized at 0, 4, and 8 weeks after implantation of bioceramics. The skull was removed for decalcification, embedding and sectioning and H&E tissue staining to assess bone regeneration.

F.包裹辛伐他汀之微脂體奈米粒子 F. Microlipid nanoparticles coated with simvastatin

以磷脂質二棕櫚酰磷脂酰膽鹼(Dipalmitoyl-phosphatidyl-choline；DPPC)包覆辛伐他汀以製備微脂體(Liposome；Lipo)奈米粒子，其結構如圖2所示。10mg DPPC與1mg辛伐他汀置物反應瓶以三氯甲烷(chloroform)溶解後進行真空抽乾。再加以純水後，用機械力將震盪回溶，最後以整粒器調整粒徑大小為約200nm。產率為87-93%，就可得含有辛伐他汀之微脂體(simvastatin/Liposome)奈米粒子。 Simvastatin was coated with dipalmitoyl-phosphatidyl-choline (DPPC) to prepare liposome (Lipo); Lipo nanoparticles, and its structure is shown in FIG. 10 mg DPPC and 1 mg simvastatin storage reaction flask were dissolved in chloroform and vacuum dried. After adding pure water, the mixture was shaken back by mechanical force, and finally the particle size was adjusted to about 200 nm by the granulator. The yield of 87-93% gives simvastatin/Liposome nanoparticles containing simvastatin.

G.包覆辛伐他汀之微脂體奈米粒子結合多孔性陶瓷之藥物釋放計算 G. Drug release calculation of viscidatin-coated microlipid nanoparticles combined with porous ceramics

將含有辛伐他汀之微脂體(simvastatin/Liposome)奈米粒子以每次40μL(Lipo 40μL)或80μL(Lipo 80μL)滴入生物陶瓷後置入抽風櫃中風乾，即可製作出含藥陶瓷。含40μL(Lipo 40μL)以及80μL(Lipo 80μL)劑量之含藥陶瓷各取3顆放入2.5mL PBS，於37℃下以60rpm進行釋放24小時，每次收取2mL並補2mL PBS，連續收取7天後，以HPLC檢測辛伐他汀的量。 The simvastatin/Liposome nanoparticle containing simvastatin is added to the bioceramic with 40 μL (Lipo 40 μL) or 80 μL (Lipo 80 μL) each time, and then placed in a draft cabinet to be air-dried to prepare a medicinal ceramic. . Three medicated ceramics containing 40 μL (Lipo 40 μL) and 80 μL (Lipo 80 μL) were placed in 2.5 mL PBS and released at 60 ° C for 24 hours at 37 ° C. Each time 2 mL was taken and 2 mL of PBS was added for 7 consecutive doses. After a day, the amount of simvastatin was measured by HPLC.

2.實驗結果 2. Experimental results

A.包覆辛伐他汀之PLGA-PEG奈米粒子結合多孔性陶瓷之藥物釋放率 A. Drug release rate of PLGA-PEG nanoparticles coated with simvastatin combined with porous ceramics

實驗組分別滴入80和160μL的包覆辛伐他汀之PLGA-PEG(simvastatin/PLGA-PEG)奈米粒子於生物陶瓷中，以形成含藥陶瓷，而由PLGA-PEG奈米粒子之包覆率為35%，可計算出含80和160μL的含有包覆辛伐他汀之PLGA-PEG奈米粒子之生物陶瓷所包裹的辛伐他汀量各別為5和10μg。而以前研究指出於辛伐他汀於體外細胞實驗及大鼠動物實驗之促進骨生成之有效濃度為0.5μM，因此，本發明發現160μL的包覆辛伐他汀之PLGA-PEG奈米粒子於前三天能釋放出達到有效作用濃度的辛伐他汀(大於0.5μM)(如圖3A所示)，且160μL的包覆辛伐他汀之PLGA-PEG奈米粒子在第5天釋放率能達到80%以上(如圖3B所示)。 The experimental group was instilled with 80 and 160 μL of simvastatin-coated PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles in bioceramics to form medicated ceramics, which were coated with PLGA-PEG nanoparticles. The rate was 35%, and the amount of simvastatin contained in 80 and 160 μL of bioceramics containing simvastatin-coated PLGA-PEG nanoparticles was calculated to be 5 and 10 μg, respectively. Previous studies have indicated that the effective concentration of simvastatin in promoting osteogenesis in vitro and in rat animal experiments is 0.5 μM. Therefore, the present inventors have found that 160 μL of simvastatin-coated PLGA-PEG nanoparticles are in the first three. Sky can release simvastatin (greater than 0.5μM) to the effective concentration (as shown in Figure 3A), and 160μL of simvastatin-coated PLGA-PEG nanoparticles can reach 80% on the 5th day. Above (as shown in Figure 3B).

另外，為了進行體外實驗評估含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷對D1細胞株的促骨生成效果，實驗組分成三組：(1)控制組：僅使用培養基；(2)辛伐他汀組：加入0.5μM辛伐他汀(SIM)於培養基中；以及(3)辛伐他汀釋放組：加入第一天含有包覆辛伐他汀之PLGA-PEG奈米粒子之多孔性生物陶瓷之辛伐他汀釋放液於培養基中。將三組培養基處理D1細胞株，並收取第三、五天培養液檢測鹼性磷酸酶(Alkaline phosphatase；ALP)活性(促骨生成效果)。由圖4的結果可知辛伐他汀釋放組的ALP活性明顯高於控制組。該結果顯示含藥陶瓷對D1細胞株確實具有的促骨生成效果。 In addition, in order to carry out in vitro experiments to evaluate the osteogenic effect of porous bioceramics containing simvastatin-coated PLGA-PEG nanoparticles on D1 cell lines, the experimental components were divided into three groups: (1) Control group: medium only (2) Simvastatin group: 0.5 μM simvastatin (SIM) was added to the medium; and (3) Simvastatin-release group: PLGA-PEG nanoparticles containing simvastatin coated on the first day were added. The simvastatin release solution of the porous bioceramic is in the medium. Three groups of medium were treated with D1 cell line, and the third and fifth day culture medium was collected to detect Alkaline phosphatase (ALP) activity (osteogenesis effect). From the results of Figure 4, it was found that the ALP activity of the simvastatin-releasing group was significantly higher than that of the control group. This result shows that the medicated ceramic does have an osteogenic effect on the D1 cell line.

B.紐西蘭白兔橈骨缺損之治療效果 B. Therapeutic effect of New Zealand white rabbit tibia defect

於紐西蘭白兔橈骨缺損的治療實驗中，由X光追蹤(如圖5A所示)與H&E染色(如圖5Bi所示)的結果可發現有奈米粒子披覆的生物陶瓷能提高骨生長效果；並在免疫染色結果中，高濃度奈米粒子陶瓷的vWF(如圖5C所示，vWF所呈顏色為褐色)表現量優於不含藥陶瓷。藉由上述結果證實含藥陶瓷確實具有修復骨缺損的功效，可看出BC+160μL SIM/PP NPs組中骨生長效果都優於其他兩組。 In the treatment experiment of the New Zealand white rabbit tibia defect, the results of X-ray tracking (as shown in Figure 5A) and H&E staining (as shown in Figure 5Bi) can be found that biomaterials coated with nanoparticle can improve bone. The growth effect; and in the immunostaining results, the vWF of the high-concentration nanoparticle ceramics (as shown in Fig. 5C, the color of the vWF is brown) is superior to that of the non-medicated ceramic. From the above results, it was confirmed that the medicated ceramics did have the effect of repairing the bone defect, and it can be seen that the bone growth effect of the BC+160 μL SIM/PP NPs group was superior to the other two groups.

另外，以各組的H&E組織染色結果(圖5Bi)，再以影像軟體(ImageJ)進行定量，以計算新生骨形成率(new bone formation；%)。由圖5Bii的新生骨形成率結果可知奈米藥物披覆陶瓷之骨生長對未披覆奈米藥物之陶瓷具有顯著促進。 In addition, the H&E tissue staining results (Fig. 5Bi) of each group were quantified by image software (ImageJ) to calculate new bone formation (%). From the results of the new bone formation rate of Fig. 5Bii, it can be seen that the bone growth of the nano drug-coated ceramics significantly promotes the ceramics of the uncoated nanomedicine.

C.大鼠頭蓋骨缺損之治療效果 C. Therapeutic effect of rat skull defect

以大鼠頭蓋骨缺損模式模擬血流量較少的部位，三組實驗組分別於植入材料後第0、4及8週後進行Micro-CT追蹤及組織染色進行骨質新生之評估，如圖6A及6B之結果所示，BC+2.5 SIM-PP及BC+5 SIM-PP兩組確實具有促進修復頭蓋骨缺損的功效。 The rat head bone defect model was used to simulate the site with less blood flow. The three groups of experimental groups underwent Micro-CT tracking and tissue staining for bone regeneration after 0, 4 and 8 weeks after implantation, as shown in Figure 6A. As shown by the results of 6B, the BC+2.5 SIM-PP and BC+5 SIM-PP groups did have the effect of promoting the repair of the skull defect.

D.微脂體奈米粒子包裹辛伐他汀之釋放量的計算 D. Calculation of the release amount of simvastatin encapsulated by microlipid nanoparticles

由圖7A的結果可發現含藥陶瓷的Lipo 40μL組及Lipo 80μL組於前2天能釋放出達到有效作用濃度的辛伐他汀(SIM)(超過0.5μM)。但是，如圖7B所示，含藥陶瓷的Lipo 40μL組及Lipo 80μL組的緩 釋能力小於含有包覆辛伐他汀之PLGA-PEG奈米粒子之生物陶瓷。 From the results of Fig. 7A, it was found that the Lipo 40 μL group containing the drug-containing ceramics and the Lipo 80 μL group were able to release simvastatin (SIM) (over 0.5 μM) at an effective concentration for the first 2 days. However, as shown in Fig. 7B, the sustained release ability of the medicated ceramic Lipo 40 μL group and the Lipo 80 μL group was smaller than that of the bioceramics containing the simvastatin-coated PLGA-PEG nanoparticles.

本發明適當的描述可以在本文未具體公開的元素或限制下實施。已被用作描述的術語並不是限制。在使用這些術語和除此之外的任何等同物的表達和描述是沒有差別的，但應當認識到本發明內的權利是可能修改的。因此，雖然本發明已說明實施例和其他情況，本文中所公開的內容可以被本領域的技術人員進行修飾和變化，並且這樣的修改和變化被認為是在本發明的權利範圍之內。 Suitable descriptions of the invention may be implemented in elements or limitations not specifically disclosed herein. The terminology that has been used for description is not limiting. There is no difference in the expression and description of the use of these terms and any equivalents, but it is to be understood that the scope of the invention may be modified. Therefore, the present invention has been described with reference to the embodiments and other aspects, and the modifications and variations of the present invention are considered to be within the scope of the present invention.

Claims (10)

一種含藥物之複合性支架，其包含一多孔性骨材以及一藥物釋放奈米載體，其中該藥物釋放奈米載體包含一促進骨生長藥物，且分佈於該多孔性骨材中，其中該藥物釋放奈米載體為一兩性分子載體。  A drug-containing composite scaffold comprising a porous aggregate and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous aggregate, wherein The drug-released nanocarrier is an amphoteric carrier.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該多孔性骨材是一多孔性陶瓷骨材。  The drug-containing composite stent according to claim 1, wherein the porous aggregate is a porous ceramic aggregate.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該多孔性骨材包含一羥基磷灰石(hydroxyapatite；HAp)、一β-磷酸三鈣(β-tricalcium phosphate；β-TCP)、一羥基磷灰石-磷酸三鈣(hydroxyapatite tricalcium phosphate；HATCP)、一α-磷酸三鈣(α-tricalcium phosphate；α-TCP)、一生物活性玻璃陶瓷(bioactive glass ceramic)、一硫酸鈣、一骨水泥(bone cement)或其上述組合。  The drug-containing composite stent according to claim 1, wherein the porous aggregate comprises hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP). ), hydroxyapatite tricalcium phosphate (HATCP), α-tricalcium phosphate (α-TCP), bioactive glass ceramic, calcium monosulfate , bone cement or a combination thereof.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該多孔性骨材的孔隙率為15-85%。  The drug-containing composite stent according to claim 1, wherein the porous aggregate has a porosity of 15-85%.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該多孔性骨材的孔洞大小為5-1000μm。  The drug-containing composite stent according to claim 1, wherein the porous aggregate has a pore size of 5 to 1000 μm.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該促進骨生長藥物為一油溶性促進骨生長藥物。  The drug-containing composite stent according to claim 1, wherein the bone growth promoting drug is an oil-soluble bone growth promoting drug.   如申請專利範圍第6項所述之含藥物之複合性支架，其中該油溶性促進骨生長藥物包含一史他汀類(statin)藥物。  The drug-containing composite stent according to claim 6, wherein the oil-soluble bone growth promoting drug comprises a statin drug.   如申請專利範圍第7項所述之含藥物之複合性支架，其中該史他汀類藥物包含一辛伐他汀(simvastatin)。  The drug-containing composite scaffold of claim 7, wherein the statin comprises a simvastatin.   如申請專利範圍第1項所述之含藥物之複合性支架，其中該藥物釋放奈米載體為為一微脂體(liposome)、一高分子微胞(polymeric micelle)、或一樹枝狀高分子聚合物(dendrimer)。  The drug-containing composite stent according to claim 1, wherein the drug release nanocarrier is a liposome, a polymeric micelle, or a dendrimer. Polymer (dendrimer).   如申請專利範圍第9項所述之含藥物之複合性支架，其中該藥物釋放奈米載體為為一高分子微胞。  The drug-containing composite stent according to claim 9, wherein the drug release nanocarrier is a polymer microcell.