Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.     

人牙周膜干细胞的分离鉴定及骨形态发生蛋白-2对其趋化效应的研究

目的研究人牙周膜干细胞（PDLSCs）对骨形态发生蛋白-2（BMP-2）的趋化反应。方法通过有限稀释法分离、培养人PDLSCs,利用免疫荧光染色检测人PDLSCs波形丝蛋白及干细胞表面标志物STRO-1的表达,检测人PDLSCs多向分化能力,通过克隆形成实验和5-溴-2-脱氧尿嘧啶核苷（BrdU）共培养的方法检测其干细胞特性。利用24孔的Transwell细胞培养室来检测人PDLSCs对BMP-2的趋化反应,光镜下计迁移至滤膜下侧面的不同视野的细胞数。结果人PDLSCs抗波形丝蛋白染色阳性,表达干细胞表面标志物STRO-1,体外诱导培养的人PDLSCs能够向成骨细胞和成脂细胞分化,具有较高的自我更新能力,并在体外呈克隆状生长。在100、200 ng·mL-1 BMP-2实验组,Transwell细胞培养室中迁移的细胞数目显著多于空白对照组（P<0.01）。结论BMP-2对人PDLSCs有趋化效应。     

胰岛素样生长因子-Ⅰ和骨形态发生蛋白-2对人牙周膜细胞增殖的作用

目的：重组人胰岛素样生长因子－Ｉ（ｒｈＩＧＦ－Ｉ）、重组人骨形态发生蛋白－２（ｒｈＢＭＰ－２）分别或联合应用对人牙周膜（ＰＤＬ）细胞增殖的影响。方法：采用组织块法体外培养人ＰＤＬ细胞,ＭＴＴ法测定ＰＤＬ细胞在不同生长因子刺激下的增殖情况。结果：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２都可促进人ＰＤＬ细胞的增殖,这种促增殖作用呈一定的浓度依赖性,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对人ＰＤＬ细胞的增殖有协同作用,且与单独应用相比相差显著。结论：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２可望作为牙周再生的生物活性介质,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对ＰＤＬ细胞的促增殖作用更强。     

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro

Background and Objective: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods: To explore the characteristics of periodontal ligament cells in greater detail, fourth‐passage, sixth‐passage and eighth‐passage human periodontal ligament cells were cultured for up to 3 wk. Ki‐67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA expression was quantified by real‐time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor‐2 and bone morphogenetic protein‐7 was examined in first‐passage and fourth‐passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast‐like cells (MG63) served as reference cell lines. Results: Proliferation decreased over time and was highest in fourth‐passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth‐passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor‐2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein‐7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.     

Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage.     

Cyclooxygenase-2 inhibitor reduces simvastatin-induced bone morphogenetic protein-2 and bone formation in vivo

BACKGROUND AND OBJECTIVE: Simvastatin, a cholesterol-lowering drug, also stimulates oral bone growth when applied topically, without systemic side-effects. However, the mechanisms involved in vivo are not known. We hypothesized that bone morphogenetic protein-2, nitric oxide synthase, and cyclooxygenase-2 are involved, based on prior in vitro evidence. MATERIAL AND METHODS: A rat bilateral mandible model, where 0.5 mg of simvastatin in methylcellulose gel was placed on one side and gel alone on the other, was used to quantify nitric oxide, cyclooxygenase-2 and bone morphogenetic protein-2 (via tissue extraction, enzyme activity or immunoassay), and to analyze the bone formation rate (via undecalcified histomorphometry). Cyclooxygenase-2 and nitric oxide synthase inhibitors (NS-398 and L-NAME, respectively) were administered intraperitoneally. RESULTS: Simvastatin was found to stimulate local bone morphogenetic protein-2, nitric oxide and the regional bone formation rate (p < 0.05), whereas NS-398 inhibited bone morphogenetic protein-2 and reduced the bone formation rate (p < 0.05). CONCLUSION: These data suggest an association between simvastatin-induced bone morphogenetic protein-2 and bone formation in the mandibular microenvironment, and the negative effect of cyclooxygenase-2 inhibitors on bone growth.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic differentiation in cells isolated from human periodontal ligament.

Periodontal ligament cells may play an important role in the successful regeneration of the periodontium. We investigated the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2), one of the most potent growth factors that stimulates osteoblast differentiation and bone formation, on cell growth and osteoblastic differentiation in human periodontal ligament cells (HPLC) isolated from four adult patients. rhBMP-2 induced no significant changes in cell growth in any of the HPLCs. rhBMP-2 at concentrations over 50 ng/mL significantly stimulated alkaline phosphatase (ALPase) activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in the HPLCs. rhBMP-2 (500 ng/mL) also slightly enhanced the level of PTH/PTH-related peptide receptor mRNA expression in these cells. While interleukin-1 beta enhanced ALPase activity stimulated with rhBMP-2, tumor necrosis factor-alpha inhibited the rhBMP-2-stimulated activity. Interleukin-6 induced no significant changes in ALPase activity stimulated with rhBMP-2. Although HPLCs, whether treated with rhBMP-2 or not, could not produce measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced osteocalcin mRNA expression and protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HPLCs at both the mRNA and protein levels. These results suggest that rhBMP-2 provides an anabolic effect on periodontal regeneration by stimulation of osteoblastic differentiation in human periodontal ligament cells, and that this stimulatory effect is differentially modulated by inflammatory cytokines during the course of periodontal regeneration.     

骨形态发生蛋白-2与碱性成纤维细胞生长因子在异位和原位成骨中的作用

目的通过骨髓基质细胞（BMSCs）的体外增殖和分化、异位成骨和原位成骨实验来观察骨形态发生蛋白-2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在成骨过程中的作用。方法分别用含BMP-2、bFGF和BMP-2+bFGF的培养液体外培养Beagle犬的BMSCs,通过甲基噻唑基四唑（MTT）比色法测定细胞增殖水平,通过测定碱性磷酸酶（ALP）活性观察细胞的分化情况。将BMSCs与多孔磷酸钙（CPC）分别在含BMP-2、bFGF和BMP-2+bFGF的培养液中复合培养,制成复合材料,一部分植入裸鼠皮下,观察异位成骨情况,另一部分植入Beagle犬的种植体周围骨缺损区,经过荧光标记观察原位成骨情况。结果含有BMP-2+bFGF的培养液促进BMSCs增殖和分化的能力最强。异位成骨情况：BMP-2+bFGF组的成骨量较其他组明显增加,其新骨形成百分比为48.79%±11.31%,高于单一BMP-2组（30.71%±10.85%）和bFGF组（27.33%±9.67%）以及对照组（10.65%±6.05%）。原位成骨术后12周,BMP-2+bFGF组的矿化沉积率高于其他组,其差异有统计学意义（P<0.01）。结论在促进成骨方面,BMP-2和bFGF共同作用优于单一因子。     

骨形态发生蛋白-2释放系统的研究进展

骨形态发生蛋白（BMP）-2是体内成骨重要的促进因子,能诱导未分化间充质干细胞向成骨细胞方向分化。BMP-2释放系统的建立与应用是目前研究关注的重点,建立一个稳定的局部释放系统是治疗性的策略,可优化骨的愈合和降低并发症的发生。本文就BMP-2释放系统建立的必要性、构建材料种类、性能要求、研究趋势等进行综述。     

超声微泡破裂法促进骨形态发生蛋白-2在小鼠骨骼肌表达的研究

目的探讨超声微泡破裂法对基因重组人骨形态发生蛋白-2（rhBMP-2）质粒pIRES-rhBMP2-EGFP在小鼠骨骼肌的转染、表达的促进作用。方法24只BALB/c小鼠随机分为4组,每组6只。A组和B组选择右侧胫前肌为注射点,其中A组注射质粒,B组注射质粒与微泡造影剂混合物后用超声辐照,质粒注射量为30 μg;C组和D组选择右侧股四头肌为注射点,其中C组注射质粒,D组注射质粒和微泡造影剂混合物后用超声辐照,质粒注射量为100 μg。7 d后处死A组和B组小鼠,取胫前肌在荧光显微镜下观察绿色荧光蛋白的表达情况;14 d后处死C组和D组小鼠,取股四头肌行免疫组化检测rhBMP-2表达情况。结果7 d后B组阳性肌纤维百分率高于A组（P<0.05）;14 d 后,C组和D组都可检测到rhBMP-2的表达,但D组rhBMP-2表达量比C组多。结论超声介导微泡破裂法能增加外源性rhBMP-2基因在小鼠体内骨骼肌转染率和表达水平,有望为牙周病的基因治疗提供一种新型方法。     

Molded bone augmentation by a combination of barrier membrane and recombinant human bone morphogenetic protein-2

OBJECTIVES: To provide the histological background to a new method of local bone augmentation, we examined the events occurring beneath a barrier membrane applied with recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIALS AND METHODS: The effects on bone augmentation of rhBMP-2, applied with a membrane mold (BMP-Memb), over surgically-induced bone defects in rat calvaria were examined histologically, and the results compared with those from application of rhBMP-2 (BMP) alone, or of a molded membrane (Memb) alone. RESULTS: At postoperative week 2, the BMP group showed the most marked bone formation. However, the bone diminished in size by week 8. The Memb group showed slow but continuous bone formation by week 8. In the BMP-Memb group, bone filled the space in the mold at week 2, and this was maintained until week 8. Moreover, the soft tissue that had intervened between newly formed bone and the membrane in the Memb group was not evident in the BMP-Memb group, in which bone had formed directly on the membrane. CONCLUSIONS: The results suggest that the combination of rhBMP-2 and barrier membrane has advantages in producing and maintaining bone in the intended shape by inducing osteoblasts directly on the inner surface of the membrane.     

骨缝牵引中联合应用骨形态发生蛋白-2和骨保护素的实验研究

目的探索上颌骨在骨缝牵引时加入缓释骨形态发生蛋白-2（BMP-2）和骨保护素（OPG）对新骨形成的影响。方法以24只杂种犬为研究对象,随机分为A、B、C组。通过手术在上颌骨腭横缝植入自制的新型牵引器。A、C组术后5 d在牵引区附近注射缓释重组人骨形态发生蛋白-2/聚乳酸-羟基乙酸共聚物/纤维蛋白胶（rhBMP-2/PLGA/FS）,B、C组在牵引3周后注射人骨保护素/纤维蛋白胶（rhOPG/FS）。牵引1、2、4、6周后处死动物,采集标本进行组织学染色,并通过组织计量学方法检测腭横缝的组织改建情况。结果A、C组骨缝区成骨细胞功能活跃,透射电镜显示细胞内有大量高尔基复合体、线粒体及粗面内质网。牵引6周时,A、B、C组成骨细胞指数分别为38.5±7.7、35.7±6.5、41.7±11.0,破骨细胞指数分别为5.9±1.0、1.2±0.3、2.8±0.4,骨小梁厚度分别为（38.36±13.28）、（66.20±9.16）、（51.85±9.92）μm;B、C组表现出骨密度增加及破骨细胞指数下降。结论本实验所用牵引器能促进新骨生成;BMP-2与OPG在骨缝牵引过程中有协同作用,可以促进新骨形成及骨改建。     

Peri-implant bone regeneration using recombinant human bone morphogenetic protein-2 in a canine model: a dose-response study

The objective of this study was to evaluate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) dose on alveolar ridge augmentation and dental implant osseointegration. Bilateral, 5 mm supraalveolar, peri-implant defects were surgically created in 6 beagle dogs. rhBMP-2 (0.05, 0.1 or 0.2 mg/ml) in an absorbable collagen sponge (ACS) carrier was molded around the fixtures and wounds were closed. Treatment variations were alternated between animals (incomplete block design). Animals were sacrificed at week 8 postsurgery. Nine of twelve jaw quadrants healed uneventfully. Two jaw quadrants exhibited wound failure by week 4 and one by week 8 postsurgery. Radiographic bone regeneration was observed in defects without wound failure from week 4 postsurgery. Radiolucent voids of variable size and shape were observed and regressed over time. In weeks 6 through 8, there was an apparent increase in bone density and trabecular structure, while bone height and volume decreased. Histometric analysis revealed limited differences in bone regeneration between experimental conditions. Bone regeneration area averaged (+/- SD) 1.0 +/- 0.5, 3.5 +/- 1.4 and 2.3 +/- 0.4 mm2 for the 0.05, 0.1 and 0.2 mg/ml dose, respectively. There were no significant differences in osseointegration. Osseointegration in newly formed bone averaged 19 +/- 4%, 18 +/- 10% and 21 +/- 6% for the 0.05, 0.1 and 0.2 mg/ml rhBMP-2 sites, respectively. Collectively, the data suggest that there are no dramatic differences in bone induction and osseointegration within the selected dose and observation interval.     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

转染人骨形成蛋白-7基因对骨髓基质细胞体外生物学活性的影响

目的：观察hBMP-7基因转染对Beagle犬骨髓基质细胞（BMSC）生物学特性的影响。方法：利用脂质体介导法将hBMP-7基因转染Beagle犬BMSC，观察细胞形态及生长情况，检测碱性磷酸酶、骨钙素及钙化结节等成骨细胞表型。结果：目的基因转染后细胞形态略有变化，增殖能力无明显改变，凋亡率无明显变化，碱性磷酸酶活性明显增高，骨钙素表达增强，钙化结节增大。结论：hBMP-7基因转染可以加速BMSC向成骨细胞表型转化。hBMP-7基因转染的BMSC有望成为牙周组织工程理想的种子细胞。     

骨形态发生蛋白-2在牙发育各阶段中的表达

目的观察骨形态发生蛋白（BMP）-2在牙发育各阶段中的表达,探讨其作用和意义。方法采用免疫组化法观察BMP-2在8～36周龄的人胎儿牙胚、出生1～22d的乳鼠和出生6周至5个月的犬乳牙发育中的表达。结果BMP-2在胎儿蕾状期和帽状期牙蕾上皮、牙板、成釉器和内外釉上皮均为阳性,在钟状期内釉细胞、中间层、成釉细胞和成牙本质细胞为强阳性,在牙乳头和牙囊为阳性;在乳牙萌出前、萌出期的乳鼠以及生后6周犬牙的成釉细胞、成牙本质细胞、赫特维希上皮根鞘和牙囊为强阳性;在犬乳恒牙替换期的牙根、牙周膜以及靠近牙周膜的成骨细胞为阳性。结论BMP-2是牙发育的重要调控因子,参与牙胚细胞的分化、牙胚的形态发生、牙的萌出和乳恒牙替换各阶段的调控。     

人骨形态发生蛋白-2基因克隆及真核表达载体的构建

目的克隆人骨形态发生蛋白- 2（hBMP2）基因片段,构建pcDNA3.1- hBMP2真核表达质粒。方法采用逆转录聚合酶链式反应（RT- PCR）技术,从人骨肉瘤中扩增出人骨形态发生蛋白- 2基因片段,通过DNA重组技术将该基因片段重组于pcDNA3.1真核表达载体上,构建pcDNA3.1- hBMP2重组质粒,通过用PCR扩增、酶切电泳分析及DNA测序的方法对重组DNA进行鉴定。结果经PCR扩增、酶切电泳分析和DNA测序证实,本实验构建的重组质粒目的基因片段为人BMP2- cDNA。结论本实验成功克隆了hBMP2基因并构建成其真核表达质粒。     

骨形态发生蛋白-2基因转染对舌癌细胞增殖活性的影响

目的 探讨腺病毒介导的人骨形态发生蛋白-2 (BMP-2)基因转染舌癌Tca8113细胞后,对Tca8113细胞形态和增殖活性的影响.方法 分别以感染复数(MOI)为0、50、100的腺病毒为载体的BMP-2基因转染体外培养的舌癌Tca8113细胞.采用荧光倒置显微镜观察转染前后Tca8113细胞形态的变化,Weste...     

Effect of recombinant human bone morphogenetic protein-2 on the osseointegration of dental implants: a biomechanics study

Background Bone augmentation procedures in combination with dental implants enhance osseointegration in areas that demonstrate localized bone deficit. Clinical confirmation of a biomechanically stable interface is essential for functional implant loading.Purpose The aim of this study was to evaluate biomechanically the effect of recombinant human bone morphogenetic protein (rhBMP)-2 on implant osseointegration and correlate it with periotest and radiographic measurements.Materials and methods Hollow cylinder implants were filled with absorbable collagen sponge soaked with rhBMP-2 or left empty and implanted in dog mandibles. The animals were followed for 4, 8, and 12 weeks, periotest assessment was performed at the end of each time interval, and specimens were collected for pullout biomechanical testing and radiographic evaluation of bone-implant contact levels.Results Periotest assessment did not provide evidence of statistically significant differences between the two groups and correlated well with the radiographic bone-implant contact levels. The pullout test revealed a higher correlation between force/displacement and displacement/energy for the experimental group, suggesting that the addition of rhBMP-2 did influence the rate of osseointegration.Conclusion The results from the pullout test support the potential role of rhBMP-2 in clinical applications by promoting a biomechanically mature interface at 12 weeks. However, radiographic and periotest assessment of the bone-implant interface did not provide evidence of the differences observed with biomechanical testing.This project is part of a dissertation prepared in partial fulfillment of Dr. Sykaras requirements for the degree of Doctor of Philosophy.