Taipei Medical University Institutional Repository:Item 987654321/4258

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(1)台北醫學大學牙醫臨床研究所碩士論文. Taipei Medical University Graduate Institute of Dentistry Postgraduate Faculty. 指導教授：林哲堂博士歐耿良博士. 不銹鋼表面功能化處理之特性研究 Characteristic Research of Functional Modification on Stainless Steel. 研究生：陳思翰撰中華民國九十八年六月 June, 2009.

(2) 致謝漫長又辛苦的日子終於熬過去了，在這幾年中不僅學到許許多多學業上的知識，也學到不少待人處事及問題處理的能力。很慶幸的在這樣艱苦的路程我能被最好的老師們所指導，由衷的感謝所有指導過我的老師林哲堂院長、歐耿良老師、陳玫秀老師、馬隆祥醫師、張維仁老師，特別感謝林院長及歐老師，因為有他們的強力幫忙下我才能夠平順的畢業。另外也要感謝許多實驗室的同學(鴨子、瀚毅、志華、豪哥、千惠、阿富)以及在附設醫院贋復組的同學(宗豪、柏超)，謝謝你們幫助我解決一堆疑難雜症。最後要感謝我的家人，爸爸、媽媽、姊姊、家綺，不管何時都一直陪伴在我的身邊加油打氣，並且幫助我度過難關。畢業了，新的路程即將開始了!. I.

(3) 論文摘要. 論文名稱：不銹鋼表面功能化處理之特性研究. 私立台北醫學大學口腔醫學院牙醫學系碩(博)士班研究生姓名：陳思翰畢業時間： 98 學年度第二學期指導教授：林哲堂博士口腔醫學院院長歐耿良博士生醫材料所暨工程研究所主任. 論文摘要內容 316 L 不銹鋼經由表面處理後，經血液相容性實驗證實不銹鋼經表面處理後對於血塊的形成有極大的影響。有諸多學者對於人造之生醫材料研究顯示：生醫材料表面必須先有血液中之蛋白質吸收於材料表面後，才可能會有後續的一些細胞反應進而達到所追求之生物相容性。血液中有數百種蛋白質，其中以血漿蛋白為最多，而血漿蛋白會使凝血的時間拉長，使血塊延後產生。對於一個牙科植體 II.

(4) 手術的區域來說，血塊的存在與骨頭生長的是植體手術成功與否的關鍵，骨頭能夠生長才能更進一步促進植體與骨頭的骨結合，即所謂之骨整合。本次研究將 316 L 不銹鋼經由表面處理後，於不同電壓及不同處理時間為參數，嘗試著去與血液中所存在的纖維蛋白及免疫蛋白相容，經由一系列的血液實驗來證實處理過的 316 L 不銹鋼不引起過多的細胞沾附及良好的血液相容性，可用做為最好的過渡期植體骨釘。. III.

(5) Abstract. Title of Thesis：Characteristic Research of Functional Modification on Stainless Steel. Author：Chi-Han Chen Thesis directed by： Professor Che-Tong Lin Professor Keng-Liang Ou. Abstract Content 316L stainless steel treated with the electrochemistry greatly influenced the rate of blood coagulation, which had been proven by series of blood compatibility tests. Previous studies have shown that the protein adsorption is the key to further induce the interactions between cells and material surfaces. In the blood, the albumin is the most abundant protein, the adsorption of the albumin tends to prolong the formation of the blood clot. However, the presence of the blood clot in the dental implant site is crucial in order for the bone formation to IV.

(6) initiate, which leads to the ultimate goal, “ osseointegration ”. In the present research, we treated the 316L stainless steel with various voltages and charged times of positive charge. We then put these samples through a series of blood test, in order to find the best hemocompatible sample for interim implant screw.. V.

(7) Table of Content 致. 謝 ……………………………………………………….. I. 中文摘要 ……………………………………………………….. II. 英文摘要 ……………………………………………………….. III. 目. VI. 錄 ……………………………………………………….. Chapter 1 Introduction ............................................................................ 1 1.1 Purpose of Current Research ........................................................ 1 1.2 Hypothesis of Current Research.................................................... 2 1.3 General Background ..................................................................... 3 Chapter 2 Literature Review................................................................... 4 2.1 History of Stainless Steel ............................................................... 4 2.2 Stainless Steel Properties............................................................... 5 2.3 The Concept of Osseointegration ................................................ 10 2.4 The Interaction between Blood and Artificial Interfaces ............ 12 2.5 The Influence of the Implant Surface Structure........................... 14 2.6 The Biomaterials Surface Pretreatment ...................................... 16 Chapter 3 Experimental Procedures .................................................... 40 3.1 Sample preparation ..................................................................... 40 3.2 Physical and chemical properties of samples with and without treatment ........................................................................................................... 41 Chapter 4 Result ..................................................................................... 51 4.1 Microstructure analysis of 316L alloy with and without treatment51 4.2 Hemocapatibility of 316L alloy with and without treatment....... 54 VI.

(8) Chapter 5 Discussion.............................................................................. 91 5.1 Microstructure Analysis............................................................... 91 5.2 Hemocompatibility ...................................................................... 93 Chapter 6 Conclusion............................................................................. 96 References ............................................................................................... 97. VII.

(9) Chapter 1 Introduction. 1.1 Purpose of Current Research In the modern days, titanium alloy bone screw is widely used among the dental implant field, since it has the mechanical characteristics that mostly fulfill the rudimentary of a bone screw materials. However, the high boost in the medical technology results in over consuming of the pure titanium and its alloys, which greatly raises the cost just to obtain the access to the titanium.. Nowadays, only. the richest and the famous are qualify for the use of the medical titanium alloys, not everyone is able to have their optimal medical treatments. Therefore, a way to solve this crisis is to develop and research another good anti-corrosion metal, the 316L stainless steel. In the current study, we explored more with the use of the electrochemistry method to the surface of the 316L stainless steel with various voltage and the length of the times charged. Then, with these treated samples we went through a series of blood compatibility tests. The goal of this research is to find the best voltages and the duration charged for the 316L stainless steel with the human blood compatibility tests.. 1.

(10) 1.2 Hypothesis of Current Research The hypothesis of this study is that by treating 316L stainless steel with electrochemistry method, to achieve the oxide layer that we expected to form. Our expectation is that the treated stainless steel surface is covered by Cr2O3 layer. With the help of this layer, when it is initially contact with the human blood, the proteins in the blood will not denature and result in massive thrombosis reaction. The treated stainless steel should be more blood compatible than the untreated stainless steel in terms of blood coagulation.. 2.

(11) 1.3 General Background Recently, the prevalence of dental implant has pursued by many companies and researchers in searching for better dental implants materials. Ever since Leon Guillet discovered stainless steel in 1904, many clinicians followed Strauss for the use of stainless steel in surgical application [1]. Stainless steel demonstrates good mechanical properties, ductility, cost effectiveness and biocompatibility. For these reasons, stainless steel are widely used for bone plates and screws in fracture fixation, spinal rods and intravascular heart stent [1]. The exposure of untreated stainless steel surface to the flowing blood, may result in the formation of thrombus and the proliferation of smooth muscle cell [2]. Factors which determine biocompatibility are corrosion resistance and toxicity of metals, in which the stainless steel must have been treated either with coating or topographically [2,3]. In order to understand the relationship between the metal surfaces and blood coagulation, a reasonable way to further investigate is electrochemistry suggested by Sawyer and Schaldach [4].. 3.

(12) Chapter 2 Literature Review. 2.1 History of Stainless Steel Today, stainless steel is one of the mostly used biomaterials for fixation due to its favorable combination of mechanical properties, corrosion resistance and cost effectiveness when compared to other metallic implant materials. The biocompatibility of implant quality stainless steel has been proven by successful human implantation for decades. The stainless steel was first discovered by Leon Guillet in 1904 [1]. Then Strauss in 1926 patented the 18Cr-8Ni stainless steel with 2~4% molybdenum, for the use of stainless steel in surgical application [table 2.1-1]. Due to its outstanding characteristics, the use of stainless steel in orthopedic surgery opened up a variety of possible treatments for bone fractures [fig. 2.1-2~4] [1]. Recently, new nickel-free stainless steels have been developed primarily to address the issue of nickel sensitivity [fig. 2.1-5]. This stainless steel also has the superior mechanical properties and yet with better corrosion resistance. With this new combination of stainless steels, it becomes more competitive to other metals for implants materials in the future [1]. 4.

(13) 2.2 Stainless Steel Properties Composition, microstructure and tensile properties of stainless steel used for internal fixation are standardized in IS0 and ASTM material specifications. Metallurgical requirements are to ensure with sufficient corrosion resistance, nonmagnetic response, and satisfactory mechanical properties. nComposition The minimum 2.25% of molybdenum content and the high chromium content ensure that the compositional requirement % Cr + 3.3 x %Mo > 26 will be met. This formula provides a quantitative measure of the resistance to localized corrosion attack.. The nominal nickel content in implant. quality stainless steel is significantly higher than in commercial quality. It is primarily responsible for maintaining a completely austenitic nonmagnetic microstructure. Low sulfur content in the stainless steel composition has a favorable effect on the volume fraction of sulfide inclusions. For example, Manganese sulfides (MnS) are undesirable because they decrease the quality of surface.. Reduced silicon content is responsible for decreasing in. silicate-type inclusions and also provides better austenite stability. Low phosphorus content provides better ductility, since the majority of the 5.

(14) surgical implants that are cold worked for increased strength.. The. limitation on copper content is a common method of controlling tramp elements that may be present in revert material that is used in the melting process. oMicrostructure Implant quality stainless steel should have a single phase austenitic microstructure. However, delta ferrite is an unacceptable secondary phase in implant stainless steel due to the inferior corrosion resistance compared to the austenitic matrix. In addition, delta ferrite is ferromagnetic and therefore increases the magnetic permeability of the stainless steel. Carbides have been observed in the higher carbon type 316 steels after prolonged heating in the 400-650°C sensitization range. These carbides tend to precipitate inter-granularly and have an adverse effect on inter-granular corrosion resistance because the high amounts of chromium are bound. The lower chromium content in the vicinity of the carbides can promote enhanced corrosive attack. The microstructure and composition will also influence the magnetic permeability of stainless steel. Stainless steel implants are completely 6.

(15) nonmagnetic. Therefore, these implants will not experience movement or create tissue heating effects during magnetic resonance imaging (MRI). However, stainless steel implants will produce signal distortions that may compromise the ability to obtain satisfactory MRI image. pPhysical and tensile properties The density of stainless steel is about 7.9 g/cm3. This is nearly twice the density of titanium. The stiffness of stainless steel is directly related to the modulus of elasticity, which is about 186 GPa and therefore 80% greater than unalloyed titanium. Stainless steel implants will be significantly stiffer than titanium implants of the same general dimensions. In addition, the implant design will also influence the stiffness or flexibility of an implant system. qTorsional properties The difference in torsional response between stainless steel and titanium is an important concept that explains some of the clinical features related to the insertion and tightening of bone screws.. When a stainless steel bone. screw is inserted and the torque reaches its maximum, the screw bottoms out then the screw stops advancing. With the continuation of tightening the head 7.

(16) of the screw leaving the torque constant until the stainless screw head breaks after about 1.5 turns. This property provides a specific tactile response or “feel” during tightening, that is the uniqueness of stainless steel bone screws. Due to the reason that torque resistance does not stabilize after the screw bottoms out. After numerous of practices, the surgeon can begin to feel the difference in the torsional response while tightening the titanium screw versus stainless steel screw. With a properly tightening sensation, implant screws can be achieved without exceeding the failure torque of the material.. 8.

(17) 2.3 The Concept of Osseointegration The concept of “osseointegration” was first proposed by Dr. Brånemark in 1981 [5], “osseo” means bone, “integration” means mixed together.. The. formation of bone around the implant area can be divided into three stages.. The. first stage is the inducing of bone cells “Osseo-induction”, as the osteoblasts spread through the surface of the implant and fixed firmly onto it. The second stage we called it “bone formation”, as the calcium phosphate gradually matures.. The. third stage is the “bone remodeling”, the bone growth continues and gradually the calcified cells deposit, along with some bone resorptions. The success of the osseointegration means there is a tightly bonded relation between the implant and the bone.. In the failure case, the implant is surrounded. by unstable soft tissues, mostly due to the lack of stability. During the time that implant is under the loading force and micro-motion have occur beyond the acceptable range, the intimate integration of the bone and the implant begins to destruct.. This situation leads to the failure of osseointegration.. The key to the stability of the implant and bone tissues, which greatly influence the progress of osseointegration has two factors; the quality of the bone tissue and the other is the contact area between the bone and implant. 9.

(18) 1. Bone Quality The density of the alveolar bone must be dense enough. After a force loaded, the bone will undergo internal structure adjustment, without any changes to the outlook for shape and size.. Then the bone will be adapted to for the changes in. the surrounding environment. Frost et al after series of researches classified the adjustment reactions of dense bone under a loaded force into four classes [fig. 2.3-1]. nMicrostrain greater than 4000 is called “Pathlogical overload zone”. Under this condition, after the bone resorbed, only the woven bone will be formed, and the formation of lamellar bone is very slow. The bone tissue is continuously being resorbed, but the newly transformed bone tissue formation is suppressed. oThe range from 2500~4000 microstrain is called the “overload zone”, this is when the bone tissue starts to absorb faster than the repaired bone tissue. pWhen 500~2500 microstrain was applied on to the bone tissues, the bone can be maintained at a “physiological homeostasis”. qLess than 500 microstrain will be called the trivial zone, in which the bone tissue under this microstrain will form “disuse atrophy”. 10.

(19) 2. Contact Area between Bone and Implant The strength of the alveolar crest bone will be weaken if the density decreases. In order to prevent microfacture of the peri-implant bones, the bones around the implant must have less stress loaded. In the initial healing period after the implant implanted into the bone, there must be a mechanical fixation to assist the implant in position.. Therefore, in the less dense bone condition, the increasing of. effective contact area between alveolar bone and implant are necessary, for better anti-forces and force distribution.. 11.

(20) 2.4 The Interaction between Blood and Artificial Interfaces The type of protein initially adsorbed from blood into the material surfaces plays a dominate role in determining subsequent events.. The pre-requisite for any. further cell function is the cell adhesion stage [fig. 2.4-1,2] [3]. First, the protein adsorbed into the artificial surface, and the cells such as platelets then adhered to the surface.. After the firm adherence, the cells started to spread along the surface,. while picking up more and more cells which led to the formation of the thrombus [fig. 2.4-3] [6]. The thrombus may cause embolization in the blood circulation, and induces severe artilleries necrosis. For the heart stent surgery this is the most unwanted crisis, but the thrombus formation is a must for dental implant surgery. The formation of the thrombus in the dental implant site not only provokes the formation of collagens and bones, also to act as a big scaffold to allow the integration with bone cells [fig. 2.4-4,5] [7]. The adsorption of the proteins depends on not only the topography but also the charge of the surface material.. In another word, conformational change and. protein denaturization are the reasons for the protein adsorption [6]. Chen et al [8] describe that when the energy gap of the material surface is near the energy gap of 12.

(21) the fibrinogen (1.8eV), the fibrinogen started to denature and decompose into fibrinmonomer and fibrinopeptides, then the irreversible thrombus will form [fig. 2.4-6]. In the blood, there are hundreds of proteins within the stream, and the most dominant protein is albumin. The albumin reduces the deposition of the platelets, and prolong the time for the formation of the blood clot. On the other hand, the fibrinogen and immunoglobulin G (IgG) tends to enhance the platelet deposition by activating platelets to release cytokines and promote thrombogenesis [6].. 13.

(22) 2.5 The Influence of the Implant Surface Structure If the implant surface is smooth, it is less possible to induce stimulation and toxicity. The bad part is that the implant will not react with the surrounding tissues, and then there will be about 0.1~ 10 μm of fibrous capsule formed around the implant area. From a long term perspective, the capsule will keep increasing its thickness, and may be possible to block the surrounding blood supply. It may further lead to the accumulation of the wastes around the implant, and causes inflammation to form a cyst. For these reasons, the purposes for having the rough surface implants are as the following: nIncrease the contact area between the implant and bones [9,10] [fig. 2.5-1] YPromote the activity of mature osteoblasts [11,12,13] ZIncrease the mechanical interlocking between implant and bones [14,15,16] [Promote the attachment molecules [17,18] \Promote the adhesive ability of osteoblasts [19,20] ]Increase the blood clot retention ability of the implant surface [21,22] ^The roughness of the implant surface has impact on the cell adherence and morphology, which affect the behavior of the cell.. 14.

(23) Other ways of implant surface treatment can be briefly classified into the following groups: nNo treatment to the implant surface, like machine surface YApplication of etching technique, to create indentation or porous surfaces; these methods are called “subtractive treatment” such as sand-blast, acid-etching, resorbable blast medium [23,24] [fig. 2.5-1]. ZImplants coating with different materials, such as HA plasma spray coating, TiO2 plasma spray coating; coating procedure are called “addictive treatment” [25- 28] [fig. 2.5-2] [Other method such as sintering particles on the implant surface; the particles used can be growth factors, bone morphological proteins [29,30] [fig. 2.5-3]. 15.

(24) 2.6 The Biomaterials Surface Pretreatment When the endothelial injury occurs, non-biocompatible stent material and inflammation usually tend to lead to thrombin generation, and the stimulation causes the release of growth factors and cytokines from activated platelets and inflammatory cells. These factors stimulate the migration and proliferation of smooth muscle cells and enhance the production of extracellular matrix which dramatically increases the risk for further vessel occlusion. In order to reach the most effective treatment for atherosclerosis, the stent material must be supportive, flexible, capable of expansion, and biocompatible. Presently, most of the stents are consisted of 316L stainless steel. However, this pure 316L stainless steel is not yet fully biocompatible and may induces high occurrence of restenosis and thrombosis. These types of the stents can be either passively or actively coated in order to improve its biocompatibility. Passive (fixed) coatings such as polymers, silicon carbide [31], carbon [32] or gold [33], provide a biologically inert barrier between the stent surface, the circulating blood and the vessel wall. Active coatings possess biologically active substances like heparin [34–37] or release drugs (e.g. paclitaxel, sirolimus or rapamycin) that prevent the occurrence of thrombosis or inhibit neointimal hyperplasia and thereby reduce restenosis [38] [fig. 2.6-1,2].. 16.

(25) Therefore in order to improve the anti-wear and the corrosion resistance abilities as well as the hardness of 316L SS, the following techniques were put in series of trial: (1) glow discharge nitrogen implantation, (2) carbon-doped stainless steel coating sputtering and (3) low temperature plasma nitriding [fig. 2.6-3]. [39] Biocompatibility of 316L SS was studied with human osteoblast and fibroblast cultures need transition. Chromium and cobalt have been shown to (a) concentrate in the nucleus and mitochondria [39,40] (b) interact with DNA and RNA, (c) inhibit oxidative metabolism and (d) induce neoplasmic cell formation [fig. 2.6-4]. Nickel, as well as chromium, has been proven to induce significant inhibition of mitosis in human fibroblasts, and Ni-Cr metallic powder has induced cell alterations and zones of lysis in cell cultures. According to Bordji et al, the stainless steel with nitrided exhibited a high degree of cytoincompatibility. Such an effect was not obvious during first few days of culture, especially during the cell proliferation cycle. Thus, if poor cytocompatibility of nitrided SS is not linked to a decrease in corrosion resistance, it may be due to the nature of the surface generated by the nitriding treatment. Early decomposition of the nitrided layer in contact with a culture medium seems improbable, since no pitting corrosion was observed. Another explanation may be the negative influence of a high concentration of nitrogen on the cells. The upper supersaturated layer could be 17.

(26) partially dissolved and a non-negligible amount of atomic nitrogen may be released into the medium. The high activity of this element could further poison the cells, leading to biological damage [39]. The long-term interfacial bond between an implant and bone may be improved by creating roughen, or porous surface coating on the implant to increase the surface area available for bone to implant apposition. Simple methods for surface roughening include grit blasting and shot peening. Also the more complex methods involve creating a surface consisting of pores of a certain size (between 100 and 300 µm) [42]; providing the implant material has good biocompatibility with bone then the bone may grow into these pores, thus providing extremely rigid fixation of the implant [43,44]. Other approaches involve the use of flame sprayed titanium powder which creates a porous surface with pores of 25-100 µm, or the use of a titanium wire mesh surface bonded to the implant to produce pores of around 100 µm [45]. In the case of titanium and aluminium, there is mounting evidence that these metal ions can affect cell function in vivo [46], as well as cell proliferation and synthesis of extracellular matrix in vitro [47-49]. Furthermore, it has been shown that titanium, aluminium and vanadium ions can inhibit apatite formation in vitro [49,50], and this could have important implications for mineralization at bone-metal implant interfaces in vivo. 18.

(27) According to Yoshimitsu et al [fig. 2.6-5], they compare the metal concentrations in rabbits’ tibia tissues with various metallic implants, which includes the SUS 316L stainless steel, Co–Cr–Mo casting alloy, Ti–6Al–4V and V-free Ti–15Zr–4Nb–4Ta alloys were implanted into the rat tibia for up to 48 weeks [fig. 2.6-6~8]. Then Fe concentrations were determined by graphite-furnace atomic absorption spectrometry. The Ti concentration in the tibia tissues with the Ti–15Zr–4Nb–4Ta implant was lower than that in the tibia tissues with the Ti–6Al–4V implant with its low metal release in vivo is considered advantageous for long-term implants. (SUS 316L implant was relatively high in concentration, and it rapidly increased up to 12 weeks and then decreased thereafter.) [51].. 19.

(28) Table I、Composition limits for implant quality stainless steel ISO 5832-1 Composition D [wt. %] ≦ 0.03 ≦ 2.0 ≦ 0.025 ≦ 0.01 ≦ 1.0 17.0- 19.0 13.0- 15.0 2.25- 3.5 ≦ 0.1 ≦ 0.5 Balance. Element C Mn P S Si Cr Ni Mo N Cu Fe. Table 2.1-1. Table II、Tensile properties of implant quality stainless steel according to ISO 5832-1 Condition. Ultimate tensile strength, Rm [MPa]. Yield strength, Rp0.2 [MPa]. Elongation A5 [%]. Annealed. 490- 690. min. 190. min. 40. Cold Worked. 860- 1100. min. 690. min. 12. Cold Drawn. 1350- 1600. -. -. Table 2.1-2. 20.

(29) Product no. 204.030 404.030 206.024 406.024 214.026 414.026. Table III Bone screw torsional properties Yield Failure Failure Description torque torque angle [Nm] [Nm] [0] 3.5 mm 1.82 (±0.11) 2.75 (±0.04) 443 (±58) Cortex ss 3.5 mm 1.59 (±0.03) 2.4 (±0.01) 230 (±6) Cortex Ti 4.0 mm 1.59 (±0.04) 1.85 (±0.02) 503 (±87) Cancellous ss 4.0 mm 1.27 (±0.09) 1.85 (±0.11) 289 (±27) Cancellous Ti 4.5 mm 4.45 (±0.29) 5.37 (±0.06) 515 (±96) Cortex ss 4.5 mm 3.62 (±0.08) 5.25 (±0.06) 249 (±4) Cortex Ti Table 2.1-3. Fig 2.1-4. Torque versus angle of rotation for stainless steel and titanium screws 21.

(30) Table IV Comparison of the mechanical properties of standard and nickel-free implant stainless steel in the annealed condition. Yield strength, Rp0.2 Tensile strength,Rm Elongation, A5 Reduction of area, Z Fatigue strength. Ni-free steel. ISO 5832-1. 600 MPa 1000 MPa 50 % 70 % 480 MPa. 250 MPa 590 MPa 57 % 88 % 180 MPa. Table 2.1-5. 22.

(31) 500. Light Loading Normal Hemostasis. 2500. Excessive Loading 4000. Pathological overloading microstrain. Fig 2.3-1. 23.

(32) Fig. 2.4-1 FESEM photograph of a hard resin embedded sample with a tainless-steel implant 26 weeks after operation. M= metal implant, FC=fibrous capsule with collagen fibres and fibroblast type cells, MT= muscular tissue. A similar tissue reaction was seen with all materials at 26 weeks (magn. *330).. 24.

(33) Fig. 2.4-2 Cell-metal interface of NITI 4 weeks after implantation. Torn cell podia and membrane structures (arrows) can be seen in the under surface of the cell. Respective focal contacts to the metal surface (asterisk) are also present (magn. *5000).. 25.

(34) Fibroblast (FB) attachment to a metal (M) surface. The 15 μm gap between metal and soft tissue is due to sample preparation. A close connection, a slender cell shape and small filopodia are seen (Ti-6Al-4V 4 weeks after implantation, magn. *1700).. Fig 2.4-3 A closely connected focal adhesion site with ruptured cell membrane structures. The gap to the metal (M) surface is under 30 nm (NiTi 4 weeks after implantation, magn. *50000) 26.

(35) Fig 2.4-4 The SEM morphologies of 316L stainless steel wire surface after in vivo thrombosis test with the administration of heparin.. 27.

(36) Fig. 2.4-5 Wound healing in adult skin. (A) hemostasis of the wound occurs; (B) thrombosis (clotting) froms plus; (C) inflammation (migration of white blood cells); (D)formation of granulation tissue with new vessels.. 28.

(37) Fig 2.4-6 Interaction of fibrinogen with a solid via the charge transfer process. 29.

(38) Fig 2.5-1 SEM micrographs of an SLA surface on a titanium dental implant (Courtesy of Straumann AG, Switzerland). 30.

(39) Fig 2.5-2 SEM micrographs of a TiO blasted surface (Courtesy of Astratech TiO blast TM, France). Fig. 2.5-3 SEM photos of scaffolds surface. Pure chitosan/collagen scaffold (A), the scaffolds with Ad-BMP7 (B), HPLCs on the scaffolds with Ad-BMP7 after 2 days culture in vitro (C).. 31.

(40) Fig 2.6-1 Schematic representation of EPC capture strategy with CD34 antibody coated stents. EPCs express CD34 antigen on cell surface. After implantation of CD34 antibody coated stents. CD34 antibody specifically targets CD34 positive cells in the blood circulation, such as EPCs. EPCs are fished out of the bloodsteam for endothelialization of the stent surface.. 32.

(41) Fig. 2.6-2 Blood stream process & Cell-Selex Process. 33.

(42) Fig 2.6-3 Scanning electron micrographs of fibroblasts after 10 d culture on a. untreated stainless steel (SS) b. N-implanted SS. C. nitrided SS and d. C-doped SS (bars= 100 μm). 34.

(43) Fig. 2.6-4 Chromium and Cobalt reaction with cells. 35.

(44) Fig. 2.6-5 Radiograph of rat tibia with the Ti-15Zr-4Nb-4Ta implant after 12 weeks. 36.

(45) Fig. 2.6-6 Changes in metal concentrations in lyophilized tibia tissue with SUS 316L stainless steel or Co-Cr-Mo coating alloy implant as a function of implantation period: (a) Fe released from SUS 316L; (b) Co released from Co-Cr-Mo; (c) Cr relseaed from SUS 316L or Co-Cr-Mo; (d) Ni released from SUS 316L; (e) Mo released from SUS 316L or Co-Cr-Mo. 37.

(46) Fig. 2.6-7 Changes in metal concentrations in lyophilized bone tissue with Ti-6Al-4V or Ti-15Zr-4Nb-4Ta implant as a function of implantation period: (a) Ti released from Ti-6Al-4V or Ti-15Zr-4Nb-4Ta; (b) Al released from Ti-6Al-4V; (c) V relseaed from Ti-6Al-4V; (d) Zr released from Ti-15Zr-4Nb-4Ta; (e) Nb released from Ti-15Zr-4Nb-4Ta; (f) Ta released from Ti-15Zr-4Nb-4Ta. 38.

(47) Fig. 2.6-8 Comparison of metal quantities (μg cm-2 per week) calculated from metal concentrations in tibia tissues with each metal implant after 6 weeks of implantation and those obtained from immersion test at 37 0C for 1 week. 39.

(48) Chapter 3 Experimental Procedures. 3.1 Sample preparation The 316L disks for the present study were prepared from 1 mm thick sheets (Hung Chun Bio-S Co. Ltd, Taiwan). The specimens were mechanically polished through 1200 grit paper and were further polished by diamond abrasives through 1 μm. The specimens were finished with colloidal silica abrasives through 0.04 μm. Prior to use, degreasing and acid pre-pickling of all disks were done by washing in acetone, processing through 2% ammonium fluoride, and a solution of 2% hydrofluoric acid and 10% nitric acid at room temperature for 60 s. Finally, the specimens were etched in an aqueous mixture of HF (2 vol%) and HNO3 (4 vol%) at room temperature for several seconds, and then washed with distilled water in an ultrasonic cleaner. Subsequently, 316 L was performed by cathodically polarization at a constant current for 20 min in 1 M H2SO4 solution at 298 K. The charging current density was kept at 0.1, 1, 3, and 5 A/dm2 (ASD). In addition, the charging current density was kept at 5 ASD for 40 min was also performed. A platinum plate was used as counter electrode in this treatment. To form the nanoporous oxide layer on the surface of the 316L, chemical etched stainless steel was treated by anodization at a constant current of 15 ASD for 10 min in 5 M NaOH solution.. 40.

(49) 3.2 Physical and chemical properties of samples with and without treatment. X Energy dispersive x-ray spectroscopy (EDXS) Electron microscopy will be performed at a 30 kV acceleration voltage. This microscope is usually equipped with an energy-dispersive X-ray spectrometer (EDS) for chemical microanalysis as Fig. 3-1. In our research, 316L discs with and without treatments will be investigated using energy dispersive X-ray spectroscopy (EDX).. Y Field-Emission Scanning Electron Microscope The surface morphology and cross-section view were analyzed with a field emission scanning electron microscope (Hitachi S-4000) shown in Fig. 3-1. The accelerating voltage and emission extracting voltage were 20 kV and around 5kV.. The grain sizes of thin films were evaluated by a plane view method.. The thickness and layer structure was evaluated by cross-sectional view.. Z Phase Identify by X-ray Diffraction Analysis Crystal structures will be determined by X-ray diffraction analysis with Cu-Kα radiation. Furthermore, in order to identify phase transformation in surface treatment process and observe the microstructure of nanoporous layer, cross-sectional transmission electron microscopy (TEM) will be used at 300 kV 41.

(50) acceleration voltages.. [ Grazing-Incidence X-ray Diffraction (GIXRD) Since x-ray path of conventional θ-2θ scanning is too short to produce sufficient diffraction signal to identify minor compound, the crystal structure and phase of the films are identified with grazing-incidence technique instead. The path in the film can be increased or the penetration depth of X-ray can be decreased by using a fixing incident angle of a smaller angle between 20 degrees.. \ X-ray Photoelectron Spectroscopy (XPS) Surface analysis by XPS is accomplished by irradiating a sample with monoenergetic soft x-ray and analyzing the energy off the detected electrons. The XPS measurement was done on Perkin Elmer model PHI 1600 by using a single Mg Kα x-ray operating at 250W. The x-ray source is at an angle of 54.7o with respect to the analyzer. An in situ 3 kV Ar+ ion gun was applied to sputter the samples. Energy calibration was done by using the Au 4f7/2 peak at 83.8 eV (as shown in Fig. 3-2). Based on the high-resolution spherical capacitor analyzer (SCA), the energy resolution is 1.6 eV for survey scan spectrum and 0.2 eV for core-level spectrum, respectively. All peaks of core-level spectrums were fitted with the same full width at half maximum (FWHM). The atomic. 42.

(51) ratio of the relevant elements were obtained by integration the core-level peak areas, corrected by the atomic sensitivity factors (ASF, C:0.296 and N:0.477) based upon empirical peak area values modified for the system’s transmission function.. ] Atomic Force Microscope (AFM) The atomic force microscope (AFM) was proved to be one of the most exciting developments in surface science over the past decade. AFM has recently been shown to be a powerful technique for the accurate measurement of the surface morphology of thin films from which the film growth processes can be elucidated. The present work strongly relies on the ongoing AFM technique. In principle, the possibility for obtaining atomic-resolution images with, in many cases, a minimum of prior preparation of samples. The AFM is a stylus-type instrument with a sharp probe. Scanned raster-fashion across the sample is employed to detect changes in surface structure on the atomic scale. The surface morphology of 316L discs with nanoporous oxide films were observed by a Digital Nanoscope 3000 operation in tapping mode under ambient conditions. All images shown are the row data, and no filtering techniques have been employed.. 43.

(52) ^ Secondary Ion Mass Spectrometry (SIMS) SIMS is one of the most powerful tools in surface analysis used to determine isotope and impurity concentration gradients in studies related to the analysis of thin film, diffusion and ion implantation for semiconductor characterization. The basic SIMS apparatus schematic in Fig. 3-3, it is the destructive removal of material from the specimen and the analysis of that material by a mass spectrometer. Sputtered species are emitted as neutral in various excited states, as clusters of particles and as ions both negative and positive, singly and multiply charged. The O+ and Cs+ activity ions were used as sputtering source. The sputtering source Analysis of sputtered species is the most sensitive of the surface analysis techniques. The collection and analysis of the ionized species, such as the secondary ions are capable to determine the composition of the analyzed specimen. The advantages of SIMS are listed below: ① the detection limit of concentration is lower than PMMA ② all of elements on the periodic table can be detected ③ the depth resolution inherent in this method is less than 5 nm ④ the resistance of the specimen are not limited. u Auger Electron Spectroscopy (AES) AES is an analytical technique used to determined the elemental 44.

(53) composition, and in many cases, the chemical state of the atoms in the surface region of a solid material. The AES analysis was conducted in a Perkin-Elmer PHI 670Xi scanning Auger microprobe. The chamber was evacuated to a pressure of ~10-10 torr after samples loaded. The beam voltage for survey scan and depth profile is 20 kV and 10kV, respectively, and the resolution is 1 eV for the survey scan. In AES, the derivative form dN(E)/dE is used more frequently. The need for differentiation arises because the number of background secondary electron is usually larger than the number of Auger electrons. In this research, A Perkin-Elmer 670Xi scanning Auger electron spectroscope (AES) shown Fig. 3-2 equipped with an ion sputtering gun was utilized for concentration-depth profile analysis. The surface composition was determined by the AES using 15 nA primary beam current with 3 keV electron in a base pressure of 8×10-10 Torr. The beam size was about 20 μm. Depth profiles were obtained by sputtering rate and rastered area are 5 nm/min and 4.0 mm2, respectively. The values of the peak-to peak height signals of the Auger electron transitions were monitored during sputtering.. `Transmission Electron Microscope (TEM) Transmission electron microscopy skill has been successfully used for studies of materials in ULSI field such as observations of stress and strain and diffusion phenomenon between substrate and thin film, etc. Specimens for the. 45.

(54) electron microscope must obviously fit into the holder and hence are usually limited to a disk of maximum external diameter approximately 3 mm and thickness about 0.5 mm (as shown in Fig. 3-4). The actual area of interest must be a few tens of nanometers thick since it will be observed at say 20,000X magnification, need only be on the order of micros or tens of micros in extent to be an adequately usable. The specimens with treatments for 0.1 ASD-20 min, 1 ASD-20 min, 3 ASD-20 min, 5 ASD-20 min and 5 ASD-40 min are as denoted as SS-1, SS-2, SS-3, SS-4 and SS-5, respectively.. 46.

(55) Fig. 3-1 Field-emission scanning electron microscope. 47.

(56) Fig. 3-2 Auger electron spectroscopy. 48.

(57) Ion gun. UHV Ion detector. Energy filter. Ti ta. ni. um. Mass filter im. pl an. t. Sample. SIMS spectrum. Fig. 3-3 Secondary ion mass spectrometry (SIMS). 49.

(58) Fig. 3-4 Transmission electron microscope (TEM). 50.

(59) Chapter 4 Result. 4.1 Microstructure analysis of 316L alloy with and without treatment A. XRD Putting the treated and untreated 316L stainless steel through the X-ray Diffraction Analysis, the results are shown as below [fig. 4-1]. The γ-phase of the untreated 316L stainless steel in the control group showed in the color black, compared with the other electrochemistry treated 316L have no obvious changes. This means that in the electrochemistry treated surfaces, the surface properties of the 316L have not changed drastically. The treated 316L have the same surface properties of untreated 316L and the crystallization still existed in the ET-316L.. B. AFM, SEM By using the Atomic Force Microscope (AFM), we can see in the control group [fig. 4-2], the surface of each grain is quite smooth and with few shallow indents. The average roughness (Ra) measured is 112.84 nm, and the mean roughness (Rm) is -23.096nm. After the surface treatment by electrochemistry. 51.

(60) with the parameter of 3V-20min [fig. 4-3], many pebbles can be seen within few µm, and the value for Ra 66.53nm and Rm 110.91pm. With further treatment of electrochemistry 5V-20min [fig. 4-4], many pores were formed and created many groves with fused grain boundaries, the Ra value is 99.959nm and the Rm value is 45.423nm. Under the Scanning Electron Microscopy (SEM), the control group [fig. 4-5] shows clear and definite grain boundaries. Each grain is larger in size and with the smoother surfaces. As in the 3V-20min [fig. 4-6] the grain boundaries are not as clear. Also the sizes of each grain are smaller and with groves and pores with created rougher surfaces. With the further treatment 5V-20min [fig. 4-7], the boundaries are even less definite and the numerous pores were seen in the diagram. Nevertheless, there are little pores exists in the big pores which greatly increases its roughness and the surface area.. C. Wettability and Surface Energy We then look at the 316L to see how well a material can adhere to the surface through its contact angle and its surface energy. Glass having the lowest contact angle, the untreated and treated 316L have no significance difference in contact angle. The contact angle results are taken with CCD camera as shown 52.

(61) [fig. 4-8]. Testing the surface energy of both the treated and untreated 316L stainless steel. Acting as positive control, glass still has the highest amount of the surface energy. Both treated and untreated 316L have no significance differences in the surface energy [fig. 4-9]. . D. Cross-section In the cross-section view of Scanning Electron Microscopy (SEM), the control group of untreated 316L has shown a very thin and unorganized (fragile) outer oxide layer over a smooth inner layer [fig. 4-10]. Conversely, the treated 316L resulted a thicker and more uniformed oxide layer, with a porous inner layer throughout these surfaces [fig. 4-11]. Micro-implants are increasingly popular in clinical orthodontics to have impact on skeletal anchorage. However, biofilm formation on their surfaces and subsequent infection of peri-implant tissues can result in either exfoliation or surgical removal of these devices.. 53.

(62) 4.2 Hemocapatibility of 316L alloy with and without treatment A. Clotting Time We have demonstrated the effect of clotting time to either treated or untreated 316L, and each sample is being measured by optical density versus time. The data results are formulated into graphs shown as below [fig. 4-12]. When the uncoagulated red blood cells are immersed into the distilled water with the samples, the bilirubin inside the red blood cell burst out of the cells due to the osmotic diffusion between the distilled water and the cell. It was observed that the more of the unattached and uncoagulated blood, the redder the distilled water was seem. As shown in the data, the glass grabbed the most coagulated blood cell within the first 20 minutes compares to the other 316L alloys. After 20 minutes, the control group grabbed more red blood cells than glass; only the treated 316L especially the samples treated with 5V still maintain lesser cells adhered than glass.. B. Plasma Protein Assay (Fibrinogen) Fibrin (also called Factor Ia) is a fibrous protein involved in the clotting of blood. It is a fibrillar protein that is polymerized to form a mesh, that forms a. 54.

(63) haemostatic plug or clot (in conjunction with platelets) over a wounded site. Fibrin is made from fibrinogen, a soluble plasma glycoprotein that is synthesized by the liver. Processes in the coagulation cascade activate the zymogen prothrombin to the serine protease thrombin, which is responsible for converting fibrinogen into fibrin. Fibrin is then cross linked by factor XIII to form a clot. Therefore the data are obtained and as shown in fig. 4-13. The treatment with 2V and 5V have the highest amount of fibrinogen analyzed, the others are about the same as the non-treated stainless steel.. C. Platelet adhesion assays (CD 61) Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61), also known as ITGB3, is a human gene. CD61 is a cluster of differentiation found on thrombocytes. This protein product is the integrin beta chain beta 3. As we know, integrins are integral cell-surface proteins composed of an alpha chain and a beta chain. Any given chain may combine with multiple partners resulting in different integrins. Integrin beta 3 is found along with the alpha IIb chain in platelets. Integrins are known to participate in cell adhesion as well as cell-surface mediated signaling. So we used CD 61 to test how well the platelets can adhere to the samples we prepared, and the result is shown in fig. 4-14. 55.

(64) Glass having the least amount of platelets adhere to the surfaces and the samples with 1V and 5V have the highest amount of the platelets adhered to the surface. The other samples are only slight lower than the non-treated stainless steel, in terms of platelet adherence.. D. Platelet activation assay (p-selectin) P-selectin is a cell adhesion molecule found in granules in endothelial cells (cells lining blood vessels) and activated platelets. Other names for P-selectin include CD62P, Granule Membrane Protein 140 (GMP-140), and Platelet Activation-Dependent Granule to External Membrane Protein (PADGEM). P-selectin plays an essential role in the initial recruitment of leukocytes (white blood cells) to the site of injury during inflammation. The endothelial cells are activated by the molecules such as histamine or thrombin during inflammation, then the P-selectin moves from an internal cell location to the endothelial cell surface. Thrombin is the one that triggers and stimulates endothelial-cell release of P-selectin, and recent studies suggest an additional Ca2+ independent pathway involved in release of P-selectin. Ligands for P-selectin on eosinophils and neutrophils are similar sialylated, protease-sensitive, endo-beta-galactosidase 56.

(65) -resistant structures, clearly different than those reported for E-selectin, and suggest disparate roles for P-selectin and E-selectin during recruitment during inflammatory responses. The result of the P- selectin test is shown in fig. 4-15. The glass and the sample with 3V have the highest P-selectin in the data, which means the inflammation occurs more rapidly than the other samples, the lower voltage samples have lower inflammation than the non-treated, it may due to the etching treatment that causes the grooves to be more deeper. The 5V samples have only slight higher inflammation than the non-treated stainless steel.. E. Clotting Time Samples under SEM XPure Human Blood Under the SEM, in the Control group (Non-treated stainless steel) in fig. 4-16, the red blood cell is still in its unique shape without any deformation. Very little fibrin can be seen stretched out to other red blood cells or adhere on to the stainless steel surface. As shown in the treated sample 5V in fig. 4-17, the pores are obvious in the picture. Noted the smaller pores inside the big pores, with the red blood cells shoot out multiple fibrins to withhold its self from falling into the porous hole. This orientation allowed the trapped 57.

(66) cells to grab more cells nearby creating a huge cluster of cells. It is quite similar to the geometry of spider webs we often seen in the nature used to trap its prey on to the web. Y Pure Human Blood Clotted After 10 mins In the sample 1V [fig. 4-18], the surface of the stainless steel only formed little shallow and very wide pores compare to the 5V. Only two of the cells in the picture extended the fibrins to grab each other, the other cells are “stopped” in the crater with no fibrin extension nor cluster together. The sample 3V [fig. 4-19] have narrower and deeper grooves.. Nearly all cells. have their fibrin extended and one cell was burst and deformed. Compare to the 1V sample, the cell aggregation is more obvious. In the sample 5V [fig. 4-20], notice that the surface was very rough with pores. The fibroblast can be seen with fibrin extended out to grab onto the surface and also the red blood cells. ZPure Human Blood Clotted After 40 mins As shown in the 1V sample [fig. 4-21], compare to 10 mins, there were more fibroblasts and adhered more firmly to the stainless steel surface with more fibrin extension. In the 3V sample [fig. 4-22], more red blood cells were trapped by many fibroblasts. 58. The shape of the red blood cells.

(67) maintain in a contant stable form. The adherence ability of the 3V sample is more obvious than the 1V sample. The 5V sample [fig. 4-23] were seen very few red blood cells attached, though a big cluster of fibroblasts were noted in the picture. As a result, it was demonstrated that 3V sample would give good adherence ability than the other voltage samples. [ Platelet rich plasma (PRP) and Platelet poor plasma (PPP) with treated and non-treated stainless steel In the control group with PRP [fig. 4-24] and PPP [fig. 4-25], the amount of cells noted in the PPP were lesser than the PRP, though the fibrin can still been seen in the picture. Comparing the 1 V sample with PPP [fig. 4-26] and with PRP [fig. 4-27], still more cells were seen in the PRP sample than those with PPP. However, there were more platelets cells than the red blood cells in the PRP. As shown in the 3V sample treated with either PRP [fig. 4-28] and PPP [fig. 4-29], more platelets cells but no obvious fibrin were shown in the picture taken using PPP sample. In the other hand, less fibrin were noted in the PPP [fig. 4-30] of 5V sample picture compare to PRP [fig. 4-31]. Moreover, more platelets and red blood cells were observed in the 5V PRP picture, though most of the platelets had no fibrin extension except for the red blood cells. 59.

(68) Fig. 4-1 XRD. 60.

(69) Fig. 4-2 AFM analysis (Control Group). 61.

(70) Fig. 4-3 AFM analysis (3V-20min). 62.

(71) Fig. 4-4 AFM analysis (5V-20min). 63.

(72) 10 μm Fig. 4-5 SEM (Control). 64.

(73) 10 μm. Fig. 4-6 SEM 3V-20min. 65.

(74) 10 μm. Fig. 4-7 SEM 5V-20min. 66.

(75) Fig. 4-8 Contact Angle CCD Camera. 67.

(76) Fig. 4-9 Surface Energy and CCD Camera. 68.

(77) Fig. 4-10 Cross section View of Control Group. 69.

(78) (a). (b) Fig. 4-11 Treated 316L cross-section view (a) 3V-20 min (b) 5V-20 min 70.

(79) Fig. 4-12 Clotting time with various condition. 71.

(80) Fig. 4-13 Plasma Protein Assay (Fibrinogen). 72.

(81) Fig. 4-14 Platelet adhesion assays (CD 61). 73.

(82) Fig. 4-15 Platelet activation assay (p-selectin). 74.

(83) Fig. 4-16 Clotting Time Test (control at 0 min) in SEM, with pure human blood. 75.

(84) Fig. 4-17 Clotting Time Test (5V at 0 min) in SEM, with pure human blood. 76.

(85) Fig. 4-18 Clotting Time Test (1V at 10 min) in SEM, with pure human blood. 77.

(86) Fig. 4-19 Clotting Time Test (3V after 10 min) in SEM, with pure human blood. 78.





(91) Fig. 4-24 Clotting Time Test (Control) in SEM, with PRP. 83.

(92) Fig. 4-25 Clotting Time Test (Control) in SEM, with PPP. 84.

(93) Fig. 4-26 Clotting Time Test (1V sample) in SEM, with PPP. 85.

(94) Fig. 4-27 Clotting Time Test (1V sample) in SEM, with PRP. 86.





(99) Chapter 5 Discussion. 5.1 Microstructure Analysis As many articles have discussed the toxicity of the stainless steel gradually released after a period of time in vivo. These may be due to strength of the corrosion resistance and anti-abrasion ability [52]. Electrochemistry is a quite efficient way to increase its corrosion resistance and anti-abrasion ability. The widely used of 316L is in the coronary stent, Humbeeck et al [53], done a series of tests about the surface roughness via electrochemistry. The result of the roughness experiment showed in the data in the previous section. 5V-20 min treatment has the most porous structure with Ra 99.959 nm and unclear grain boundaries were observed. The porous also appeared as multiple layers compared to the lower voltage samples. In the contact angle experiment, the angle of all the samples are determined to be almost identical, with the exception of 5V-20 min treatment has a lower contact angle than others. Nevertheless, it is interesting to find out the 5V-40 min treatment resulted a greater contact angle than the 5V-20 min treatment. Prolong the voltage time may cause the number of porous to increase, the numerous pores. 91.

(100) then act like the lotus leafs with nano-scale fibers which can repel liquid easily. Comparing the SEM data with those taken by Humbeeck [53], the electrochemically treated samples appear to be far smoother than the other techniques used, such as laser cut or acid-pickling. Electrochemical polishing is a method of brightening and smoothering the surface of metals [54, 55] by immersing the parts in an electrolyte and applying direct positive current to the sample. The main electrical parameters of electrochemical polishing process are the anodic potential, the anodic current density and the applied voltage.. The nature and rate of any. electrochemical reaction are both determined by the electrode potential.. 92.

(101) 5.2 Hemocompatibility In the first 20 minutes, the treated samples of the 316L stainless steel performed astonishing result in the blood compatibility experiment. The red blood cells were not deformed or denatured, but the effect was not obvious to the fibrins. However, the blood cells started to denature after 5V-40 min treatment, and significant differences cannot be seen among various treatment except the 5V-40 min one showed good blood compatibility better than 5V-20 min ones. It still may due to its main limitation is the tendency to corrode when in contact with blood [56]. 316L stainless steel contains non-negligible amounts of chromium (16%~18%) and nickel (10%-14%); the release of these metallic ions into human tissue and fluids must be regarded as a likely source of long-term problems owing to their known carcinogenic and toxic effects. Particularly, chromium and cobalt have been shown to (a) concentrate in the nucleus and mitochondria [57, 58], (b) interact with DNA and RNA, (c) inhibit oxidative metabolism and (d) induce neoplasmic cell formation. In the fibrinogen test, the 2V and 5V are the two samples that have the most fibrinogens detected. The 2V treated sample still have a smoother surface and the 5V sample have the most porosity, and these criteria caused the fibrins. 93.

(102) cannot be easily adhered to the surface of the treated 316L stainless steel. As in the platelet adhesion test, the 5V sample was consistent detected the most platelets, with the same reason as the fibrinogen. Greater amount of the platelets in sample are also detected when 1V was applied. However, the roughness between the 1V and 2V samples were not significantly different, since the smooth surface of these samples prevent the platelet to adhere to the sample surface. Interestingly, the 3V sample showed the most obvious result among other sample in the platelet activation test. Subsequently, from the AFM data, 3V sample also showed to have the narrow and deep grooves which may stimulate the platelets to release more P-selectin [59]. Clark et a1. [60, 61] and Wojciak-Stothard et al. [62] have shown that a complex situation, varying with cell type as well as with several of the topographical measures. Cells react to a single cliff and demonstrated good reactivity cliff height in the range of l-20 pm according to Clark et a1. [61]. Clark et al. [60, 61] showed that on the groove and ridge topography that the extent of reaction is related to the groove width and depth and possibly also due to the number of adjacent grooves. There is a general evidence that the extent of orientation increases with groove depth up to about 25 pm from topographies of about 1 pm in height. Below this degree of relief results are 94.

(103) less available, partly owing to the difficulty of quantifying the etch depth in earlier years, and perhaps partly due to the previous reasoning that there would not be any effect on the cells. Wojciak-Stothard et al. [62, 63] have demostrated that P388Dl macrophage-like cells react with cliff height down to dimensions at least as small as 44 nm. Other cell types so far studied do not appear to be as reactive, nevertheless epitena, epithelia, fibroblasts and endothelia would still react to depths as shallow as 70 nm. When the grooves or ridges are appreciably wider than the cells, the impact on orientation are not remarkable, although cells may still align to one edge as according to Clark et al [64]. As the width of grooves and ridges is reduced to the width of the cells and less, effects on orientation become more remarked. When more than two ridge grooves meet, lines of actin condensation from a cell will mark out each discontinuity [62]. Clark et al. [65] showed that baby hamster kidney (BHK) cells will react to groove/ridge topography with a pitch (repeat) at 260 nm. These grooves were relatively deep at 500 nm. No work has yet been discovered about the minimum width of topography to which a cell can react. Conversely, there are many reports suggested that rough, often grossly rough surfaces could aid cell adhesion, for instance that of Lydon and Gray [66]. 95.

(104) Chapter 6 Conclusion The biocompatiblity of the 316L stainless steel material was significantly enhanced by electrochemical surface modification. The anodization of the electrolyte applied with enough voltage has successfully allowed the 316L stainless steel to create more porosity and smoother surface to the cells. With the higher voltage applied, the stainless steel surface could become more biocompatible than lower voltage applied. In addition, the voltage with different time applied samples should also be further investigated. Thus, the usage of treated 316L stainless steel should be well-acceptable to allow biocompatibility taken place drastically in the implant system.. 96.

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