Enhancement Osteogenic Capacity By Intermittent Administration Parathyroid Hormone (1-34) In An Ovariectomized Rat Model

PartⅠEnhancement of fracture healing by intermittent administration of PTH(1-34) in ovariectomized rats femoral fracture modelAim: 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 graftAim: 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.