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Flame Spray Coating of Strontium- and Magnesium-doped Hydroxyapatite on Dental Implant
Strontium,Magnesium,Hydroxyapatite,Flame Spraying Technology,Dental Implant,Osseointegration,Biocompatibility,
|Publication Year :||2017|
|Abstract:||氫氧基磷灰石在現今被廣泛地使用在骨整合研究，對於人體骨骼的組成元素非常相似。過往研究中已證明鍶與鎂微量元素摻雜混入氫氧基磷灰石中，會促進骨母細胞與骨細胞的貼附與增生。本實驗研究macro等級之多孔性的氫氧基磷灰石結構中，鈣離子位置會被鍶與鎂部分取代，利用化學濕式合成法來合成所需微摻元素之粉末，鍶與鎂以不同莫耳比例(0.5, 1, 5, 10 mol%)摻雜其中(xSr-yMg-HA)，接著將粉末進行材料鑑定。|
運用火焰噴塗薄膜技術，將粉末噴塗覆蓋於細胞所需之純鈦基材與動物實驗所需之牙根表面，來進行生物相容性試驗。實驗細胞株為人類胚胎上顎間質細胞(HEPM cells)，利用此細胞來研究本實驗中其合成材料的生物相容性與生物活性。在細胞存活率分析(MTT assay)與鹼性磷酸酶試驗(ALP assay)，實驗天數為第3、7、11天，發現在第11天時5Sr-5Mg-HA組(Sr0.5Mg0.5Ca9 (PO4)6(OH)2)的細胞生長與礦化情況最佳，ALP蛋白含量高達65 ng/µl；即時定量聚合酶鏈鎖反應(Real-time PCR)去分析基因的層次，可以發現上游轉錄因子RUNX2與礦化調控基因OPN、OCN，5Sr-5Mg-HA組之DNA產物量在第11天時最為明顯；偵測蛋白質表現含量的西方墨點法(Western blot)，5Sr-5Mg-HA組的RUNX2與OPN蛋白質表現，在時間點第11天時表現含量增加；利用人類骨鈣素即時定量ELISA試劑組(Human Osteocalcin Instant ELISA Kit)將OCN蛋白質表現定量，也發現在第11天時與各組比較，具有顯著差異，OCN蛋白質含量約18.5 ng/ml，也印證基因分析表現的一致性。
除了材料鑑定和體外細胞分析測試(in vitro)之外，本實驗也進行了相關動物實驗(in vivo)的研究，各別為市售Ti植體、HA-Ti植體與5Sr-5Mg-HA-Ti植體等三組進行實驗。在植牙實驗的時間點2、4、8週分別皮下注射骨螢光染劑標定，以觀察不同時間點之骨新生，可以發現5Sr-5Mg-HA組在各時間點與他組比較，骨新生情況最佳；而觀察期間之植體動搖度，其測量處為頰舌側與近心遠心側部份，各組都符合臨床上的植體動搖度範圍；在動物犧牲後取得之標本，以X光與micro CT計算骨覆蓋率(BC%)、骨量(BV)、骨密度(BMD)、植體-骨組織接觸面積(BIC)；在第8週的骨覆蓋率比較，對照組HA-Ti植體(84.46±5.27%)和實驗組5Sr-5Mg-HA-Ti植體(86.61±3.18%)兩者無顯著差異，但與空白組Ti植體(73.67±3.43%)比較則有顯著差異；而骨量、骨密度、植體-骨組織接觸比率，在時間點2、4、8週觀察，三組皆無顯著差異；再以脫鈣(Hematoxylin-Eosin Staining)、非脫鈣研磨染色(Stevenel’s blue Alizarin red S Staining)與骨螢光染色(Bone Fluorescence Label)實驗觀察組織切片，可以發現5Sr-5Mg-HA組之骨整合最佳。
Hydroxyapatite (HA) is widely used in osseointegration research because its compositionis similar to with natural bone. Incorporation of strontium (Sr2+) and magnesium (Mg2+) to HA has been demonstrated to enhance pre-osteoblasts and osteoblasts adhesion. Porous macro-granules of HA with Ca2+ partially co-substituted with Sr2+ and Mg2+ indifferent mole percent ratios (xSr-yMg-HA). In this study, we used wet chemical method to synthesize HA powder which included different ratios of Sr2+and Mg2+ (x=0.5, 1, 5, 10). Human embryonic palatal mesenchyme (HEPM) cells were used to study the biocompatibility and biological activity of materials. MTT assay and alkaline phosphatase activity test were performed to examine the cell proliferation and mineralization. The results showed that the best ratio of Sr2+ and Mg2+ co-doped HA were 5Sr-5Mg-HA (Sr0.5Mg0.5Ca9 (PO4)6(OH)2). ALP protein concentration of 65 ng/μl to be detected.
The synthesized powders were further characterized using X-ray diffractometer (XRD) at diffraction angles from 10o to 60o. Moreover, Fourier Transform Infrared Spectroscopy (FTIR) and Raman Spectroscopy were used to examine the functional group changes. X-ray photoelectron spectroscopy (XPS) was used to measure Ca2+, Sr2+, Mg2+ contents and Scanning Electron Microscope (SEM) was used to observe the surface morphology.
In addition, we used 5Sr-5Mg-HA powder to coat with coating the pure-Ti implant using the flame spraying technology. We tested the expressions of osteogenesis-related genes, including runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and osteopontin (OPN), using real-time PCR. Several previous studies have reported that RUNX2 was a master transcription factor and OPN and OCN were late osteogenic markers. They were studied for chondrocyte and osteoblast differentiation and bone formation. Furthermore, Western blot was used to examine protein expression levels. RUNX2 and OPN proteins in the 5Sr-5Mg-HA group showed the high expression level at the 11th day. We measured OCN protein was quantified by the Human Osteocalcin Instant ELISA Kit, and 5Sr5Mg-HA-Ti group had a significant difference. OCN protein had 18.5 ng/ml compared with each group at the 11th day.
Besides examining material identification and in vitro assay, we also took out other biocompatibility tests, in vivo, to determine the bio-functionality in a more overall way. We conducted on the related animal experiments about osseointegration function. In this study, self-developed implants in which were coating 50-55 μm particles which have HA-Ti or 5Sr5Mg-HA powder for surface treatment were enrolled in experimental group, and commercial dental Ti implants (OsseoSpeedTM) were enrolled in the control group. Implants in each group were according to the different observation intervals (2, 4 and 8weeks) with randomly distribution at bilateral mandible edentulous area in four beagle dogs. Non-invasive evaluation including clinical observation on the implant status and implant stability analyzer, Periotest, it was used for accessment of degree of osteointegration in the observation period. After animal sacrificed and specimens harvested, X-ray and micro CT could use to measure bone coverage (BC%), bone volume (BV), bone mineral density (BMD) and bone-implant contact (BIC). At the 8th week, bone coverage were no significant differences between the HA-Ti implant (84.46 ± 5.27%) and 5Sr-5Mg-HA-Ti implant (86.61 ± 3.18%), but Ti implant (73.67 ± 3.43%) were significant differences in comparison. However, bone mass, bone density, and bone-implant contact at the time point 2, 4, 8 weeks were no significant difference with the three groups. Bone fluorescence labeling technique with dyes injected at 2, 4 and 8 weeks was used to observe new bone formation. Decalcified and non-decalcified staining could observe the direct contact of the bone tissue with the part of the implant area. We confirmed 5Sr-5Mg-HA group to carry out the best bone formation, remodeling and osseointegration.
The comprehensive biocompatibility tests and bone osseointegration analyses were performed by in vitro and in vivo experiments to accurately assess the pros and cons of synthetic materials in biomaterials to prepare future clinical studies.
|Appears in Collections:||口腔生物科學研究所|
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