Biochemical Markers(N-terminal Telopeptide Of Type Ⅰ Collagen, C-terminal Telopeptide Of Type Ⅰ Collagen, Osteocalcin, Bone-specific Alkaline Phosphatase)and Quantitative Computer Tomography Detection Of Bone Mineral Density For Early Diagnosis Of Exp

The diagnosis and treatment of bone nonunion have been extensively studied. The diagnosis and follow-up of nonunion rely mainly on the interpretation of X-ray findings, which depend on the clinician’s experience and the degree of bone callus mineralization during the fracture healing process. X-ray findings are also affected by the projection, film processing conditions, and subjective factors, resulting in poor accuracy. Furthermore, the X-ray resolution may be compromised when the bone mineral content is <25%. The bone formation is a complicated process including the granulation tissue connection, callus formation and bone remodeling in which osteoblasts, osteoclasts, granulocytes et al coordinate to each other. Ordinary X-ray imaging cannot bring this process clearly. It is due to inconsistency among the X-ray imaging signs of fracture nonunion, biochemical and histological data. Development of a novel and feasible method of monitoring bone healing is therefore urgently needed. B-mode ultrasound, response to vibration, mechanical impedance analysis, and the creation of artificial neural networks have been extensively studied with respect to the function, mechanical status, and changes of bone. However, the role of measuring serum biomarker concentrations and quantitative computer tomography detection of bone mineral density for the monitoring of fracture healing remains unclear.This study investigated the diagnosis of fracture healing at the molecular level with the aim of developing a method for early diagnosis and outcome prediction of fracture nonunion, and of providing an objective method of assessing the efficacy of bone-growth drugs. As changes in bone metabolism precede changes in morphology, changes in biomarker levels can be detected much earlier than changes in bone density and bone mass. However, the balance between bone formation and bone resorption in fracture nonunion remains unclear. The coupling of bone resorption and formation is closely related to collagen metabolism, growth factors, vascular mediators, and osteoblast activity. This study measured the serum concentrations of markers of bone turnover during the bone healing process. The serum biomarker concentrations were compared with the morphological changes on X-rays and quantitative computer tomography detection of bone mineral density to determine whether they reflected bone nonunion, and to establish a database of serum biomarker concentrations. In this study, a rabbit model of bone nonunion was monitored by regular X-ray examinations and quantitative computer tomography detection of bone mineral density. Serum biomarker concentrations were measured to investigate their usefulness for the early diagnosis of bone nonunion, and the changes in concentration during early bone nonunion. The role of biochemical markers in the metabolism of bone will be revealed through the comprehensive analysis and study, as a tool to help clinicians detect fracture healing and provide a new promising application field for fracture healing monitoring.Section I Development of a nonunion modelObjectiveTo develop a reliable nonuion model in rabbits and explore the dynamical changes of the biochemical markers and the bone mineral density. The exact pathogenesis of bone nonunion is still under discussion yet, but many factors such as defection at the fracture ends, unstable fixation, soft tissue interposition, microcirculation obstruction, infection et al could all lead to bone nonunion which were used to develop a nonunion animal model. The bone formation of the nonunion animal model was intervened during the experiment condition, and the model was widely used in the study of the mechanism of the bone nonunion and in new therapies to assess bone nonunion. In addition to the fractures, a perfect nonuion model should imitate the clinical environment to the utmost including the surrounding soft tissue injuries, and should also have good repeatability.MethodsTwenty purebred New Zealand rabbits were divided into two groups. In the bone defect group, a 15-mm length of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken preoperatively and at 2,3,4,5,6,7,8,10, and 12 weeks after surgery.ResultsAll the bone defect group and fracture group were builded successfully and all rabbits entered the result analysis. In the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks, but none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6-8 weeks. In the defect group, the fracture ends were connected by the fibrous tissues and obvious medullary sclerosis could be seen by the X-ray detection. Experimental rabbit fracture healing time generally is 6 weeks,12 weeks will be treated as bone nonunion. This experimental observation time up to 12 weeks after surgery.All cases in defect group showed nonunion. It was a successful and stable bone nonunion animal model and could be easy to operate and repeat easily.ConclusionsFibrous tissues filling the fracture ends and medullary sclerosis were proved by the X-ray detection in the defect group. This experimental observation time up to 12 weeks after surgery.All rabbits in defect group showed nonunion. These results show that a bone nonunion model can be established in New Zealand rabbits by resecting a 15-mm length of bone from the mid-radius with bone wax blocking. It was a successful bone nonunion animal model and could be repeated easily.Section II The application of NTX、TX、OC and BSAP in the early diagnosis of nonunion in the rabbit animal modelObjectiveTo develop an animal bone nonunion model and explore the change rules of the biochemical markers during the process of nonunion. The bone nonunion includes 3 phases such as callus formation, callus resorption and quiescence. During the bone formation, the osteoblastic cells produce collagen and deposit bone mineral while the osteoclast absorb the callus which is a dynamic balance regulated by the osteoblastic cells. The quantity of the callus depends on the activity of both osteoblasts and osteoclasts. The bone nonunion occurred when the callus resorption exeeded the callus formation which could be reflected by the change of the biochemical markers.MethodsTwenty purebred New Zealand rabbits were divided into two groups. In the bone defect group, a 15-mm length of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken and blood samples were collected preoperatively and at 2,3,4,5,6,7,8,10, and 12 weeks after surgery. The serum concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP) as markers of bone formation, and of C-terminal telopeptide of type I collagen (CTX), N-terminal telopeptide of type I collagen (NTX), and tartrate-resistant acid phosphatase 5b (TRACP 5b) as markers of bone resorption, were measured using biotin double-antibody sandwich enzyme-linked immunosorbent assay.ResultsIn the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks, but none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6-8 weeks. In the bone defect group, the serum BSAP and TRACP 5b concentration increased after surgery, maximum value at 4 weeks, began to decrease at 5 weeks, and stabilized after 6 weeks. The BASP concentration in the defect group was lowest 5 weeks postoperation and significant lower than in the fracture group. The concentration of of the two groups was not significant differently. There was no significant differences in the serum TRACP 5b concentration within each group at different time points.The concentration of OC in the defection group began to decline 4 weeks postoperation, and was significant lower than in the fracture group. The serum CTX concentration fluctuated during the first 4 weeks, peaked at 5 weeks, then decreased and stabilized after 6 weeks. There was significant differences between the serum CTX in the defection group 5 weeks postoperation and the fracture group. In the bone defect group,the serum NTX concentration fluctuated during the first 4 weeks, was significantly lower at 5 weeks than at all other time points before and after surgery. There were significant differences between the serum NTX concentration at 7,8,10, and 12 weeks and at 3,4, and 5 weeks. The serum NTX concentration stabilized after 6 weeks.The concentration of NTX in the defect group 6-12 weeks postoperation was significant lower than in the fracture group.ConclusionsThe bone turnover markers in the defect group maintained high turnover rate postoperation and the combination of measurement of the biochemical markers would help monitor the activity of bone formation. It was observed that the serum CTX concentration in the defect group was highest 5 weeks postoperation,serum concentration of OC, BSAP and NTX began to decline 4 or 5 weeks postoperation, while the serum TRACP5b concentration did not change significantly.It could be concluded that the serum concentration of CTX、OC、BSAP and NTX could sensitively reflect the bone turnover in vivo. Further studies are needed to determine the feasibility of systematic monitoring of the biochemical markers which could reflect the bone turnover effectively be used in the early diagnosis of nonunion in the rabbit animal model.Section III Application of quantitative computer tomography detection of bone mineral density in the early diagnosis of nonunion in the rabbit animal modelObjectiveTo develop an animal bone nonunion model and explore the changes of the bone mineral density(BMD) during the process of nonunion. Quantitative computer tomography permits non-invasive assessment of cortical and cancellous bone density with the muscle and fat tissue nearby as internal reference. It has a high resolution, accuracy and reproducibility and it is an unique technique available to differentiate cortical and cancellous bone structures thereby making it an useful tool for the diagnosis of nonunion. QCT can provide the data of bone volume ratio, bone surface area ratio, structural model parameters, inter-trabecula connecting parts, and thickness, interval, length of trabecula by measuring the BMD. The data of QCT BMD is in good linear correlation with bone ash density.MethodsTwenty purebred New Zealand rabbits were divided into two groups. In the bone defect group, a 15-mm length of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays and quantitative computer tomography detection of bone mineral density were taken preoperatively and at 2,3, 4,5,6,7,8,10, and 12 weeks after surgery.ResultsIn the bone defect group, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks, but none of the bones had healed at 8 weeks. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6-8 weeks. In the bone defect group, the BMD value was significantly decreased at 5 weeks postoperatively compared with that of the bone fracture group and the preoperative control values. There were significant differences between the BMD value at 7,8,10, and 12 weeks (combined) and at 3,4, and 5 weeks (combined). The BMD value stabilized after 6 weeks.ConclusionsSystematic measurement of the bone mineral density could reflect the process of the nonunion.QCT BMD changed significantly in the animal model which could be useful for the early detection of bone nonunion.Section IV Pathological (NTX immunohistochemistry) detection of experimental rabbit bone nonunionObjectiveTo explore the change rules of the biochemical markers by immunohistochemistry during the process of nonunion through an experimental model. As soon as the fracture occurrs, the biomarkers such as BMP, PDGH, FGF, NTX, some nonspecific cytokines and hormones coordinated during the process of fracture healing.Procollagen type I amino-terminal propeptide(NTX) has been shown to be a reliable bone formation marker, and it is released to the blood circulation after the degradation of collagen type I. It can reflect the activity of the osteoclast as a sensitive bone resorption marker.In addition, the change of bone tissue in experimental rabbit bone nonunion can confirm the reliability and repeatability of the model.MethodsAnother twenty purebred New Zealand rabbits were divided into two groups. In the bone defect group, a 15-mm length of bone (including the periosteum) was removed from the mid-radius, and the medullary cavities were closed with bone wax. In the bone fracture group, the mid-radius was fractured. X-rays were taken and each group executed a rabbit preoperation and 2,3,4,5,6,7,8,10,12 weeks postoperation respectively. Tissue of the operation site including 5mm wide normal bone nearby was extracted, decalcified, histotomied and stained by hematoxylin-eosin and by NTX immunohistochemistry(Shanghai Yaji Shengwu, Shanghai, China). Bone regeneration of the two groups was observed respectively.ResultsIn the bone defect group, the fracture ends clear in the operation day, bone callus was observed on X-ray at 2 weeks in three rabbits and the bone calluses stabilized at 5 weeks, but none of the bones had healed at 8 weeks. The callus vanished in the defect site, large bone defects and medullary sclerosis of the fracture ends 12 weeks postoperation meanted bone nonunion occured. In the bone fracture group, the fracture line was distorted at 2 weeks and bone calluses formed at 6-8 weeks.Histochemical Changes:In the defect group, there is no sign of bone connection between the fracture ends 4,6 and 8 weeks postoperation. The defect site was filled with hematoma early which was transformed into fibrous tissue scar gradually. There was little newborn capillary and islands of reactive new bone early which were replaced by the fibrous tissue scar at last.The fracture was still unstable with the medullary sclerosis and large bong defects without any callus 12 weeks postoperation. In the fracture group, new trabecula and bone tissue formed 5 weeks postoperation and plenty of callus occurred 8 weeks postoperation.Weak NTX-positive showed in the granulation tissue, fibrous tissue, fibrous callus and endochylema in the polykaryocyte during the first 4 weeks postoperation in the defect group. The granulation tissue in the defect site was NTX-negative from 5 weeks to 12 weeks postoperation. The endochylema in the polykaryocyte was NTX-positive 3,4 and 6 weeks postoperation in the fracture group. The newborn bone trabecula and osteoid tissue were NTX-negative except the osteoid tissue connecting the fibrous tissue and the cartilaginous tissue. Most of bone trabecula including the osteocyte and bone matrix was NTX-negative although the endochylema in the osteoblasts was NTX-positive.ConclusionsThe 15 mm wide defect in the radius did not heal 12 weeks postoperation by histological examination which meant the rabbit bone nonunion model was successfully developed with a good reliability. Part of pathophysiologic changes during the nonunion can be reflected by immunohistochemical analysis of the NTX activity. Considering together with the results of biochemical markers and QCT BMD, NTX immunohistochemical analysis may be useful in the early diagnosis of nonunion in rabbits.