Influences Of Osteoporosis On The Middle And Late Periods Of Fracture Healing & Influences Of Simvastatin On Fracture Healing In A Rat Ovariectomized Model

Osteoporosis is defined as a systemic disease characterized by loss of bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture. Fracture is the ultimate and most catastrophic consequence of fracture. The demographic changes that are being predicted will increase the total number of fractures associated with osteoporosis and, hence, the cost to society. With the life expectancy and the number of elderly people increase in China, osteoporosis has become one of the major health problems countrywide. Because osteoporotic fracture mainly occurs in the senile population, who generally incorporated with other systemic disease, it requires to be healed with less bed rest so as to minimize the deadly complications. While on the contrary, clinical practices have suggested that osteoporosis is well associated with the increased fixation failure and the recurrence of fracture. So there exists a contradiction between the requirement of healing acceleration and high rate of fixation failure. It is a puzzle leave to be solved by orthopaedics surgeons.The treatment of fractures associated with osteoporosis is still not solved satisfactorily today. As fracture healing concerned, it can be affected by many factors, which could be mainly divided into exterior factors and inferior factors. The application of conventional implants for fracture fixation to osteoporotic bone is limited, and fixation failure occurs as a consequence of the weak bone structure. Otherwise, under the same force, osteoporotic bone is easier to be comminuted than bone with normalquality. These factors make up the exterior factors impairing fracture healing in osteoporotic bone. The primary principles in the manipulation of osteoporotic fracture are similar to that of normal fracture. Due to the poor bone quality and the insufficient of healing ability in osteoporotic subjects, different methods have been established to cope with this dilemma. Besides the development of new stabilization instruments, biological stimulation of osteoporotic fracture healing process is also of grate interesting to increase clinical outcomes.Internal factors refer to the inherent healing capacity of bone. It is essential to have a comprehensive knowledge on the characteristics of healing capacity in osteoporotic bone before effective strategies could be established. Many efforts have always been put on fracture prevention and development of therapies aimed at conserving bone mass and thus reduceing fracture risk, but little emphasis has been given to the investigation of fracture healing in osteoporosis. The influence of osteoporosis on the fracture healing remains poorly understood and controversial. In about the recent 5 years, investigations on osteoporotic fracture healing have been accumulated. It has been accepted that osteoporosis could impair the early stage of fracture healing, which manifested as small callus area and decrease of mechanical properties. While its influence on the later stage of fracture healing remains controversial. Compared with early stage, the late stage has closer relationship with the fixation time span and recurrence of fracture, which play important role in the treatment performance.Base on reasons above, we pulled out investigation on the influence of osteoporosis to the middle and late period of fracture healing via an animal model. Furthermore, we attempt to search out a new method to improve fracture healing in osteoporotic bone. In recent years, some statins (Cholesterol-lowering Drugs) have been indicated as anabolic agents for the treatment of osteoporosis, and can reduce the occurrence of fracture. But as a new finding, problems remains as below: 1) consentient conclusion on its anabolic effect on bone have not been established;2) the underlying mechanism of its effect on bone remolding has not been well interpreted;3)its effect on fracture healing has not been conducted. So we carried out a second study aiming on theeffect of statins on the fracture healing process. Besides, the anabolic effect of statins on bone remolding and the possible mechanism were also been explored our study.This dissertation is composed up by two sections: 1) Influences of osteoporosis on the middle and late periods of fracture healing;2) Influences of simvastatin on fracture healing in a rat ovariectomized model. Four objectives were aimed as follow: 1) To evaluate the influence of osteoporosis on the middle and late periods of fracture healing process;2) To validate the anabolic effect of simvastatin on bone via investigating the histological and cellular changes of callus;3) To investigate the effect of simvastatin on the quality of fracture healing;4) To explore the underlying mechanism of statin on bone remodeling via observing the influence of simvastatin on the expression of OPGSection one: Influences of osteoporosis on the middle and late periods of fracture healing in a rat ovariectomized modelFemale SD rats of 4 months old were randomly divided into Sham group and OVX+LCD (ovariectomized + low calcium diet) group. Rats in OVX+LCD group were ovariectomized and fed on LCD(Ca: 0.20%;P: 0.14%) to the day fracture was established, while those in Sham group with sham operation and fed on normal diet(Ca: 0.46%;P: 0.38%). To avoid weight gain due to OVX, diet of OVX+LCD group was controlled to the amount of those to the Sham group. Midshaft tibia fracture model was established 10 weeks after ovariectomy, when BMD of OVX+LCD group was detected to be decreased obviously than that of Sham group by DEXA. Tibias were harvested 2, 4, 6, 12, 18 weeks after fracture for bone mineral density, histomorphological and biomechanical evaluation. Results showed that 1) despite diet control, OVX+LCD rat gain more weight than Sham rats;2) no obvious difference was observed on the callus area between the two groups;3) compared with the Sham group, callus BMD was decreased by 12.8%, 18.0%, 17.0% in OVX+LCD group 6, 12, 18 weeks after fracture, respectively (i*<0.05);4) Callus failure load lowed about 24.3%, 31.5%, 26.6%, 28.8% in OVX+LCD group, and callus failure stress lowed about 23.9%, 33.6%, 19.1%, 24.9% OVX+LCD group 4, 6, 12, 18 weeks after fracture, respectively (/><0.05). 5) In OVX+LCD group, endochondral bone formation was delayed, more osteoclast cellcould be seen around the trabecula, and the new bone trabecula arranged loosely and irregularly.Section two: Influences of simvastatin on fracture healing in a rat ovariectomized modelFemale SD rats of 2 months old were randomly divided into Sham+Vehicle group, OVX+Vehicle group and OVX+SVS group. OVX and diet control were performed the same way to section one, no LCD any more here. 12 weeks after ovariectomy, obvious bone loss was detected by DEXA in OVX rats. Tibia fracture model fixed with intermedullary pin was established then the same way to section one. SVS or Vehicle drugs were injected subcutaneously to the fracture local once on the day of fracture and twice on the 5 days thereafter. Each injection contained 5 mg/kg of 5%SVS to the SVS group and equal volume of vehicle to the Vehicle group. Tibias were harvested 1, 2, 4 weeks after fracture. To rats which were planted to get the undecalcification section, 25mg/kg tetracycline (20mg/ml) was injected subcutaneously in the neck -14,-13 and -4, -3 days before killed. Results showed that: l)cross-section area: no obvious difference exist between Sham+Vehicle group and OVX+Vehicle group;cross-section area in SVS+Vehicle group increased 21.3% and 21.5% in 1 (20.22±3.42mm2)> 2weeks(23.63±3.25mm2) after fracture than OVX+Vehicle group(16.67±4.02mm2, 19.45±3.19mm2,respectively)(p<0.05);difference disappeared 4 weeks post fracture;2)X plain film: no differences exist in three groups 1 weeks after fracture;OVX+Vehicle had less bridge callus than Sham+Vehicle and OVX+SVS groups in 2,4weeks;3)maximal load: no differences exist in three groups 1 weeks after fracture;maximal load in OVX+Vehicle group decreased 25.9% and 14.5% in 2 (16.33±6.32N)> 4weeks(38.05±8.51N) after fracture than Sham+Vehicle group(22.03±6.55N, 44.49±9.35N,respectively)(p<0.05);maximal load in OVX+SVS group increased 52.3% and 24.4% in 2 (24.87±6.67N)> 4weeks(47.32±9.59N) after fracture than OVX+Vehicle group(p<0.05);4) Ratio of maximal load to the unfractured tibia: no differences exist in three groups 1 weeks after fracture;ratio of maximal load to the unfractured tibia in OVX+Vehicle group decreased 28.8% and 19.5% in 2(31.66±13.13%) ,. 4weeks(66.41±13.25%) after fracture than Sham+Vehicle group(44.44±10.10%, 82.47±16.65%,respectively)(p<0.05);ratio of maximal load to the unfractured tibia in OVX+SVS group increased 57.5% and 31.4% in 2 (49.88+10.60%) , 4weeks(87.25±19.93%) after fracture than OVX+Vehicle group(p<0.05);5)maximal stress: no differences exist in three groups 1,2 weeks after fracture;maximal stress in OVX+Vehicle group decreased 18.9% in 6weeks(71.91±11.53N/mm2) after fracture than Sham+Vehicle group(88.70±9.32N/mm2) (p<0.05);maximal stress in OVX+SVS group increased 25.2% in 4weeks(90.00±15.41N/mm2) after fracture than Sham+Vehicle group(p<0.05);6) HE and toluidine blue stain: in 1 week post fracture, trabecula could be seen in OVX+SVS group, while less or not in Sham+Vehicle and OVX+Vehicle group, osteoblast strap in OVX+SVS group was thicker than that in OVX+Vehiclegroup;in 2 and 4 weeks post fracture, the new bone trabecula arranged loosely and irregularly in OVX+Vehicle than that in Sham+Vehicle and OVX+SVS group;more osteoclast cell could be seen around the trabecula of OVX+Vehicle group;7) Semi-quantitative immunohistochemistry of PCNA showed that OVX+Vehicle group decreased 17.3%(40.10±1.64), 32.1%(41.10±1.43) and 26.1%(16.84±2.14) (pO.Ol) in 1, 2 and 4week post fracture than Sham+Vehicle (48.51+-4.12, 53.45±5.68 and 22.80+-2.16, respectively);and OVX+SVS group increased 60.1%(64.21+4.89), 67.7%(68.93±0.64) and 67.7%(28.24±1.67) (p<0.01) in 1, 2 and 4week post fracture than OVX+Vehicle group;8)serumal total cholesterol: no differences exist between Sham+Vehicle group and OVX+Vehicle group;serumal total cholesterol in OVX+SVS group decreased 22% (83.2±8.16mg/dL) (pO.Ol), 14.0% (89.1±13.9mg/dL)(p<0.05)in land 2 weeks after fracture than OVX+Vehicle group( 106.2+11.24 mg/dL,103.6±15.4 mg/dL, respectively);no differences exist between OVX+SVS group and OVX+Vehicle group in 4 weeks after fracture;9) serumal ALP in OVX+Vehicle group increased 22.4% (127.1±15.1U/L),24.5%(122.0±18.78U/L) (pO.Ol) and 15.8%(109.3±21.49U/L) (p<0.05)inl, 2 and 4 weeks after fracture than Sham+Vehicle group (1O3.8±13.5, 98.5+.17.68U/L and 92.U19.50U/L, respectively);serumal ALP in OVX+SVS groupincreased 20.0%(152.6±14.6U/L) (p<0.01), and 12.4%(137.1±16.6U/L) (p<0.05)in 1 and 2 weeks after fracture than OVX+Vehicle group;11) Bone histomorphometry: MLW and MLV in OVX+Vehicle group is less than that in Sham+Vehiclegroup;and O.Wi> OV, OS> AOS R MAR in OVX+Vehicle group is greater than that in Sham+Vehicle group;all the parameters above in OVX+SVS group were great than that in OVX+Vehicle group;12) Semi-quantitative immunohistochemistry showed that express level of OPG was the lowest in OVX+Vehicle, and the highest in Sham+Vehicle group;there was obvious difference between OVX+SVS group and OVX+VehicleConclusions:1. Osteoporosis influences the middle and late periods of fracture healing in the rat osteoporostic model. The impairment is considered to be the result of the combined effects of the prolonged endochondral calcification, high activated osteoclast cell and the deceleration of minimization in callus;2. The anabolic effect of simvastatin on bone was supported by current study;3. Local application of simvastatin(10mg/kg/day) could prevents the impairment of ovariectomy on fracture healing in a rat model;4. Simvastatin could prevent the reduction of OPG secretion result from ovariectomy, this might be the mechanism of Simvastatin on the bone remolding.