臺灣博碩士論文加值系統

治療長骨的粉碎骨折常帶給骨科醫師很大的挑戰，因為這類的損傷常合併嚴重的骨粉碎及相當的骨膜剝離損傷，這樣的傷害常導致傷口感染、軟組織缺損、及骨癒合不良等後遺症。骨膜是一個面狀的結構覆蓋在長骨的周圍，因為結構裡的高度血管化、富含骨再生細胞、擁有骨引導再生因子及具備骨傳導結構，使得骨膜成為一個骨癒合修復過程中不可或缺的結構。可惜的是，以往的文獻報告比較少提到有關如何處裡骨膜來達到促進長骨分節粉碎骨折並骨膜剝離損傷時的治療。
本研究的目的就是希望能利用生物可吸收性的聚己內酯(PCL）支架來做為骨膜結構用以治療長骨分節性骨折。我們建立了一個白兔大腿股骨分節性骨折並利用克式釘(Kirscher-wire)固定的模型，我們比較在這個白兔股骨分節性粉碎骨折的情形下，有使用或沒有使用支架來重建骨膜，是否會帶來不一樣的臨床結果。一個由聚乳酸聚甘醇酸共聚物(PLGA)所製成的帶藥奈米薄膜，其中富含利多卡因(lidocaine)止痛藥、萬古黴素(vancomycin)及頭孢曲松鈉(ceftazidime)抗生素的也被製備作為骨膜的一部分，我們假設這樣複合的仿生骨膜能提供骨引導因子的功能，建立血管增生及抑制感染的發生，來促進粉碎骨折的癒合。
這個奈米薄膜的藥物釋放情形是利用高效能液相層析儀(HPLC)定量分析及微生物盤狀擴散法來檢測。而臨床的結果包括術後第二、第六及第十二周的X光檢查檢測骨癒合情形。動物的活力是在一定時間內紀錄動物在特製的動物活力行為柵欄中的總活動量而定。當最後動物犧牲後，他的大腿股骨再進行扭力及硬度測試。
實驗結果顯示在一個半月及三個月的不同時段的組別中，有使用骨膜支架的白兔都擁有較好的骨折斷端處骨痂形成、較強的扭力及較好的骨硬度。在藥物釋放方面，實驗結果顯示奈米薄膜釋放利多卡因可以維持有效劑量達三週以上，在生物活力測試上顯示奈米薄膜釋放萬古黴素(vancomycin)及頭孢曲松鈉(ceftazidime)抗生素達有效制菌量達三十天以上，活力測試上也顯示有帶藥的白兔組別比沒有帶藥的組別擁有更好的總活動量。
總結來看，這樣的一個人工複合藥物釋放骨膜可以有效地用於長骨分節性骨折並骨膜剝離損傷的治療。

The management of comminuted long bone fractures brings several challenges to orthopedic surgeons because these injuries are always associated with bone comminution and periosteum stripping injuries which would induce unpredictable sequela like infection or bony nonunion. The periosteum is a membrane that covers the outer surface of the long bone. As a consequence of its high level of vascularization and the presence of osteogenic progenitor cells, osteoinductive growth factors, and an osteoconductive structure, the periosteum comprises a critical component of the bone-healing process. Unfortunately, few publications address the management of periosteum during the treatment of segmental fractures with periosteal stripping injuries.
The purpose of this study was to identify the biodegradable polycaprolactone stent playing as a periosteum during treating the segmental bony fracture. We set up a segmental long bone fracture model on the left femoral shaft of rabbits treating by an intramedullary Kirscher-wire fixation. Using this rabbit fracture model, we compared the bone healing conditions between those with and without stents coverage. The biodegradable poly([d,l]-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with lidocaine , vancomycin, and ceftazidime was also developed as an component of the artificial periosteum in the treatment of these segmental femoral fractures. We hypothesized that an ideal biomimetic periosteum would provide delivery of osteoinductive growth factors, restore angiogenic potential, and inhibit microbial infection on the bone surface, to allow better bone union.
The nanofibrous membrane’s drug release behavior was assessed in vitro using high-performance liquid chromatography and the disk-diffusion method. The clinical outcomes included radiographs obtained at 2, 6, and 12 weeks postoperatively to assess the bone unions. The total activity counts in animal behavior cages were also examined to evaluate the clinical performance of the rabbits. After the animals were euthanized, both femoral shafts were harvested and assessed for their torque strengths and toughness.
The results showed that those rabbits with stents coverage achieved a better bridging callus formation, better torque strength, and better toughness recovery than those without stents in 1.5-and 3-month following-ups. The daily in vitro release curve for lidocaine showed that the nanofibers eluted effective levels of lidocaine for longer than 3 weeks. The bioactivity studies of vancomycin and ceftazidime showed that both antibiotics had effective and sustained bactericidal capacities for over 30 days. The findings from the in vivo animal activity study suggested that the rabbits with the artificial drug-eluting periosteum exhibited statistically increased levels of activity and better clinical performance outcomes compared with the rabbits without the artificial periosteum.
In conclusion, this artificial drug-eluting periosteum may eventually be effectively used for the treatment of the long bone fractures with periosteal stripping injuries.

指導教授推薦書
口試委員審定書
致謝............................................................iii
中文摘要..........................................................v
Abstract.......................................................vii
Table of Contents...............................................ix
Legends of photographs..........................................xi
Legends of Tables...............................................xv
Chapter 1 Background.............................................1
1.1 Study background.........................................1
1.2 Introduction of periosteum...............................3
1.3 Introduction of polycaprolactone.............................7
1.4 Introduction of PLGA [Poly (lactic-co-glycolic acid)].......10
1.5 Introduction of clinical medicine...........................12
1.5.1 Lidocaine..............................................12
1.5.2 Vancomycin.............................................14
1.5.3 Ceftazidime............................................16
1.6 Study hypothesis and target.................................18
Chapter 2 Materials and Methods.................................19
2.1 Experimental flow chart.....................................19
2.2 Manufacture of biodegradable Polymers.......................20
2.2.1 Fabrication of biodegradable PCL stents................20
2.2.2 Preparation of drug-eluting nanofibrous membrane.......26
2.2.3 Scanning electron microscopy (SEM) of the nanofibers...30
2.3. In vitro drugs releasing study.............................31
2.3.1. HPLC assessment of Lidocaine release..................31
2.3.2. Bioactivity test of the antibiotics...................33
2.4 In vivo Animal Study........................................35
2.4.1. Surgical procedure....................................35
2.4.2. Postoperative animal care and activity assessment.....40
2.4.3. Animal euthanasia and mechanical strength test........44
2.5 Statistical analysis........................................47
Chapter 3 Results...............................................48
3.1 The results of in vitro studies.............................48
3.1.1 The results of the SEM micrographs.....................48
3.1.2 In vitro release characteristic of lidocaine...........49
3.1.3 In vitro release of antibiotics........................51
3.2 The results of in vivo animal studies.......................55
3.2.1 Body weight variation..................................55
3.2.3 The animal activity study..............................59
3.2.4 In vivo radiographic results of bone healing...........61
3.2.5 In vivo mechanical strength............................63
Chapter 4 Discussion and conclusions............................64
4.1 Discussion..................................................64
4.2 Conclusions.................................................68
4.3 Future works................................................69
Acknowledgements................................................70
References......................................................71
Appendix........................................................81
CURRICULUM VITAE................................................81
Publications....................................................83

Legends of photographs
[Figure 1] Staged operations for open fractures with periosteal stripping injury.................................................2
[Figure 2] Structure of periosteum...............................4
[Figure 3] A set of diagrams illustrating the high vascularization of the periosteum................................................5
[Figure 4] Publications using PCL in the field of Biomaterials or Tissue Engineering during the last 20 years......................7
[Figure 5] Structures made from PCL..............................8
[Figure 6] The degradation of PCL via hydrolysis intermediates 6-hydroxyl caproic acid and acetyl coenzyme A, which are then eliminated from the body via the citric acid cycle (a). Schematic visualization of how crystalline fragmentation could have taken place(b). Accelerated degradation of PCL over 5 weeks in NaOH (c) .................................................................9
[Figure 7] Structure of poly (lactic-co-glycolic acid)..........11
[Figure 8] 2D and 3D structure Ball-and-stick model of the lidocaine molecule, as found in the crystalline state...........13
[Figure 9] Chemical structure and Stick model of the vancomycin molecule, C66H75Cl2N9OO24, as found in the crystal structure of vancomycin acetate dichloride hydrate...........................15
[Figure 10] Skeletal formula and ball-and-stick model of сeftazidi
memolecule......................................................17
[Figure 11] Commercial products of PCL (Sigma-Aldrich, USA).....21
[Figure 12] Engineering draw of the stent scaffold model........22
[Figure 13] Lab-made micro-injection molding machine............22
[Figure 14] 3D-stereogram of the mold box drawing by Pro/Engineer 5.0 and final metal mold box with both cope and drag components.23
[Figure 15] A set of diagrams illustrating the steps of interconnection procedure to transform the stent element into stent mesh......................................................24
Figure 16] (A) Stent element design(B) Each stent element, interconnected with another stent element(C) Final assembled biodegradable stent(D)The end-product-a polycaprolactone stent..25
[Figure 17] poly[(d,l)-lactide-co-glycolide] (PLGA).............26
[Figure 18] Lidocaine hydrochloride [Sigma-Aldrich (St Louis, MO, USA)]...........................................................26
[Figure 19] Syringe pump and needle.............................28
[Figure 20] High voltage power supply machine...................28
[Figure 21] The collection plate................................29
[Figure 22] The electrospinning apparatus for nanofibers production......................................................29
[Figure 23] The nanofibrous membrane produced by the electrospinning procedure.......................................30
[Figure 24] The connection diagram of the HPLC analysis system..31
[Figure 25] The diagram of the steps of the antimicrobial susceptibility test.............................................34
[Figure 26] Diagram of the inhibition zones measurement in the antimicrobial susceptibility testing............................34
[Figure 27] The rabbit was induced of general anesthesia in a lab-made anesthesia chamber.........................................35
[Figure 28] The inhalation anesthesia system of Isoflurane vaporizer.......................................................36
[Figure 29] The surgical procedures in group A..................37
[Figure 30] The surgical procedures in group B..................38
[Figure 31] The surgical procedures in group C..................39
[Figure 32] A diffuse reflecting sensor HP100-A1 (Glotec, Taiwan.) and a diagram of all 9 sensors in three bars separated the cage into 9 symmetrical areas........................................41
[Figure 33] The photograph of the lab-made Animal Behavior Cage (ABC) built up with barbed iron wire to form a 1.2 m X 1.2 m squared network base and four 1.2m X 0.7 m network sidewalls....41
[Figure 34] A photograph of an individual unrestricted cage with standard rabbit chow and sterilized drinking water ad libitum for further postoperative care......................................42
[Figure 35] A photograph of a rabbit arranged for the X-ray radiographic assessment of the left thigh.......................43
[Figure 36] A photograph of the mechanical strength machine.....44
[Figure 37] a diagram and a photograph of a femoral specimen was eradicated residual muscles and removed the intramedullary K-wire ................................................................45
[Figure 38] A diagram and a photograph illustrating the procedure of the rotational torque and toughness assessment...............46
[Figure 39] SEM micrographs of the drug-eluting PLGA nanofibers under 20,000 magni¬fication.....................................48
[Figure 40] In vitro daily release and accumulated characteristic of lidocaine....................................................50
[Figure 41] In vitro release characteristic of vancomycin.......52
[Figure 42] In vitro release characteristic of ceftazidime......53
[Figure 43] Bioactivity of the eluted antibiotics...............54
[Figure 44] Body weight variations at various postoperative days ................................................................56
[Figure 45] Food intake at different post-operative days........58
[Figure 46] Water consumption at different post-operative days..58
[Figure 47] The photoelectric switch sensors of the animal behavior cage were 4484±366, and 6856 ±716 counts for groups A and B respectively..................................................60
[Figure 48] The total activity counts of rabbits in the ABC for one week........................................................60
[Figure 49] The comparison of radiographic results between two groups..........................................................62

Legends of Tables
[Table 1] Parameters of polycaprolactone (PCL)..................20
[Table 2] the major component of HPLC...........................32

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