具生物可分解性注射式高分子聚合物及磷酸鈣鹽混合骨水泥之研究

Studies of Biodegradable and Injectable Composite Bone Filler Based on Polymer and Calcium Phosphate Ceramic Powder

吳長晉(Chang-Chin Wu)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.00036

摘要

雖然已有許多不同的填充材料(filler materials)已應用脊椎椎體整形手術(vertebral augmentation procedures)，但就生物力學和生物學特性上來看，沒有一個是完美的。為了盡量減少可能出現的缺點，我們新合成了一種可生物降解的、可注射且可術中混合的聚丙烯富馬酸(poly(propylene fumarate), PPF)和雙相α-磷酸三鈣/羥基磷灰石陶瓷粉(biphasic α-tricalcium phosphate/hydroxyapatite ceramics powder, α-TCP/HAP)所製成的複合材料，並評估這化合物在體外測試的材料特性。混合不同比例的PPF液體和雙相α-TCP/HAP粉末，並以N-乙烯基吡咯烷酮(N-Vinyl pyrrolidinone, NVP)為交聯劑、過氧化苯甲酰(benzoyl peroxide, BP)為起始劑，來製成檢測用的圓柱體。記錄硬化的時間和溫度，雖然這兩樣可以經過不同濃度的對苯二酚(hydroquinone)和N,N-二甲基甲苯胺(N,N-dimethyl-p-toluidine, DMT)來調控。之後再分析固化後複合物圓柱體的的降解狀況、細胞相容性、力學分析和顯影度。我們還將另兩種材料，市售的聚甲基丙烯酸甲酯 (poly(methyl methacrylate), PMMA)和聚丙烯富馬酸(poly(propylene fumarate), PPF)與純羥基磷灰石顆粒(PPF/HA)與我們自製的複合材料做比較。結果發現新複合材料在固化過程中(38-44°C)的最高溫度較低、具有足夠的初期力學壓縮斷裂強度(61.1±3.7MPa)、並可以逐步分解。至於其顯影度，是以電腦斷層掃描(computed tomography scan, CT) 所得之亨斯菲爾德單位(Hounsfield units, HU)來評估，70％ α-TCP/HAP的與PMMA的相近。而生物相容性測試，包括酸鹼值的變化和細胞毒性，都在生物可容忍的限度之內。在不同比例中，以70％ α-TCP/HAP粉的混合物優於其他組，故以70％複合材料用在動物體內研究。
我們使用豬脊椎模式，來評估這複合材料所製成的骨水泥在動物體內的效用。除了前面提到的70％α-TCP/HAP/PPF混合物外，還因四鈣磷酸鹽(tetracalcium phosphate, TtCP)及二鈣磷酸鹽(dicalcium phosphate, DCP)的骨水泥已在臨床上廣泛應用，且其在水合之後亦會有雙相變換的性質，故同時也將70％ TtCP/DCP/PPF的複合材料加入實驗，並將這兩種可生物降解的骨水泥與市售PMMA比較。共有十二頭豬納入實驗。動物實驗手術中，豬經全身麻醉後，在腹部施以前側開，將其腰椎椎體外側骨頭清出。再以鑽頭在不同椎體各鑽出直徑5毫米長10毫米的圓柱空洞，並隨機注入不同骨水泥。術後飼養三及六個月後，各犧牲六隻個體，並將取下的腰椎做放射線檢查，以評估整體圓柱塊外形及放射顯影之變化。之後除去腰椎上附著的軟組織，用以觀察外觀及組織切片檢查。
術中PMMA的最高硬化溫度明顯比實驗組高。術後飼養過程中並無神經系統的問題或其它併發症，既使其中有兩個椎體在CT檢查中發現有水泥滲漏脊髓管中。
雖然用普通X光檢驗無法清楚分辨檢體間的差異，但CT掃描可提供更好的形態學、體積測量和量化各組的顯影度的比較。PMMA 在3個月和6個月的CT檢測中發現其體積、形態和HU的變化很小，這顯示PMMA惰性穩定的性質。在PMMA 組中12個中只有2個有周圍有顯影透亮線(radiolucent line)，而這些在組織學上是纖維組織，而其餘沒有顯影透亮線的為骨頭與PMMA直接接觸。相比之下，兩種複合水泥有明顯降解，儘管HU的減少並不顯著,但可觀察到沿著水泥外緣的不規則硬化外環、水泥體積顯著減少和形態學變化。在TtCP/DCP組可觀察到較多的水泥分層裂片、外透光亮暈和脊椎畸形，這顯示在TtCP/DCP組因其降解產物造成周圍宿主組織明顯的影響。至於對照組的空洞明顯縮小，且其HU的顯著增加，這導因於年輕豬隻良好的骨癒合特性。
在組織學檢查，可發現在複合水泥這兩組有新骨形成取代水泥，但我們無法量化各組間的差異，這是導因於檢體的取樣差異、處理時樣品的損耗、染色問題和觀察者的誤差所致。而沿著這一些複合水泥塊外圍，除了可觀察到新骨形成和骨重塑外，但在其他一些脊椎骨的水泥塊外緣，有看到大量的纖維組織，甚至長入複合水泥塊中。這些放射線和組織學上的變化，可以在所有複合水泥塊慢慢減少變小的外緣觀察的到。這些顯示分解下來的複合材料漸漸由新骨所取代，而這無法在PMMA 組看到。在未脫鈣的組織學，PMMA組的結果較穩定，既沒有新骨形成，也無沿PMMA塊邊緣有複合材料替代現象。
而比對外觀、組織學和影像學發現的結果時，找到之間互相對應的關係。如CT 所見外圍的顯影透亮線是代表組織學上的纖維組織，而硬化環代表重塑的形成新生骨頭。雖無法做出與組織切片完全相同的CT掃片影相，但CT掃描可能用來當成一個初步評價注射骨水泥在椎體內的方法。
為此可顯示我們開發出的可注射式、可降解式骨水泥，具優於現有PMMA的水泥的一些特性。可望未來在施行脊椎整形術時，提供醫師一個不同的選擇。但未來仍在其組成比例及製程上，仍需再調整，以增加穩定性並方便量產。

關鍵字

骨質疏鬆症 ； 椎體 ； 聚甲基丙烯酸甲酯 ； 聚丙烯富馬酸 ； 磷酸三鈣 ； 可生物降解的水泥 ； 骨水泥注射 ； 動物實驗 ； 電腦斷層掃描

並列摘要

While many different filler materials have been applied in vertebral augmentation procedures, none is perfect in all biomechanical and biological characteristics. To minimize possible shortages, we synthesized a new biodegradable, injectable and premixed composite made from poly(propylene fumarate) (PPF) and biphasic α-tricalcium phosphate (α-TCP)/hydroxyapatite (HAP) ceramics powder and evaluated the material properties of the compound in vitro. We mixed the PPF cross-linked by N-Vinyl pyrrolidinone and biphasic α-TCP/HAP powder in different ratios with benzoyl peroxide as an initiator.
The setting time and temperature were recorded, although they could be manipulated by differing the concentrations of hydroquinone and N-N-Dimethyl-p-Toluidine. Degradation, cytocompatibility, mechanical properties, and radio-opacity were analyzed after the composites were cured by a cylindrical shape. We also compared the study materials with polymethyl methacrylate (PMMA) and PPF with pure HAP particles. Results showed lower temperature during curing process (38-44oC), sufficient initial mechanical compression fracture strength (61.1±3.7MPa), and gradual degradation were observed in the newly developed bone filler. Radio-opacity in Hounsfield units was similar to PMMA as determined by computed tomography scan. Both pH value variation and cytotoxicity were within biological tolerable limits based on the biocompatibility tests.
Mixtures with 70% α-TCP/HAP powder were superior to other groups and were applied in vivo study.
To evaluate these cements in vivo, a porcine vertebral model was applied. Besides the before-mentioned 70% α-TCP/HAP/PPF mixture, 70% TtCP/DCP/PPF composite was also recruited in vivo for commercial availability and similar biphasic transformation properties after hydration to compare these two biodegradable cements with PMMA. Twelve miniature pigs had been enrolled in this study. Four cylindrical holes were drilled at the center of lateral cortex of vertebral bodies of lumbar spine with 5mm in diameter and 10mm in length through lateral retroperitoneal approach. The premixed cements were injected into the holes randomly and hardened in situ with setting time and highest setting temperature recorded. After 3months and 6months, the pigs were sacrificed. The retrieved spines specimens were scanned with X-ray and computed tomography. Samples were prepared for histological studies with/without de-calcification. The different appearances of solidified cements and the interaction zones between cement and bone were analyzed and compared.
Setting temperatures of PMMA were significantly higher than composite groups. There were two leakages of cement in spinal canals without significant neurological complication.
The differences were difficult to be identified by plain X-ray films and CT scans provided better resolutions for morphologic comparisons, volumetric measurement and quantified radiopacity among groups. Little volume, morphology and Hounsfield unit variation of PMMA is detected between 3 months’ and 6 months’ groups and implies inert nature of PMMA cement. Only 2 of 12 PMMA blocks were surrounded by radiolucent lines, and those lines were fibrous tissues in histology. While the remainder without radio-lucent lines were direct bone-PMMA contact. In contrast, irregular sclerotic ring along the cement blocks, significant decreases of cement volume and morphologic changes showed the degradability of both composite cements, despite the decreases of HU were not significant. More cement block laminations, radiolucent halos and vertebral deformities were observed in TtCP/DCP group, and this implied better degradability with obvious influences on surrounding host tissues by released products. Regarding control groups, the defects shrunk significantly with significant increase in Hounsfield units and this indicates good bone healing property of young porcine individuals.
New bone formation with substitution of cement were observed in histological specimens of both composite cements groups, but we are unable to quantify the differences between groups for sampling divergences, processing losses, staining errors and observer’s biases. Besides new bone formation and remodeling along outer surfaces of some composites blocks, however in some other blocks, abundant fibrous tissues surrounding, or even invading into blocks, was also observed in some vertebra. Radiological and histological changes were observed in all composite groups and these modifications were along diminished block boundaries. These suggested gradual substitution of decomposed composite by new bone formation, which could not be inspected around PMMA block.
In non-decalcified histology, results of PMMA group were more reliable but neither new bone formation nor composite substitution was identified along the PMMA blocks. When combining the results of gross, histological and radiographic finding, each radiologic finding corresponding to a specific histological illustration, such as radio-lucent line indicating histological fibrous tissue and sclerotic rings representing new bone formation with remodeling. Even though it is unable to make histological slices identical completely to CT scan slices, CT scanning could be a preliminary and easy way for evaluation of injectable bone cements in vertebral bodies.
This study indicated that these composites of PPF and biphasic CPCs powder are promising, premixed, injectable biodegradable bone fillers for these two composites possessing characteristics better than PMMA’s, but there are still some improvements should be done to increase reliability of the composite in the future.

並列關鍵字

osteoporosis ； vertebroplasty ； poly(methylmethacrylate) ； poly(propylene fumarate) ； calcium phosphate ； biodegradable cement ； injectable bone cement ； animal study ； computed tomography

參考文獻

1. Pearson D, Miller C. Clinical Trials in Osteoporosis. London: Springer-Verlag London Ltd. 2007, p189-233.

2. Peterlik M. Aging, neuroendocrine function, and osteoporosis. Exp Gerontol 1997;32:577-86.

3. Lindsay R. The menopause and osteoporosis. Obstetrics & Gynecology 1996;87:16-9.

4. Riggs BL, Melton LJ. The worldwide problem of osteoporosis: Insight afforded by epidemiology. Bone 1995;17:505-11.

5. A Devine. Who are candidates for prevention and treatment for osteoporosis? Osteoporosis Int 1997;7:1-6.