低劑量骨成形蛋白二及其短鏈胜肽於人工植體周圍齒槽骨再生試驗

Abstract

人工植牙乃重建缺牙區的主流之一，臨床上常遇到齒槽骨缺損不足的病人而無法直接置放人工植體，需要進行額外手術來增加骨量。大範圍骨缺損及植體周圍骨缺損在如今仍是一項困難的挑戰。本研究旨在測試以人工合成骨塊加入骨成形蛋白二或其抗原決定位之短鏈胜肽，修復植體周圍大範圍骨缺損的潛力及效益。 研究分為兩個單元。第一單元的主要目的在建立實驗犬下顎骨之植體周圍臨界骨缺損模型，以此模型測試人工合成骨塊是否能作為良好的支架以及骨成形蛋白二之載體，並找尋骨成形蛋白二之最低有效劑量，證實此最低有效劑量能成功誘導植體周圍大範圍齒槽骨再生。第二單元的主要目的則在測試位於骨成形蛋白二抗原決定位之短鏈胜肽是否同樣具有誘導齒槽骨再生之功能，將短鏈胜肽之功效及使用劑量和骨成形蛋白二相互比較，期待未來能取代昂貴及有副作用之骨成形蛋白二。 實驗首先建立實驗犬下顎骨之植體周圍臨界骨缺損模型。於米格魯雄性成犬之下顎做出高度四毫米、長度十毫米之齒槽骨缺損，並將人工植體（ø 4.0 x 8.5mm; Brånemark MkIII）的根部四毫米植入骨缺損中央，冠部四點五毫米則懸空於齒槽骨缺損中。此模型在無介入治療的組別中，於實驗終點無法達成植體周圍骨再生，因此證實為植體周圍之臨界骨缺損模型。 本實驗採用HAp (Hydroxyapatite)/TCP (Tri-calcium Phosphate)/Col (Collagen) 複合骨材當作支架及載體，加入0.02 mg/mL至0.2 mg/mL之骨成形蛋白二，測試其在無再生膜的情況下，於四週及八週癒合期內促進植體周圍骨再生的能力。植體的初級和次級穩定度由植體共振頻率作分析。植體周圍齒槽骨再生的效能則由放射影像、微電腦斷層影像、不脫鈣研磨骨組織切片之螢光骨標記及脫鈣骨組織染色來做分析測定。 實驗結果證實，植體周圍骨再生的能力受到骨成形蛋白二的劑量影響，骨成形蛋白二的濃度越高則齒槽骨再生的速度越快、新生骨質密度越高、植體穩定度也越好。HAp/TCP/Col 複合骨材加上 0.2 mg/mL 骨成形蛋白二可以誘導顯著優異的齒槽骨再生效能，並在八週內達到大範圍植體周圍骨缺損之完全癒合。因此，我們定義出骨成形蛋白二之最低有效劑量為0.2 mg/mL，較FDA核可之INFUSE Bone Graft低7.5倍。以本研究的實驗模組同時可證實，在HAp/TCP/Col 複合骨材加上 0.2 mg/mL 骨成形蛋白二且不使用再生膜的模式下，齒槽脊增高手術可以和人工植體植入同時進行。 接著我們使用同樣的植體周圍臨界骨缺損模型與相關分析方法，以高於骨成形蛋白二最低有效劑量20至100倍之濃度，來測試骨成形蛋白二抗原決定位之短鏈胜肽是否同樣具有誘導齒槽骨再生的功能。實驗證實 4–20 mg/mL 短鏈胜肽確實具有誘導植體周圍骨再生之能力，但其骨導引再生的效能皆不及 0.2 mg/mL 骨成形蛋白二。20 mg/mL 短鏈胜肽的表現類似於 0.02 mg/mL 骨成形蛋白二的表現。因此推測短鏈胜肽和骨成形蛋白二在質量比1000:1時能達成類似的骨誘導能力。 總結來說，骨成形蛋白二應用於植體周圍齒槽脊增高手術之最低有效劑量為0.2 mg/mL，HAp/TCP/Col 複合骨材加上 0.2 mg/mL 骨成形蛋白二能取代自體骨移植的需求並達成植體周圍大範圍骨再生的困難任務。相信未來研究改良及劑量調整後，骨成形蛋白二抗原決定位之短鏈胜肽有潛力可以作為骨成形蛋白二的替代產品。

Dental implant therapy has become a standard of care for the treatment of edentulous patients. Sufficient bone height and width is a prerequisite for implant insertion. However, a reduced alveolar ridge contour often occurs after tooth extraction and jeopardizes the long-term survival of dental implants. Various techniques have been attempted to increase the amount of alveolar bone, however, managing large alveolar defects remain difficult tasks. In this study, we combined osteoconductive scaffolds (HAp/TCP/Col) with osteoinductive signals (rhBMP-2 protein/or a synthetic 73–92-residue BMP-2 peptide) aiming to achieve the difficult task of peri-implant bone augmentation without the use of autogenous grafts. The study was divided into two parts. The aim of the first part was to establish a peri-implant critical size defect model at the mandibles of beagle dogs. The compatibility of the HAp/TCP/Col composite as a scaffold and carrier of rhBMP-2 was assessed using the defect model. Then we search for the lower bound for rhBMP-2 doses that could successfully induce peri-implant bone regeneration. The second part aimed to investigate the bone inductive ability of a synthetic 73–92-residue BMP-2 peptide and compare its efficacy with rhBMP-2, exploring the possibility that BMP-2 peptide could be a substitute for rhBMP-2 in the future as osteogenic molecules. Large saddle-type alveolar defects (10 mm mesiodistally, and 4 mm apicocronally) were surgically created in post‐extraction regions of the mandible in beagle dogs. Dental implants were placed into the prepared osteotomies at the center of the defects. Each implant fixture was placed with apical 4 mm of implant inserted into the bone, leaving cervical 4.5 mm exposed to the large through-and-through defects. The blank group without treatment intervention did not show obvious bone growth around the dental implants throughout the study, so the defect model could be considered a critical-sized defect model. HAp/TCP/Col composite was used as scaffold and rhBMP-2 carrier. RhBMP-2 at concentrations of 0.02–0.2 mg/mL was applied and tested using the critical-sized peri-implant defect model. After healing for 4 or 8 weeks, bone regeneration and mineralization were assessed through radiography, micro-CT, fluorescence labeling, and histologic analyses. Implant stability was measured through resonance frequency analysis. It was evident that bone regenerative ability was influenced by rhBMP-2 doses. HAp/TCP/Col with 0.2 mg/mL rhBMP-2 has presented significantly better new bone formation and was able to fulfill the bone repair of large peri-implant defects in 8 weeks. The lower bound for rhBMP-2 dose was then defined at 0.2 mg/mL, which is 7.5 times lower than the commercial INFUSE Bone Graft. Our results also implied that alveolar ridge augmentation could be performed simultaneously with dental implant placement by using HAp/TCP/Col composite combined with 0.2 mg/mL rhBMP-2 without barrier membranes. The second part of this study was to investigate the osteogenic ability of a synthetic 73–92-residue BMP-2 peptide with the same animal model and similar analyses. The BMP-2 peptide doses were set at 20 to 100 times higher than the 0.2 mg/mL rhBMP-2. Our results demonstrated the BMP-2 peptide at 4–20 mg/mL could facilitate bone regeneration in vivo, however, the bone inductive ability was inferior to 0.2 mg/mL rhBMP-2 and the mineralization level did not show a significant difference compared with the control group. The performance of 20 mg/mL BMP-2 peptide was similar to 0.02 mg/mL rhBMP-2, therefore, a mass ratio of 1000:1 was thus suggested for the BMP-2 peptide and rhBMP-2 to achieve similar osteoinductive performance. In conclusion, 0.2 mg/mL rhBMP-2 was defined as the lowest effective dose in peri-implant bone regeneration. Our results highlight the constructs of HAp/TCP/Col + 0.2 mg/mL rhBMP-2 without barrier membranes as a promising tool for peri-implant ridge augmentation. The synthetic 73–92-residue BMP-2 peptide has the potential to become an alternative to rhBMP-2. Further refinement and dose estimation are required.