Fracture is a break in bone which usually results from trauma but underlying bone diseases such as osteoporosis may cause atraumatic fracture and contribute to the severity of traumatic fracture. Osteoporosis is a bone metabolic disease characterized by low bone mineral density resulting in bone fragility and an increased risk of fracture.1 Fracture healing is a complex regenerative process that is still not fully understood. It involves the coordinated participation of hematopoietic and immune cells within the bone marrow in conjunction with vascular and skeletal cell precursors that are recruited from the surrounding tissues and the circulation. Histologically, fracture healing can occur by primary or secondary healing. In primary healing, the cortex reestablished new Haversian systems when the fracture fragments were reduced by an anatomical rigid internal fixation.2 This type of healing does not involve callus formation.3 Most fracture healing occur by secondary healing which is characterized by six stages of hematoma and inflammation, angiogenesis and formation of cartilage, calcification of cartilage, removal of cartilage, bone formation and bone remodeling.3
This process is further complicated in the healing of osteoporotic fractures. Studies have shown that osteoporosis can delay callus maturation and consequently decelerate fracture healing.4 Consequently, agents used for prevention and treatment of osteoporosis such as calcium, vitamin D, estrogens and bisphosphonates may also affect fracture healing. Due to these complexities, animal osteoporotic models, such as the rat, rabbit, or dog, may be more appropriate than human subjects to study the effects of osteoporosis on fracture healing process.5 Besides ethical issues, studies using human subjects are limited by difficulty to attain control group and create homogeneous study groups.6 As a result, there are too few studies on the differences of bone healing in normal and osteoporotic individual to suggest reduced bone remodeling and bone healing in osteoporosis.7–9
We have carried out a study on the effect of calcium supplementation on fracture healing of osteoporotic bone using ovariectomized rats. Although there are plenty of information regarding the influence of ovariectomy on bone mass and metabolism,10–12 very few attempts have been made to investigate the effects of established osteoporosis on fracture healing.13, 14 This is surprising considering the clinical importance of osteoporotic fractures and the wealth of information regarding osteoporotic animal models.
It is well accepted that calcium supplements may prevent osteoporosis and prevent fractures. According to the results of the Women's Health Initiative,15 calcium and vitamin D supplements in healthy postmenopausal women resulted in a small but significant improvement in hip bone density, did not significantly reduce hip fracture, and increased the risk of kidney stones. Later, the authors have made corrections that their data seem to support the current recommendations for adequate calcium intake in older women only (more than 60 years old). Would the beneficial role of calcium supplements in preventing osteoporosis be extended to healing of osteoporotic fractures? We would expect calcium to play a major role in fracture healing of osteoporotic bone as it is the main mineral in the callus. However, there are no studies to date on the effects of calcium in fracture healing of osteoporotic bones, while, there are limited studies on the effects of calcium in fracture healing of normal bone. In one of these studies, the calcium content of the callus was found to determine its mechanical strength.16 Another study showed that the fractured tibias of rats treated with calcium were stronger than nontreated rats.17
The purpose of this study is to investigate the effects of calcium supplements in the healing of osteoporotic fractures using the ovariectomized rat as an osteoporotic fracture model. The fracture healing was compared to the sham-operated rat which represented the traumatic fracture model.
MATERIALS AND METHODS
Twenty-four female Sprague–Dawley rats weighing between 250 and 300 g were divided into three groups. The first group was sham-operated but the rest of the groups were ovariectomized. These rats were left without any treatment for 2 months to allow the bones of the ovariectomized rats to become osteoporotic. The right femurs of all the rats were then fractured aseptically according to Vialle et al.18 Briefly, the rats were anaesthetized using ketamine and xylazil (1:1) at the dose of 0.1 mL/100 g rat weight. The right patella was dislocated and a Kirshner wire (K wire) (1.0 mm in diameter) was inserted with a drill into the intramedullary canal of the femur for internal fixation. The right patella was relocated and the incision closed with sutures (nylon 4.0).
The fracture device was then used to fracture the right femur. It fractures femur using the guillotine principle to produce a closed and standardized fracture. A 500 g steel blunt bar was dropped on the mid-diaphysis of femur supported by two metal bases, forming a three-point system. Baytril was injected intramuscularly for perioperative antibiotic prophylaxis. The rats were then injected subcutaneously with buprenorphine at the dose of 0.3 mg/kg rat weight every 12 h as analgesics. X-rays of the right femurs were taken immediately using X-ray machine (Proteus XR/a, GE, Buckinghamshire, UK) to make sure that fractures had occurred at mid-diaphysis and the K wires were inserted correctly. The rats were allowed unrestricted weight bearing after recovery from anesthesia. Postfracture, the ovariectomized rats were randomly divided into two groups of ovariectomized-control (OVXC) and calcium treated (Ca) groups. The rats were housed in individual cages at room temperature of 27°C and 12 h natural light/dark cycles. The rats were fed on rat-chow (Gold Coin, Port Klang, Malaysia) and given deionized water ad libitum. Rats in the Ca group was supplemented with calcium by mixing 1% lactic-acid-hemicalcium salt (Sigma, St. Luis, MO) in drinking water and given ad libitum. This method of calcium supplementation does not stress the rats compared to oral gavage or intramuscular injection and has been shown to be optimal for bone growth.19
After 2 months of treatment, the rats were sacrificed humanely and the fractured right femurs were dissected out. The K wires were removed carefully to avoid damage to the femur. All the tests were performed immediately on the dissected femurs. X-rays were performed on the dissected femurs in anteroposterior and mediolateral planes using a high-resolution digital radiography system (Philips Digital Diagnost/Optimus 80 system) at 46 kV, 2.5 mAs and 10.6 ms exposure. The dissected right femurs were scanned on a Computer Tomograph (Somatom Sensation 64, Erlangen, Germany) which produces a narrow fan beam by means of an X-ray tube (120 kV; 40 mAs). CT scans were performed at slice thickness of 0.6 mm, an in-plane voxel size of 0.234 mm, and matrix size of 512 × 512 pixels. The manufacturer's software package (ver 2006A) was used for image processing and data evaluation. Calibration of the scanner was performed with a water phantom with 0 Hounsfield Unit (HU) and density of 1.0 g/cm3.
The axial callous volumes of the fractured bone were measured at the level of 1.0 cm above and below the diaphyseal fracture using the CT scan software (Software Version Syngo CT 2006A). Fracture healing was staged on the X-ray images using a modified 5-point radiographic scoring system according to Warden et al.20 (Table 1). We have also devised a 5-point scoring system to assess the callus density which appeared in the X-ray films (Table 2). All radiographs were randomized and independently assessed by a qualified radiologist who was unaware of the treatment the rat had received.
Table 1. Fracture Healing Stage Was Assessed Using a 5-Point Radiographic Scoring System (Modified Warden et al.20)
No evidence of healing
Callus formation evident but fracture gap not bridged
Callus formation evident with bridging of the fracture gap but fracture line evident
Callus formation evident with bridging of the fracture gap with only faint fracture line
Table 2. Callus Staging Was Assessed Using a 5-Point Radiographic Scoring System
Callus + (very minimal callus)
Callus ++ (minimal callus)
Callus +++ (moderate callus)
Callus ++++ (exuberant callus)
The fractured femurs were then prepared for biomechanical testing. They were kept moist all the time by wrapping them with gauze soaked in phosphate buffered solution and aluminum foil while waiting for the testing. The study groups were numbered to blind the biomechanical measurements. The femurs were placed on the Instron machine (Instron Microtester 5848, Instron Corp., Norwood, MA) in a 3-point bending configuration. The load was applied at the mid-span in an anteroposterior direction with a span length of 10 mm and loading speed of 5 mm/min. This corresponds to the fracture site at the femur mid-epiphyses. The load was applied until the femur re-fractured. The load, stress, strain-deflection curves were automatically calculated by the computer using Bluehill software. The study method was approved by the UKM Animal Ethics Committee (FP/FAR/2008/NAZRUN/13-FEB/217-FEB-2008-FEB-2010).
The results were expressed as mean ± SEM. For normally distributed data, the statistical test used was ANOVA followed by Tukey's HSD. Data that were not normally distributed data were analyzed using Mann–Whitney followed by Kruskal–Wallis test if more than two groups were compared. The level of significance was taken as p < 0.05.
After 8 weeks of fracture healing, CT scanning showed that the fractured femurs of rats in the OVXC group have significantly higher callus volume compared to the SO group (Fig. 1). The abundance of callus compared to that of normal bone (SO group) indicates impaired fracture healing of osteoporotic bone. This is because at the late stage of fracture healing, most of the callus should have been replaced with lamellar bone through bone remodeling. The callus volume of the Ca group was found to be similar to the SO group. Therefore, calcium supplementation seemed to be able to improve fracture healing of osteoporotic bone (Fig. 8). The fracture healing score from the X-ray images showed that the OVXC group has significantly lower healing score compared to the SO group. The healing score for the Ca group was similar to the SO group (Fig. 2). The callus staging of the X-ray images was consistent with the CT-scan findings. At the late-phase of fracture healing, the OVXC group had significantly higher callus score than the SO group. The callus scoring for the Ca group was similar to the SO group (Fig. 3). Both these parameters indicate poor fracture healing of osteoporotic bone while calcium supplementation has improved healing (Fig. 9).
Biomechanical testing was carried out to determine the strength of the healed fractured femur. All the groups have similar stress value. OVXC and Ca groups have significantly lower strain and load values compared to SO group. The Young's modulus of the Ca group was significantly lower than both the SO and OVXC groups. These biomechanical test results indicate that the healing of the osteoporotic femurs of ovariectomized rats resulted in weaker bones. Calcium supplementation did not improve the strength of the healed fractured femurs in osteoporosis (Figs. 4–7).
It is well accepted that calcium is one of the most important nutrients in the healing of bone fracture. There is a need to study its effects on fracture healing of bone with osteoporosis, a condition which may impair its healing. We have conducted a study using the ovariectomized rat, an accepted postmenopausal osteoporosis model21, 22 and an osteoporotic fracture model.23, 24 There are also various techniques for performing experimental fractures.25–28 We have chosen the guillotine method by Vialle et al.,18 as it produces a consistent fracture of the femur with fewer trauma to the surrounding tissues. This mimics the condition of osteoporotic fractures in which the fractures occur with trivial force. Surgical fracture results in significant bleeding and trauma to the surrounding tissues which will affect fracture healing.
During normal fracture healing, calluses are formed in the intermediate phase and are mostly replaced by lamellar bone in the late-phase of fracture healing. About 5–10% of the 6 million fractures that occur annually in the United States show delayed or impaired healing. Conditions such as infection, poor nutrition or osteoporosis, may impair fracture healing as indicated by the abundance of calluses due to delay in callus maturation.6 This was seen in our osteoporotic fracture model during the late-phase healing of fractured femur. Studies have shown that the ovariectomized rat model is useful for studying fracture healing if the end points are more than 30 days29 as ovariectomy did not markedly affect the early healing process, but largely affected the bones in the later period of healing.30
Calcium is one of the main minerals in bone, in the form of calcium hydroxyapatite crystals. Previous studies have suggested that during the early phase of fracture healing, calcium is deposited in the callus. The required calcium is drawn from the skeleton and is independent of dietary calcium. Only in the later stages is dietary calcium important for fracture healing.31, 32 In our study, half of the ovariectomized rats were given calcium supplements throughout the healing phases and this may be responsible for the better radiological assessment of fracture healing which was comparable to the normal healing of sham-operated rats.
Mechanical strength of the healed fractured bone is a reliable test of the bone's recovery to its normal strength. In normal rat bones, tibial fractures are usually considered healed in 90 days when the strength has reached the values of intact bone.33 In another study on fracture healing of rat tibia, it was found that after 80 days, the maximum load and stiffness were 81% and 118%, respectively, of intact bone values.34 The fracture healing of osteoporotic bone in animal models was associated with poor recovery of bone strength.14, 35–37 Namkung-Matthai et al.13 had concluded that the inferior mechanical properties of fractured femur in the ovariectomized rat may be contributed by poor bone quality, less bone mineral and alterations in the bonding interactions between the mineral and organic constituents of the bone matrix. Dai and Hao4 had suggested that the reduced mechanical strength in the hard callus of osteoporotic bones is caused by the disorganized and irregular collagen fibers with regard to the direction of the principal stress.
Based on the normalization of fracture healing in osteoporotic bone with calcium supplementation, we would expect the strength to be similar to sham-operated rats. However, for the first time, we reported that the healed fractured femurs of ovariectomized rats supplemented with calcium are inferior in strength compared to sham-operated rats and were not different from ovariectomized-control rats. A similar pattern was reported in the fracture healing of a sciatic neurectomy rat model, where the callus formation was accelerated and bone mineral density was high but there was no improvement in strength. Melhus et al.38 had also found no difference in the mechanical properties of callous between ovariectomized and vitamin D-deficient diet group and sham group. However, there was a high but insignificant rate of incomplete healing in both groups which was different from our results. We cannot compare directly our results with that of Melhus et al. because of the differences in the species of rats used, the fracture method and the type of bone fractured. Furthermore, we had scored fracture healing according to the method by Warden et al.,20 but we are unsure of the method used by Melhus et al. In a different study using an ovariectomized rat model, calcium-depletion resulted in decreased femoral bending strength although the bone mineral density and cortical thickness were not affected.39 The study has shown that calcium deficiency may compromise femoral strength of ovariectomized rats but it did not explore fracture models to determine the effects of calcium deficiency on callous strength. Another interesting finding in our study is that the Young's modulus of the calcium supplemented group, which measures the stiffness of isotropic bone material was the lowest compared to other groups. These findings have raised questions on the benefit of calcium supplements in promoting bone healing.
There are studies which do not support the benefit of calcium in bone health. Tordoff et al.40 found that there was no significant correlation between bone mineral density and content to calcium intake in mice. In a cohort study, it was found that calcium intake of 500–1,500 mg/day by adolescents was not associated with better hip bone mineral density.41 It was also found that although the average daily calcium intake for South African blacks is markedly lower than African-Americans, the hip fracture rate for African-Americans is nine times greater than the South African blacks.42
The conclusion of this study is that calcium supplements may appear to improve the fracture healing of osteoporotic bone based on radiological assessment but they failed to restore the strength of the healed fractured bone. Further investigations are required.
We thank the Ministry of Higher Education and UKM for providing the grant GUP-SK-07-21-202 for this study.