The effect of strontium ranelate on the healing of a fractured ulna with bone gap in rabbit

The treatment of delayed unions, nonunions, malunions and bone loss pose great challenges for orthopedic surgeons [1]. Fracture healing disorders negatively affect the patient’s quality of life and result in high healthcare costs, as a second surgery is necessitated to stabilize the fracture and stimulate bone biology. In most cases, successful union will be achieved by restoration of the alignment and stable fixation of the fracture. However, many cases call for adjunctive measures such as bone-grafting or bone transports to stimulate bone-healing and fill bone defects. The gold standard for bone defect repairs is autograft but their usage is affected by morbidity at the donor site and limited supply [2]. Limitations of the allograft include the immunogenic response by the host to the graft and the potential for disease transmission. To overcome these limitations an increasing number of new orthobiologic materials are being developed to aid in the management of bony defects. In future, advanced biotechnologies that accelerate fracture healing may be useful tools, which might also prevent the onset of these disorders [3]. The several options available for bone replacement are autologous bone or allogeneic cancellous or cortical bone, demineralized bone matrix, calcium phosphate-based bone-graft substitute or autologous bone marrow. The biology of each of these grafts varies but they perform one or several essential functions such as osteoconduction, osteoinduction, osteogenesis and structural support [4, 5].

Besides bone graft and bone graft substitutes, orthopedic surgeons at present use anabolic agents such as teriparatide and strontium ranelate to promote fracture healing and increase bone quality. An anabolic agent is one that raises the bone strength by increasing the bone mass due to an overall increase in bone remodeling combined with a positive bone balance [6]. Several case reports on strontium promoting fracture healing have been reported in the literature. Strontium ranelate accelerates filling of the bone defect and improves the properties of bone healing in rats [7]. Strontium nucleus is almost similar in size to that of calcium, the body easily absorbs it, incorporating it into the bones and tooth enamel instead of calcium [8]. Strontium ranelate acts via a dual action mode with its bone-forming and bone-resorbing properties, resulting in improved bone microarchitecture [9]. This agent is approved for the prevention of vertebral and hip fracture in postmenopausal osteoporosis. Preclinical studies have demonstrated enhanced fracture healing and osseo-integration with strontium ranelate, coupled with the improvement in bone microarchitecture [9]. Studies have also showed contradicting results in which 3 weeks of treatment with strotium increased the thickness of the healing mid-femoral cortical bone defects in rats, but did not influence volume, mechanical properties, periosteal callus volume, either 3 or 8 weeks. Furthermore, strontium had no effect on the microstructure and mechanical properties of the vertebral bodies [10, 11].

The ulna is found to have similar function, in both humans and four-footed animals. If the ulna breaks, it will most commonly occur at either the point where the radius and ulna form a joint or where the ulna forms a joint with the hand's carpal bones. Ulnar fractures cause severe pain, difficulty in moving the affected joint and even lead to deformity of the arm if the fracture is compound. So far reported studies have focused on the development of biocomposite materials or scaffold on different type of bone fractures and as well as ulna bone, while the effect of strontium ranelate has not been tested on ulna bone. The maximum period for spontaneous healing of the bone fracture is around 6 weeks, so we anticipated the treatment with strontium ranelate would trigger faster healing in rabbits compared with control [12].

Radiographic examination of the radius-ulna of the strontium treated rabbits was performed at the end of study. None of the samples showed radiological union at the end of the six-week study (Fig. 1). However, excessive callus formation was observed in between the radius and ulna rather than in the fracture gap. From the findings mentioned above it was evident that the strontium treatment did not significantly improve fracture healing in rabbit ulna with bone gap. The union and nonunion rates using x-ray in the test and control groups were assessed by Cheung’s method (Fig. 2a & b) and the p-value was calculated as 0.44 using Fisher Exact Test. There is no association of healing of the ulnar fracture with strontium.

An obvious increase in the union rate was recorded using Cheung’s scoring method. However, the control group showed a better union rate on scoring method. All the rabbits were reviewed and the CT scans were taken within the first week post-surgery. No evidence of any callus formation was observed and the 0.5 cm gap was clearly seen. At around three weeks, evidence of minimal callus formation was seen. The callus began to form from the edges of the osteotomized bones. More calluses were noted in between the radius and ulna rather than within the bone gap. At six weeks, only two samples showed radiological union.

The CT scans of the rabbit fore-arm at the end of study in Group A (with strontium) and Group B (without Strontium) at the end of study (without strontium) are shown in Fig. 3. Post-surgery all the rabbits in both groups were reviewed and the CT scans were done within the first week of surgery. No evidence of any callus formation was initially found. At three weeks post-surgery, patchy calcifications were noted around the center of the defect and bone edges. The patchy calcifications moved towards the center of the bone defect. The callus in the center of the defect enlarged gradually and expanded proximally and distally. At around four weeks post-surgery, the proximal callus, which grew faster, met and united with the center callus. Then, it slowly grew distally and met the distal callus at around six weeks. At this point in time, no fracture gaps were observed. The bone growths were noted to fill up the whole osteotomy site. At six weeks, the fracture had united and there were continuity between the bone trabeculae but no remodeling noted. The diameter sizes of the newly formed bone were smaller than the actual defect. The p-value by Fisher Exact Test is 0.52. In the control group, the union rate is 100 % with x-ray and 80 % with the CT scan using Cheung’s scoring methodology, which is almost two times higher than that of the test group. A comparison of the union rates from the x-rays and CT scans by Cheung’s Method are shown in Fig. 4.

The biochemical markers including the levels of serum alkaline phosphatase, serum calcium, serum phosphate and serum osteocalcin were taken at weeks 1, 3 and 6 post surgery (Figs. 5a-d). The level serum markers were compared as the callus grew old. Serum levels of calcium and phosphate showed no change at weeks 1, 3 and 6. However, a significant statistical difference was observed in the serum levels of ALP and osteocalcin at week six, where the serum ALP was higher and osteocalcin was lower in the strontium treated group. Based on the histological examination, the grading and percentage of lamellar bone, woven bone, cartilage and fibrous tissue are shown in (Tables 1 & 2). The presence of lamellar bone in the specimen with radiological nonunion may have been contributed by the radius. However, no significant difference in the healing was observed between the strontium treated and untreated groups.

Grade	Radiographic description
Grade 1	Indicates no calcification at the fracture site.
Grade 2	Indicates patchy calcification
Grade 3	Indicates that the calcification takes on the appearance of the callus
Grade 4	Shows callus bridging across the fracture gap
Grade 5	Indicates continuity of bone trabeculae
Grade 6	Demonstrates remodeling to normal bone.

	Lamellar bone	Woven bone	Cartilage	Fibrous tissue
Control group	70 %	20 %	5 %	4 %
Test group	58 %	28 %	7 %	7 %
p-value	0.53	0.64	0.17	0.30

Fracture healing in the presence of bone defect is a significant clinical issue. Non-union is a well-known complication of segmental loss in bone defect [10]. One of the main challenges in successful bone healing and new bone formation is the in-growth of soft tissue which may disturb the osteogenesis and cause non-union. Such defects greatly impact the patient’s quality of life due to limb length discrepancy and prolonged postoperative treatment. Furthermore, it will become an added financial load to the patient causing a socio-economic burden to the family as treatments for this condition are complicated. The current treatment option involves the use of the Ilizarov technique by distraction osteogenesis [11]. However, this treatment includes complications of its own; pin site infection, bulky implant, expensive cost, long hospital stay etc. Recently, anabolic bone-forming agents on fractures in animal models have been reported. The ulnar bone was selected in this study, as it is one of the common sites for fracture and segmental bone loss in humans, especially in trauma cases. Rabbit ulnar bone is easily accessible and the bone size is also appropriate to use. It is relatively easier to perform the surgical procedure in the ulnar bone as it is subcutaneous in location with less soft tissue coverage. Furthermore, it has a splinting effect from the radius and no implant is required.

A 0.5 cm bone defect was created in this study as we desired to see the difference in the physiological repair process through the fracture gap between the group undergoing strontium ranelate treatment and the control group. The ulnar diameter on average was 6–8 mm, measured at the midshaft region. The 0.5 cm bone defects were made in the rabbits at the midshaft of the bone to assess the osteogenesis capacity of the rabbit ulna through the fracture gap. According to the earlier report, in the short experimental time, bone healing was partial and involved the immature bone and fibrous connective tissues [13]. Under such conditions, it became possible to evaluate the effect of an agent in the healing process and whether the healing would be satisfactory as far as tissue quantity and quality were concerned. The callus formation in relation to the repair period was required to assess the healing bone quantitatively and qualitatively; however, the treatment was not significant as observed in the group given strontium ranelate when compared with the control group [14]. In fact, the best tissue quality should be achieved within the shortest time period, assuming that the bone will be remodelled during the animal’s entire lifetime. In general, bone regeneration will be initiated from the periosteum (periosteal callus) and grow in a circumferential pattern from the outside into the centre of the defect. Bone regeneration without periosteum will occur from the primary response callus initially, later the medullary callus and follow a linear growth pattern of growth. The proximal bone edge callus will grow faster than the distal bone edge callus. In our study, the periosteum was removed in both the control group and test group which will exert a negative effect on bone healing. However, in the control group, the fracture unites even without the periosteum when compared with the test group, which received the bone stimulant, strontium ranelate. Therefore, we may conclude that strontium may not be effective if the periosteum is removed. Studies have previously demonstrated that periosteum is important for the acceleration of bone growth after stripping. The grown acceleration has been reported after transection of periosteal and resection [15–17]. Reports has also compared the different procedures head to head in the same mammalian model, transverse transection of the longitudinal periosteal fibers appears to be the most important aspect in accelerating growth [18–20].

From the X-ray findings, four of the five samples in the control group fulfilled the criteria for union using cheung’s scoring method, all five samples from the control group and three samples from the strontium group showed bony union. At the end of the six-week study, four samples from the control group and two samples from the strontium group achieved radiological union from the CT scan findings using Cheung’s method. The radiological findings revealed an association between the use of strontium ranelate and delayed unions or non-unions. Fibrous tissues (fibroblast) which grow faster than the bone (osteoblast) were observed to rapidly migrate and repopulate into the bone defect; however, no significant effect was observed after six weeks between the strontium ranelate treated group and the untreated group [21].

All the samples showed 80 % bone formation within the fracture gap created. The bones that formed were either lamellar bone or bone woven with the minimal fibrous tissue formation, between 5 and 10 %. We expected to see more fibrous tissues in the specimens from the radiological findings of the big bone gaps. Instead, we observed various stages of callus formation. The seven slides were prepared initially by cutting the radius and ulna longitudinally in the sagittal plane to facilitate the comparison of the callus and surrounding normal bones. When the results showed that 80 % of the bones in all the slides were in contrast to our imaging studies, we suspected a mixing of the callus with the surrounding normal bones. We removed the radius in the last three slides prepared, but we still observed the same results. These results demonstrated that the strontium ranelate treatment could interfere with the mineralization process, even though it did not inhibit callus formation. The results of the blood parameters showed no significant statistical difference in the serum levels of calcium and phosphate at weeks 1, 3 and 6. Statistical difference was noted in the serum alkaline phosphatase and osteocalcin at week six with the serum ALP being higher and serum osteocalcin dropping lower in the strontium treated group. This shows that strontium has slow action onset on the bone markers.

Reports have shown strontium ranelate has bone anabolic and anti-resorption effect which probably increase healing of bone defects [22–24]. In addition some reports have shown strontium ranelate seems to decrease bone resorption as well preserves or increases bone formation. Preclinical studies on fracture healing have shown that it has a positive effect on hard callus formation and bone strength [25, 26]. From our assessment using the radiographs and CT scans, strontium ranelate does not promote fracture healing within the first six weeks, especially a fracture with a bone gap, as none of the fractures were observed to unite in the treatment group. However, the HPE report revealed that the gap became filled with lamellar bone or woven bone of differing maturity. It may have initially delayed the fracture healing or impaired the mineralization process. However, changes in the serum levels of the bone markers such as osteocalcin and alkaline phosphatase showed that strontium has a slow onset of action on bone activity. We are, however, still unsure about the long-term effect of the strontium on fracture healing with bone gap. However, based on the x-ray and CT scan findings, strontium did not have a significant impact on fracture healing in the acute setting compared with the control group. One of the reasons why the callus was unable to bridge the gap could be the absence of the perisoteum. The surrounding soft tissue could have prolapsed and blocked the bone gap thus inhibiting the osteogenesis process. But, in specimens with the strontium supplement, the bone grew faster in any case. One of the possibilities is that the periosteum is essential for the strontium to act.

In conclusion, strontium ranelate is not found to promote bone healing and in fact may delay bone formation, especially during the early stages of fracture healing with bone gap, in rabbit. Further studies are warranted using bigger animal models to confirm the role of strontium ranelate.