Development of a tibial experimental non-union model in rats.

BACKGROUND: Many non-union animal models have been developed to explore the problems surrounding fracture healing. However, the existing models are not perfect and cannot satisfy all non-union studies. This study aimed to make a non-union model of the tibia in rats by cauterization of the posterior of 2 mm on both sides of the fracture end after open osteotomy of the tibia and fixing the fractured tibia with a Kirschner wire 0.8 mm in diameter. METHODS: For this study, 96 female adult Sprague-Dawley (SD) rats were used. The rats underwent surgery to produce a tibial open fracture and were fixed with a 0.8-mm diameter Kirschner wire. In 48 of the rats, the periosteum proximal and distal to the fracture end was cauterized. RESULTS: At 2, 4, 6, and 8 weeks after surgery, radiological and histological analysis showed typical physiological healing in the control group, and the healing rate was 100% at 6 weeks. But the non-union group was characterized by resorption of the fracture ends with few callus formations and no bridging callus formation, and the healing rate was 0% at 8 weeks. CONCLUSIONS: This method represents a reproducible model to create atrophic non-unions. This model provides a new option for studying the basic healing mechanisms and evaluating new therapies for bone regeneration and treatment of non-unions.

Deficiency of inducible and endothelial nitric oxide synthase results in diminished bone formation and delayed union and nonunion development.

BACKGROUND: Between 5% and 10% of all fractures fail to heal adequately resulting in nonunion of the fracture fragments. This can significantly decrease a patient's quality of life and create associated psychosocial and socio-economic problems. Nitric oxide (NO) and nitric oxide synthases (NOS) have been found to be involved in fracture healing, but until now it is not known if disturbances in these mechanisms play a role in nonunion and delayed union development. In this study, we explored the role of endothelial and inducible NOS deficiency in a delayed union model in mice. MATERIALS AND METHODS: A 0.45mm femur osteotomy with periosteal cauterization followed by plate-screw osteosynthesis was performed in the left leg of 20-24week old wild type, Nos2(-/-) and Nos3(-/-) mice. Contralateral unfractured legs were used as a control. Callus volume was measured using micro-computed tomography (µCT) after 28 and 42days of fracture healing. Immuno histochemical myeloperoxidase (MPO) staining was performed on paraffin embedded sections to assess neutrophil influx in callus tissue and surrounding proximal and distal marrow cavities of the femur. After 7 and 28days of fracture healing, femurs were collected for amino acid and RNA analysis to study arginine-NO metabolism. RESULTS: With µCT, delayed union was observed in wild type animals, whereas in both Nos2(-/-) and Nos3(-/-) mice nonunion development was evident. Both knock-out strains also showed a significantly increased influx of MPO when compared with wild type mice. Concentrations of amino acids and expression of enzymes related to the arginine-NO metabolism were aberrant in NOS deficient mice when compared to contralateral control femurs and wild type samples. DISCUSSION AND CONCLUSION: In the present study we show for the first time that the absence of nitric oxide synthases results in a disturbed arginine-NO metabolism and inadequate fracture healing with the transition of delayed union into a nonunion in mice after a femur osteotomy. Based on these data we suggest that the arginine-NO metabolism may play a role in the prevention of delayed unions and nonunions.

Matrix metalloproteinases that associate with and cleave bone morphogenetic protein-2 in vitro are elevated in hypertrophic fracture nonunion tissue.

OBJECTIVES: A delayed union or a nonunion of a fracture is a potentially adverse complication. Understanding the mechanisms of nonunion development may lead to improved treatment modalities. Proteases such as the matrix metalloproteinases play important roles in bone remodeling and repair, in which an imbalance or a nonfunctioning enzyme may lead to defects in bone healing (nonunion). The purpose of this pilot study was twofold: first to define an mRNA expression profile of all the matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases with thrombospondin motif (ADAMTS) enzymes, and their inhibitors (TIMPs) within fracture nonunion tissue, and second to compare this profile with mineralized fracture callus. METHODS: Using a systematic real-time polymerase chain reaction, we screened the gene expression profiles of all members of the MMPs, ADAMTS, and their inhibitor TIMPs on human fracture nonunion tissue and matched mineralized callus tissue. Significant results were further analyzed using Western immunoblotting, immunohistochemistry, and in vitro protein interaction assays with bone morphogenetic protein-2. RESULTS: This analysis confirmed MMP-7 and MMP-12 as two unidentified enzymes expressed in fracture nonunion tissue. Both MMP-7 and MMP-12 mRNAs were significantly elevated in nonunion tissue when compared with local mineralized callus from the same site (P < 0.001); the elevated protein levels of interest were visualized through immunoblotting and immunohistochemistry. In addition, these two MMPs were found to directly bind to and degrade bone morphogenetic protein-2 in vitro. CONCLUSION: Collectively, our findings indicate that tissue present at the site of hypertrophic nonunions commonly expresses significantly higher levels of MMP-7 and MMP-12 in relation to mineralized fracture callus. Both were found to directly inactivate bone morphogenetic protein-2 in vitro, the best established growth factor in bone formation and repair.

Correlation of serum alkaline phosphatase activity with the healing process of long bone fractures in dogs.

BACKGROUND: Bone healing is monitored mainly by physical and serial radiologic examinations of the fracture site. However, it is sometimes difficult to distinguish a delayed union from a nonunion, and advanced imaging techniques may not be available. Serum biochemical markers of bone formation, such as alkaline phosphatase (ALP) activity, may be clinically useful in evaluating the progress of healing. OBJECTIVE: The purpose of this study was to correlate serial values of serum ALP activity with the process of fracture healing in dogs and to assess its potential as a postsurgical prognostic indicator. METHODS: Changes in serum ALP activity were studied in 83 dogs with closed long bone diaphyseal fractures treated surgically. Physical and radiologic examinations of the fracture site and determination of serum ALP activity and calcium (Ca) and phosphate (P) concentrations were performed on admission (day 0); postoperatively on days 10, 20, and 30; and subsequently on a monthly basis until bone union was completed or signs of nonunion were evident. The dogs were allocated into 3 groups with respect to the fracture healing progress as documented by physical and serial radiologic examination. RESULTS: Group A dogs (n=35) developed a medium-sized callus that led to bone union within 2 months. Group B dogs (n=36) had a hypertrophic callus and delayed union, within 3-5 months. Group C dogs (n=12) had slow progress in fracture healing, with minimal callus formation during a 2-month period. Changes in mean serum ALP activity followed the same pattern in groups A and B, reaching a maximum level on day 10. Group A values returned to normal within 2 months, at which point bone union was complete, whereas group B values remained increased and returned to normal within 3-5 months, thus correlating with delayed union. In Group C, mean serum ALP activities showed no significant changes during the 2-month follow-up period, consistent with failure of bone union (nonunion). Serum P and Ca changes followed a proportional and inverse pattern to ALP changes, respectively. CONCLUSION: Serial determination of serum ALP activity during fracture healing could be an additional tool in predicting fractures at risk of developing a nonunion, helping the clinician to choose the appropriate intervention.