间断甲状旁腺素[PTH（1-34）]治疗促进去势雌性大鼠成骨能力及其机理的实验研究
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摘要
第一部分间断甲状旁腺素[PTH(1-34)]治疗促进去势雌性大鼠股骨干骨折愈合的实验研究
目的:研究PTH在小剂量间断使用时对卵巢去势状态下大鼠骨折愈合的作用,了解PTH在治疗骨质疏松时能否同时促进骨折愈合。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次行右侧股骨干开放截骨克氏针内固定术。实验组和对照组各18只,术后当天起实验组皮下注射人甲状旁腺素[PTH(1-34)]30μg/kg/天;对照组注射等量体积生理盐水。在术后4周和6周时每组处死9只大鼠,取股骨骨折标本行X片、Micro-CT、骨密度、生物力学和组织学检查来判断PTH对于去势大鼠骨折愈合的作用。结果:标本骨痂直径测量结果显示在两时间点治疗组骨痂直径都高于对照组,6周时差异有统计学意义(P<0.05)。骨密度测量显示治疗组高于对照组,两时间点P<0.05。4周时治疗组和对照组X片都能见到骨折线,治疗组稍显模糊,对照组明显,骨痂密度低于治疗组,有骨痂不连续区域存在。治疗组骨折愈合7/9,对照组为4/9(P<0.05)。6周时治疗组和对照组骨折线都消失,治疗组骨密度明显高于对照组,骨痂横径大于对照组,两组X片骨痂测量6周时治疗组骨痂横径和对照组相比有统计学意义(P<0.05)。显微CT检查显示治疗组骨折周围有丰富的骨痂形成,骨痂成熟,皮质骨壳厚度明显高于对照组,骨折愈合进程较对照组提前,愈合质量更高。4周和6周时BV/TV高于对照组且有统计学意义(P<0.05)。生物力学测定结果显示4周和6周时治疗组最大载荷和对照组比较有统计学意义,p<0.05;4周时最大桡度优于对照组,P<0.05。4周时PTH组标本组织切片中发现骨折断端有新骨形成,可见成熟骨样组织,相反,在对照组无成熟骨形成,软骨和纤维组织占大多数。6周时PTH组标本组织切片发现骨折两端之间形成丰富的骨桥和成熟骨组织,骨组织互相连接形成骨小梁,周围皮质完整且较厚。对照组显示新骨成熟形成相对较晚,骨痂由细的骨小梁形成、皮质薄。组织形态测量学分析结果显示两者矿化沉积率(MAR)在4周时有统计学意义,P<0.05。结论:基于影像学、组织形态学、生物力学的研究结果显示在去势雌性大鼠股骨骨折间断使用甲状旁腺素治疗可以加速骨折的愈合进程、增加软骨痂和硬骨痂的量、提高骨痂的质量、增加骨痂矿化、加速骨痂的重建、提高骨痂的生物力学性能。研究结果支持临床使用间断PTH治疗用于绝经后骨质疏松性骨折以缩短骨折治疗时间和提高骨折愈合质量。
第二部分间断甲状旁腺素[PTH(1-34)]治疗促进去势雌性大鼠脊柱后外侧植骨融合的实验研究
目的:评价间断甲状旁腺素使用是否可以增强和促进脊柱植骨融合,对于需要行脊柱融合的骨质疏松患者有无促进融合和同时抗骨质疏松的作用。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次经wiltse入路手术建立脊柱L4-5后外侧横突间自体髂骨植骨融合动物模型。实验组和对照组各18只。术后当天起实验组皮下注射人甲状旁腺素[PTH(1-34)]30μg/kg/天;对照组注射等量体积生理盐水。分别于术后4周和6周时每组随机选9只大鼠采血后处死,取融合脊柱节段标本行手工触诊、X片、Micro-CT、组织学检查和血清骨钙素(OC)、NTX检查来判断PTH对于去势大鼠脊柱融合的作用。结果:标本手动触诊检查判断植骨融合结果为4周时治疗组(PTH)为5/9,对照组(CNT)为2/9;6周时PTH组为8/9,CNT组为5/9。融合率有统计学意义,P<0.05。X线影像学分析显示PTH组4周和6周时融合影像学评分为2.03和3.66;对照组分别为1.45和2.56,P<0.05和P<0.01。Micro-CT扫描影像结果:PTH组新形成的骨量丰富,横突间融合骨连续,冠状面和横断面融合骨面积大于对照组,融合提前,融合骨质量提高。骨小梁结构参数分析结果和计量分析融合节段部位(L4横突上端至L5横突下端之间)的骨量都优于对照组,P<0.01或P<0.05。组织学检查结果:4周时PTH组脊柱标本组织切片中发现:横突间有新骨形成,可见成熟骨组织,骨小梁结构,骨髓腔。相反,在对照组无成熟骨形成,软骨和纤维组织占大多数。6周时PTH组脊柱标本组织切片发现:横突之间形成丰富的骨桥,成熟骨组织,互相连接形成骨小梁,并由发育良好的骨髓腔所包围,周围皮质完整且较厚。对照组新骨成熟形成相对较晚,融合物由细的骨小梁形成,皮质薄。组织形态学测量结果显示两者矿化沉积率(MAR)有统计学意义。4周时P<0.01;6周时P<0.05。4周和6周时骨钙素(OC)水平都明显高于对照组,P<0.05。4周时NTX水平PTH组显著高于对照组,P<0.05。结论;间断低剂量PTH使用在去势大鼠后外侧横突间髂骨植骨脊柱融合模型能缩短融合所需时间加速融合进程,获得更为坚强的融合效果,增加了脊柱融合的成功率,融合骨的数量和质量都得以增强。有望成为加速和增强脊柱融合和其他需要植骨的骨科手术的一种有效的辅助用药。
第三部分间断甲状旁腺素[PTH(1-34)]治疗促进去势雌性大鼠脊柱后外侧植骨融合机理的实验研究
目的:目前对甲状旁腺素促进骨折愈合或植骨融合的分子生物学机制研究少见,为进一步明确其作用机制我们采用去势大鼠脊柱融合模型设计了以下实验研究。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次经wiltse入路手术建立脊柱L4-5后外侧横突间自体髂骨植骨融合动物模型。实验组和对照组各18只。术后当天起实验组皮下注射人甲状旁腺素30μg/kg/天;对照组注射等量体积生理盐水。实验组和对照组分别于术后4、7、14天放血处死。各时间点每组处死大鼠6只,取出腰椎植骨骨痂行Real Time PCR实时定量检测成骨和软骨细胞等相关基因表达。检测基因为ALP、OC、COL-I、COL-Ⅱ、COL-Ⅹ、IGF-Ⅰ、BMP2。结果1、成骨细胞分化相关基因实时定量检测结果:治疗组各时间点ALP表达量都高于对照组,但前两时间点无统计学差异,在14天时两者有显著统计学差别。P<0.01。骨钙素(OC)在7天和14天时治疗组和对照组相比有统计学差别,其中7天时P<0.05,14天时P<0.01。COL-Ⅰ在术后7天和14天时两组比较P<0.05。2、软骨细胞分化相关基因实时定量检测结果:COL-Ⅱ在术后4天、7天和14天时治疗组相比于对照组都有明显升高,其中7天时P<0.01,4天和14天时P<0.05。Col-Ⅹ于术后7天时治疗组较对照组高,但P>0.05。14天时高于对照组且有统计学意义,P<0.05。3、IGF-Ⅰ、BMP2基因实时定量检测结果:治疗组IGF-Ⅰ在术后4天和7天时相对于对照组都有明显升高,均有显著统计学意义(P<0.01)。在14天时治疗组仍高于对照组但无统计学区别。治疗组BMP2在术后14天时和对照组相比升高明显,P<0.05。其它时间点无区别。结论:1 PTH间断使用对于去势大鼠脊柱后外侧横突间自体髂骨移植促进了成骨细胞的增殖分化,COL-Ⅰ、ALP、OC的表达加强。2该模型中软骨细胞分化的基因COL-Ⅱ、COL-Ⅹ的表达增加说明PTH使用后促进了软骨细胞的分化成熟,与早期形成较大的软骨痂有关。3 IGF-Ⅰ是PTH合成作用的重要因子,该模型中的表达早于其它基因,有可能参与调节成骨和软骨细胞的分化,促进植骨修复。4 PTH使用后BMP2的表达增强说明在脊柱融合的进程中间断使用PTH有可能间接或者直接加强BMP2基因的表达。
PartⅠEnhancement of fracture healing by intermittent administration of PTH(1-34) in ovariectomized rats femoral fracture model
Aim: In the present study we evaluated the underlying mechanism by intermittent PTH would affect fracture healing in ovariectomized female Sprague-Dawley rats, and appreciation whether it could enhance fracture healing while administration for osteoporosis. Method: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized; unilateral femoral fractures were produced by open osteotomy and fixed with Kirschner wire six weeks later. Rats were divided into experimental group (n=18) and control group (n=18), the experimental group was treated with PTH(1-34) at a dose of 30μg/kg/day and the control group received same volume vehicle solution (normal saline).treatment were administered daily by subcutaneous injection since the day of fracture were produced. Nine rats from each group were killed and samples harvested for investigation on four and six weeks later, examination items were appreciated for affect of PTH included X ray, Micro-CT, bone mineral dentist, biomechanics and histology. Result: the diameter of callus of experimental group were larger than controls and it had significantly different between two groups at six weeks(P<0.05); bone mineral density(BMD) of the callus in the experimental group were significantly increased compared with controls at four and six weeks(P<0.05). results showed on four weeks after fracture the fracture line could be found, but it were more clear in control group and density of callus were low, seven fractures of nine in experimental group had healing and control group was four of nine(P<0.05).all fractures of two group had healed at six weeks with the callus density were high in experimental group, callus diameter of experimental group in X ray exceeded those of control group(P<0.05). Result of Micro-CT showed that PTH treatment induced a larger and more maturated callus than control group; the cortical thick of callus is significantly exceeded that of controls; the healing processes were early than controls and the quality of callus were improved; The BV/TV of experimental group were larger than that of controls(P<0.05). results of biomechanics show the maximum load of experimental group was significantly increased than that of controls at four and six weeks(P<0.05 ); the deflection under maximum load had significantly difference at six weeks (P<0.05). Histology results showed new mature bone were found at fracture site in experimental group at four weeks; however there were less bone formed in control group which main composed of cartilaginous and fibrosis like tissue. The experimental group showed there had plenty of callus with complete and thick cortical bone; trabecular bone were connecting each other. However, the trabecular and cortical bone were thin in control group, and the processes was lagged than experimental group. histomorphology showed the mineral apposition rate(MAR)between groups had significantly difference at four weeks(P < 0.05). Conclusion: these results based on radiography, histology and biomechanics suggest that intermittent treatment with low dose PTH(1-34) enhances the processes of femoral fracture healing in ovariectomized rat,induces a larger cartilaginous and hard callus formation; Improves the quality of callus; increases the mineralization procedure of callus; promotes the remodeling of callus and augments the biomechanical strength of fracture femoral. Results support opinion of treatment osteoporosis fracture of menopause female with intermittent administration PTH, which can reduce the time of fracture healing and improve the quality of callus.
PartⅡIntermittent PTH(1-34) augments Spinal Fusion in an Ovariectomized rat posterolateral Spinal Fusion Model with iliac crest bone graft
Aim: In the present study we tested the hypothesis that intermittent administration parathyroid hormone(PTH) improves spinal fusion outcomes in the ovariectomized female Sprague-Dawley rat posterolateral spinal fusion model,and appreciation whether it could enhance graft bone healing while administration for osteoporosis. Method: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized and underwent a bilateral posterolateral L4-5 spine fusion procedure by wiltse approach six weeks later; the graft material is a morselized auto-iliac crest bone graft. the animals were divided into 2 groups of 18 animals. Starting on postoperative day, experimental group was received daily subcutaneous injections of recombinant PTH(1–34) at a dose of 30μg/kg/day, whereas control group received a daily subcutaneous injection of same volume vehicle solution (normal saline). Nine rats from each group were killed and samples harvested for investigation on four and six weeks later, L4-5 vertebral segments were removed and analyzed with manual bending, X ray, Micro-CT and histomorphometry. Serum bone metabolism markers (OC, NTX) were analyzed. Results: Manual palpation identified fusion in two of nine (control) versus five of nine (PTH) animals at four weeks, and five of nine (control) versus eight of nine (PTH) in six weeks (P<0.05). A radiographic scoring system of PTH group resulted in an average score of 2.03 and 3.66 at four and six weeks, correspondence scores of control group were 1.45 and 2.56(P<0.05 and P<0.01).Result of Micro-CT showed that the continuous fusion mass between L4-5 transverse process of experimental group were larger than controls and fusion area had significantly different in both cross section and coronal section. PTH(1–34) accelerated the spinal fusion and acquired solid fusion mass, Improve the quantity and quality of fusion callus. parameter of Fusion bone mass(L4-5) by Micro-CT analysis were also improved with PTH treatment (P<0.01 or P<0.05). Histology results showed new mature bone, trabecular bone and marrow cavity were found at fusion site in experimental group at four weeks; however, there were less bone formed in control group which main composed of cartilaginous and fibrosis like tissue. The experimental group showed there had plenty of fusion callus with complete and thick cortical bone between L4-5 transverse process; trabecular bone were connecting each other and enclosing by marrow cavity. The trabecular and cortical bone were thin in control group, and the processes was lagged than experimental group. histomorphology showed the mineral apposition rate(MAR)between groups had significantly difference at four weeks and six weeks(P<0.01 and P<0.05). Serum OC levels were significantly higher in the PTH group than in the control group at two time point(P<0.05). The CTX levels in the PTH group were higher than in the control group at four weeks(P<0.05). Conclusion: these results suggest that intermittent treatment with low dose PTH(1-34) augmented posterolateral spine fusion with auto-iliac bone graft in ovariectomized female Sprague-Dawley rats. PTH(1–34) enhanced bone formation leading to the acceleration of the spinal fusion, which reduce the time of spine graft healing and acquires solid fusion mass, increase posterolateral fusion success and Improve the quantity and quality of fusion callus. Based on the results of this study, these findings suggest that PTH has promise for use as an adjunctive agent to improve spinal fusion and all other skeletal reconstruction surgeries requiring bone grafts in clinical medicine.
PartⅢMechanisms for the enhancement of posterolateral spine fusion in ovariectomized rats treated with intermittent PTH(1-34)
Aim: In the present study, we analyzed the underlying molecular mechanism by which exogenous PTH would affect osteogenesis and chondrogenesis in an ovariectomized rat spine arthrodesis model. Methods: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized and underwent a bilateral posterolateral L4-5 spine fusion procedure by wiltse approach six weeks later; the graft material is a morselized auto-iliac crest bone graft. the animals were divided into 2 groups of 18 animals. Starting on postoperative day, experimental group was received daily subcutaneous injections of recombinant PTH(1–34) at a dose of 30μg/kg/day, whereas control group received a daily subcutaneous injection of same volume vehicle solution (normal saline). six rats from each group were killed on four, seven, fourteen days later , graft callus was harvested for gene expression investigation on osteogenesis and chondrogenesis by RT-PCR, the level of expression of ALP, OC, COL-I, COL-Ⅱ, COL-Ⅹ, IGF-Ⅰ, BMP2 were analyzed. Results: 1、mRNA expression of osteoblast-related gene. The expression of ALP in PTH group was increased than control group significantly at fourteen days(P<0.01) and without difference at four and seven days; osteocalcin expression in PTH treated was increased markedly than controls at seven and fourteen days (P<0.05 and P<0.01); The expression of COL-Ⅰin PTH group was significantly increased than controls at seven and fourteen days(P<0.05). 2、mRNA expression of chondrocyte-related gene. The expression of COL-Ⅱwas up-regulated markedly in PTH treatment group at three experimental point(7days:P<0.01,4 and 14days:P<0.05).the expression of Col-Ⅹwas increased in PTH treated rats at seven and fourteen days, which has statistical difference at fourteen days(P<0.05). 3、mRNA expression of IGF-Ⅰand BMP2. gene expression of IGF-Ⅰin PTH treated were significantly increased at four and seven days(P<0.01), there had much expression but without difference at fourteen days. The expression of BMP2 in PTH group was up-regulated at fourteen days(P < 0.05)and without difference at other time. Conclusion: 1 intermittent administration PTH enhances proliferation and differentiation of osteoblast in an ovariectomized rat spinal fusion model with auto-iliac bone graft, the expression of COL-Ⅰ, ALP and OC were up-regulated. 2 the level of chondrocytes -related gene COL-Ⅱand COL-Ⅹwere increased markedly in the PTH treated group, it suggest treatment with PTH in spine fusion facilitates proliferation and differentiation of chondrocyte, which leads to a larger soft callus formation. 3 IGF-Ⅰis an important factor for the anabolic effect of PTH, because of the early expression of IGF-Ⅰthan other gene in this model, it may augment graft bone healing by stimulated proliferation and differentiation of chondrocyte and osteoblast. 4 the increased BMP2 expression in PTH group suggest that the expression of BMP2 maybe enhance directly or indirectly in the processes of spine fusion treatment with PTH.
目的:研究PTH在小剂量间断使用时对卵巢去势状态下大鼠骨折愈合的作用,了解PTH在治疗骨质疏松时能否同时促进骨折愈合。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次行右侧股骨干开放截骨克氏针内固定术。实验组和对照组各18只,术后当天起实验组皮下注射人甲状旁腺素[PTH(1-34)]30μg/kg/天;对照组注射等量体积生理盐水。在术后4周和6周时每组处死9只大鼠,取股骨骨折标本行X片、Micro-CT、骨密度、生物力学和组织学检查来判断PTH对于去势大鼠骨折愈合的作用。结果:标本骨痂直径测量结果显示在两时间点治疗组骨痂直径都高于对照组,6周时差异有统计学意义(P<0.05)。骨密度测量显示治疗组高于对照组,两时间点P<0.05。4周时治疗组和对照组X片都能见到骨折线,治疗组稍显模糊,对照组明显,骨痂密度低于治疗组,有骨痂不连续区域存在。治疗组骨折愈合7/9,对照组为4/9(P<0.05)。6周时治疗组和对照组骨折线都消失,治疗组骨密度明显高于对照组,骨痂横径大于对照组,两组X片骨痂测量6周时治疗组骨痂横径和对照组相比有统计学意义(P<0.05)。显微CT检查显示治疗组骨折周围有丰富的骨痂形成,骨痂成熟,皮质骨壳厚度明显高于对照组,骨折愈合进程较对照组提前,愈合质量更高。4周和6周时BV/TV高于对照组且有统计学意义(P<0.05)。生物力学测定结果显示4周和6周时治疗组最大载荷和对照组比较有统计学意义,p<0.05;4周时最大桡度优于对照组,P<0.05。4周时PTH组标本组织切片中发现骨折断端有新骨形成,可见成熟骨样组织,相反,在对照组无成熟骨形成,软骨和纤维组织占大多数。6周时PTH组标本组织切片发现骨折两端之间形成丰富的骨桥和成熟骨组织,骨组织互相连接形成骨小梁,周围皮质完整且较厚。对照组显示新骨成熟形成相对较晚,骨痂由细的骨小梁形成、皮质薄。组织形态测量学分析结果显示两者矿化沉积率(MAR)在4周时有统计学意义,P<0.05。结论:基于影像学、组织形态学、生物力学的研究结果显示在去势雌性大鼠股骨骨折间断使用甲状旁腺素治疗可以加速骨折的愈合进程、增加软骨痂和硬骨痂的量、提高骨痂的质量、增加骨痂矿化、加速骨痂的重建、提高骨痂的生物力学性能。研究结果支持临床使用间断PTH治疗用于绝经后骨质疏松性骨折以缩短骨折治疗时间和提高骨折愈合质量。
第二部分间断甲状旁腺素[PTH(1-34)]治疗促进去势雌性大鼠脊柱后外侧植骨融合的实验研究
目的:评价间断甲状旁腺素使用是否可以增强和促进脊柱植骨融合,对于需要行脊柱融合的骨质疏松患者有无促进融合和同时抗骨质疏松的作用。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次经wiltse入路手术建立脊柱L4-5后外侧横突间自体髂骨植骨融合动物模型。实验组和对照组各18只。术后当天起实验组皮下注射人甲状旁腺素[PTH(1-34)]30μg/kg/天;对照组注射等量体积生理盐水。分别于术后4周和6周时每组随机选9只大鼠采血后处死,取融合脊柱节段标本行手工触诊、X片、Micro-CT、组织学检查和血清骨钙素(OC)、NTX检查来判断PTH对于去势大鼠脊柱融合的作用。结果:标本手动触诊检查判断植骨融合结果为4周时治疗组(PTH)为5/9,对照组(CNT)为2/9;6周时PTH组为8/9,CNT组为5/9。融合率有统计学意义,P<0.05。X线影像学分析显示PTH组4周和6周时融合影像学评分为2.03和3.66;对照组分别为1.45和2.56,P<0.05和P<0.01。Micro-CT扫描影像结果:PTH组新形成的骨量丰富,横突间融合骨连续,冠状面和横断面融合骨面积大于对照组,融合提前,融合骨质量提高。骨小梁结构参数分析结果和计量分析融合节段部位(L4横突上端至L5横突下端之间)的骨量都优于对照组,P<0.01或P<0.05。组织学检查结果:4周时PTH组脊柱标本组织切片中发现:横突间有新骨形成,可见成熟骨组织,骨小梁结构,骨髓腔。相反,在对照组无成熟骨形成,软骨和纤维组织占大多数。6周时PTH组脊柱标本组织切片发现:横突之间形成丰富的骨桥,成熟骨组织,互相连接形成骨小梁,并由发育良好的骨髓腔所包围,周围皮质完整且较厚。对照组新骨成熟形成相对较晚,融合物由细的骨小梁形成,皮质薄。组织形态学测量结果显示两者矿化沉积率(MAR)有统计学意义。4周时P<0.01;6周时P<0.05。4周和6周时骨钙素(OC)水平都明显高于对照组,P<0.05。4周时NTX水平PTH组显著高于对照组,P<0.05。结论;间断低剂量PTH使用在去势大鼠后外侧横突间髂骨植骨脊柱融合模型能缩短融合所需时间加速融合进程,获得更为坚强的融合效果,增加了脊柱融合的成功率,融合骨的数量和质量都得以增强。有望成为加速和增强脊柱融合和其他需要植骨的骨科手术的一种有效的辅助用药。
第三部分间断甲状旁腺素[PTH(1-34)]治疗促进去势雌性大鼠脊柱后外侧植骨融合机理的实验研究
目的:目前对甲状旁腺素促进骨折愈合或植骨融合的分子生物学机制研究少见,为进一步明确其作用机制我们采用去势大鼠脊柱融合模型设计了以下实验研究。方法:10周龄SPF级雌性SD大鼠行卵巢去势,6周后去势大鼠再次经wiltse入路手术建立脊柱L4-5后外侧横突间自体髂骨植骨融合动物模型。实验组和对照组各18只。术后当天起实验组皮下注射人甲状旁腺素30μg/kg/天;对照组注射等量体积生理盐水。实验组和对照组分别于术后4、7、14天放血处死。各时间点每组处死大鼠6只,取出腰椎植骨骨痂行Real Time PCR实时定量检测成骨和软骨细胞等相关基因表达。检测基因为ALP、OC、COL-I、COL-Ⅱ、COL-Ⅹ、IGF-Ⅰ、BMP2。结果1、成骨细胞分化相关基因实时定量检测结果:治疗组各时间点ALP表达量都高于对照组,但前两时间点无统计学差异,在14天时两者有显著统计学差别。P<0.01。骨钙素(OC)在7天和14天时治疗组和对照组相比有统计学差别,其中7天时P<0.05,14天时P<0.01。COL-Ⅰ在术后7天和14天时两组比较P<0.05。2、软骨细胞分化相关基因实时定量检测结果:COL-Ⅱ在术后4天、7天和14天时治疗组相比于对照组都有明显升高,其中7天时P<0.01,4天和14天时P<0.05。Col-Ⅹ于术后7天时治疗组较对照组高,但P>0.05。14天时高于对照组且有统计学意义,P<0.05。3、IGF-Ⅰ、BMP2基因实时定量检测结果:治疗组IGF-Ⅰ在术后4天和7天时相对于对照组都有明显升高,均有显著统计学意义(P<0.01)。在14天时治疗组仍高于对照组但无统计学区别。治疗组BMP2在术后14天时和对照组相比升高明显,P<0.05。其它时间点无区别。结论:1 PTH间断使用对于去势大鼠脊柱后外侧横突间自体髂骨移植促进了成骨细胞的增殖分化,COL-Ⅰ、ALP、OC的表达加强。2该模型中软骨细胞分化的基因COL-Ⅱ、COL-Ⅹ的表达增加说明PTH使用后促进了软骨细胞的分化成熟,与早期形成较大的软骨痂有关。3 IGF-Ⅰ是PTH合成作用的重要因子,该模型中的表达早于其它基因,有可能参与调节成骨和软骨细胞的分化,促进植骨修复。4 PTH使用后BMP2的表达增强说明在脊柱融合的进程中间断使用PTH有可能间接或者直接加强BMP2基因的表达。
PartⅠEnhancement of fracture healing by intermittent administration of PTH(1-34) in ovariectomized rats femoral fracture model
Aim: In the present study we evaluated the underlying mechanism by intermittent PTH would affect fracture healing in ovariectomized female Sprague-Dawley rats, and appreciation whether it could enhance fracture healing while administration for osteoporosis. Method: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized; unilateral femoral fractures were produced by open osteotomy and fixed with Kirschner wire six weeks later. Rats were divided into experimental group (n=18) and control group (n=18), the experimental group was treated with PTH(1-34) at a dose of 30μg/kg/day and the control group received same volume vehicle solution (normal saline).treatment were administered daily by subcutaneous injection since the day of fracture were produced. Nine rats from each group were killed and samples harvested for investigation on four and six weeks later, examination items were appreciated for affect of PTH included X ray, Micro-CT, bone mineral dentist, biomechanics and histology. Result: the diameter of callus of experimental group were larger than controls and it had significantly different between two groups at six weeks(P<0.05); bone mineral density(BMD) of the callus in the experimental group were significantly increased compared with controls at four and six weeks(P<0.05). results showed on four weeks after fracture the fracture line could be found, but it were more clear in control group and density of callus were low, seven fractures of nine in experimental group had healing and control group was four of nine(P<0.05).all fractures of two group had healed at six weeks with the callus density were high in experimental group, callus diameter of experimental group in X ray exceeded those of control group(P<0.05). Result of Micro-CT showed that PTH treatment induced a larger and more maturated callus than control group; the cortical thick of callus is significantly exceeded that of controls; the healing processes were early than controls and the quality of callus were improved; The BV/TV of experimental group were larger than that of controls(P<0.05). results of biomechanics show the maximum load of experimental group was significantly increased than that of controls at four and six weeks(P<0.05 ); the deflection under maximum load had significantly difference at six weeks (P<0.05). Histology results showed new mature bone were found at fracture site in experimental group at four weeks; however there were less bone formed in control group which main composed of cartilaginous and fibrosis like tissue. The experimental group showed there had plenty of callus with complete and thick cortical bone; trabecular bone were connecting each other. However, the trabecular and cortical bone were thin in control group, and the processes was lagged than experimental group. histomorphology showed the mineral apposition rate(MAR)between groups had significantly difference at four weeks(P < 0.05). Conclusion: these results based on radiography, histology and biomechanics suggest that intermittent treatment with low dose PTH(1-34) enhances the processes of femoral fracture healing in ovariectomized rat,induces a larger cartilaginous and hard callus formation; Improves the quality of callus; increases the mineralization procedure of callus; promotes the remodeling of callus and augments the biomechanical strength of fracture femoral. Results support opinion of treatment osteoporosis fracture of menopause female with intermittent administration PTH, which can reduce the time of fracture healing and improve the quality of callus.
PartⅡIntermittent PTH(1-34) augments Spinal Fusion in an Ovariectomized rat posterolateral Spinal Fusion Model with iliac crest bone graft
Aim: In the present study we tested the hypothesis that intermittent administration parathyroid hormone(PTH) improves spinal fusion outcomes in the ovariectomized female Sprague-Dawley rat posterolateral spinal fusion model,and appreciation whether it could enhance graft bone healing while administration for osteoporosis. Method: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized and underwent a bilateral posterolateral L4-5 spine fusion procedure by wiltse approach six weeks later; the graft material is a morselized auto-iliac crest bone graft. the animals were divided into 2 groups of 18 animals. Starting on postoperative day, experimental group was received daily subcutaneous injections of recombinant PTH(1–34) at a dose of 30μg/kg/day, whereas control group received a daily subcutaneous injection of same volume vehicle solution (normal saline). Nine rats from each group were killed and samples harvested for investigation on four and six weeks later, L4-5 vertebral segments were removed and analyzed with manual bending, X ray, Micro-CT and histomorphometry. Serum bone metabolism markers (OC, NTX) were analyzed. Results: Manual palpation identified fusion in two of nine (control) versus five of nine (PTH) animals at four weeks, and five of nine (control) versus eight of nine (PTH) in six weeks (P<0.05). A radiographic scoring system of PTH group resulted in an average score of 2.03 and 3.66 at four and six weeks, correspondence scores of control group were 1.45 and 2.56(P<0.05 and P<0.01).Result of Micro-CT showed that the continuous fusion mass between L4-5 transverse process of experimental group were larger than controls and fusion area had significantly different in both cross section and coronal section. PTH(1–34) accelerated the spinal fusion and acquired solid fusion mass, Improve the quantity and quality of fusion callus. parameter of Fusion bone mass(L4-5) by Micro-CT analysis were also improved with PTH treatment (P<0.01 or P<0.05). Histology results showed new mature bone, trabecular bone and marrow cavity were found at fusion site in experimental group at four weeks; however, there were less bone formed in control group which main composed of cartilaginous and fibrosis like tissue. The experimental group showed there had plenty of fusion callus with complete and thick cortical bone between L4-5 transverse process; trabecular bone were connecting each other and enclosing by marrow cavity. The trabecular and cortical bone were thin in control group, and the processes was lagged than experimental group. histomorphology showed the mineral apposition rate(MAR)between groups had significantly difference at four weeks and six weeks(P<0.01 and P<0.05). Serum OC levels were significantly higher in the PTH group than in the control group at two time point(P<0.05). The CTX levels in the PTH group were higher than in the control group at four weeks(P<0.05). Conclusion: these results suggest that intermittent treatment with low dose PTH(1-34) augmented posterolateral spine fusion with auto-iliac bone graft in ovariectomized female Sprague-Dawley rats. PTH(1–34) enhanced bone formation leading to the acceleration of the spinal fusion, which reduce the time of spine graft healing and acquires solid fusion mass, increase posterolateral fusion success and Improve the quantity and quality of fusion callus. Based on the results of this study, these findings suggest that PTH has promise for use as an adjunctive agent to improve spinal fusion and all other skeletal reconstruction surgeries requiring bone grafts in clinical medicine.
PartⅢMechanisms for the enhancement of posterolateral spine fusion in ovariectomized rats treated with intermittent PTH(1-34)
Aim: In the present study, we analyzed the underlying molecular mechanism by which exogenous PTH would affect osteogenesis and chondrogenesis in an ovariectomized rat spine arthrodesis model. Methods: ten-weeks-old SPF scale female Sprague-Dawley rats were ovariectomized and underwent a bilateral posterolateral L4-5 spine fusion procedure by wiltse approach six weeks later; the graft material is a morselized auto-iliac crest bone graft. the animals were divided into 2 groups of 18 animals. Starting on postoperative day, experimental group was received daily subcutaneous injections of recombinant PTH(1–34) at a dose of 30μg/kg/day, whereas control group received a daily subcutaneous injection of same volume vehicle solution (normal saline). six rats from each group were killed on four, seven, fourteen days later , graft callus was harvested for gene expression investigation on osteogenesis and chondrogenesis by RT-PCR, the level of expression of ALP, OC, COL-I, COL-Ⅱ, COL-Ⅹ, IGF-Ⅰ, BMP2 were analyzed. Results: 1、mRNA expression of osteoblast-related gene. The expression of ALP in PTH group was increased than control group significantly at fourteen days(P<0.01) and without difference at four and seven days; osteocalcin expression in PTH treated was increased markedly than controls at seven and fourteen days (P<0.05 and P<0.01); The expression of COL-Ⅰin PTH group was significantly increased than controls at seven and fourteen days(P<0.05). 2、mRNA expression of chondrocyte-related gene. The expression of COL-Ⅱwas up-regulated markedly in PTH treatment group at three experimental point(7days:P<0.01,4 and 14days:P<0.05).the expression of Col-Ⅹwas increased in PTH treated rats at seven and fourteen days, which has statistical difference at fourteen days(P<0.05). 3、mRNA expression of IGF-Ⅰand BMP2. gene expression of IGF-Ⅰin PTH treated were significantly increased at four and seven days(P<0.01), there had much expression but without difference at fourteen days. The expression of BMP2 in PTH group was up-regulated at fourteen days(P < 0.05)and without difference at other time. Conclusion: 1 intermittent administration PTH enhances proliferation and differentiation of osteoblast in an ovariectomized rat spinal fusion model with auto-iliac bone graft, the expression of COL-Ⅰ, ALP and OC were up-regulated. 2 the level of chondrocytes -related gene COL-Ⅱand COL-Ⅹwere increased markedly in the PTH treated group, it suggest treatment with PTH in spine fusion facilitates proliferation and differentiation of chondrocyte, which leads to a larger soft callus formation. 3 IGF-Ⅰis an important factor for the anabolic effect of PTH, because of the early expression of IGF-Ⅰthan other gene in this model, it may augment graft bone healing by stimulated proliferation and differentiation of chondrocyte and osteoblast. 4 the increased BMP2 expression in PTH group suggest that the expression of BMP2 maybe enhance directly or indirectly in the processes of spine fusion treatment with PTH.
引文
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