Many pre-clinical models of atrophic non-union do not reflect the clinical scenario, some create a critical size defect, or involve cauterization of the tissue which is uncommonly seen in patients.
Introduction:
Introduction. what size of defect is optimal for creating an atrophic nonunion animal model has not been well defined. Our aim in this study was to establish a clinically relevant model of atrophic nonunion in rat femur by creation of a bone defect to research fracture healing and nonunion. Materials and methods. We used 30 male Fischer 344 rats (aged 10–11 weeks), which were equally divided into six groups. The segmental bone defects to a single femur in each rat were performed by double transverse osteotomy, and different sized defects were created by group for each group (1 mm, 2 mm, 3 mm, 4 mm, 5 mm and 6 mm). The defects were measured and maintained strictly by using an original external fixator. The periosteum for each defect was stripped both proximally and distally. Thereafter, these models were evaluated by radiology and histology. Radiographs were taken at baseline and at intervals of two weeks over a period of 8 weeks.
We describe a new surgical treatment of atrophic nonunion of the clavicle. The nonunion is excised by cuts at 45° to the long axis and repair uses 3.5 mm pelvic reconstruction or dynamic compression plates, with a lag screw to provide interfragmentary compression. The site is grafted with cancellous bone. We have been successful in all seven patients, with early return to normal function. The consequent narrowing of the shoulder girdle is fully acceptable for appearance and function.
The role of mesenchymal stem cells (MSCs) in enhancing healing process has been examined with allogeneic and xenogeneic cells in transplantation models. However, certain factors might limit the use of allogeneic cells in clinical practice, (e.g. disease transmission, ethical issues and patient acceptance). Adipose tissue represents an abundant source for autologous cells. The aim of this study was to evaluate adipose-derived autologous cells for preventing non-union. Adults male Wistar rats (n=5) underwent a previously published surgical procedure known to result in non-union if no treatment is given. This consisted of a mid-shaft tibial osteotomy with peri/endosteal stripping stabilised by intramedullary nail fixation with a 1mm gap maintained by a spacer. During the same operation, ipsilateral inguinal subcutaneous fat was harvested and processed for cell isolation. After three weeks in culture, the cell number reached 5×106 and were injected into the fracture site. At the end of the experiment, all tibias (injected with autologous fat-MSCs) developed union. These were compared with a control group injected with PBS (n=4) and with allogenic (n=5) and xenogeneic (n=6) cell transplantation groups. The amount of callus was noticeably large in the autologous cell group and the distal-callus index was significantly greater than that of the other groups, P-value =<0.05, unpaired t-test, corrected by Benjamini & Hochberg. We report a novel method for autologous MSCs implantation to stimulate fracture healing. Local injection of autologous fat-MSCs into the fracture site resulted in a solid union in all the tibias with statistically significantly greater amounts of callus.
In atrophic non-union models, a minimally invasive technique is used to deliver stem cells into the fracture site via percutaneous injection. This technique is significantly affected by a backflow leakage and the net number of cells might be reduced. The Z-track method is a technique used in clinical practice for intramuscular injections to prevent backflow leakage. We evaluated the potential of the Z-track injection technique for preventing cell loss in non-union models by determining the behaviour of observable marker fluids. Firstly, toluene blue stain was used as an injection material to allow visual detection of its distribution. Rat's cadaver legs were used and tibias were kept unbroken to ensure intact skin and overlying soft tissue. Technique includes pulling the skin over the shin of tibia towards the ankle and injection of the dye around the mid-shaft. The needle was then partially pulled back, the skin was returned to its normal position and a complete extraction of the needle was followed. Secondly, a mixture of contrast material and toluene blue was used to allow direct visual and radiological detection of the injected material into the fracture site. Ante-grade nailing of tibia via tibial tuberosity was carried out followed by a 3 point closed fracture. Injection was performed into the fracture gap similarly to the steps above. X-rays were taken to visualise the location and distribution of the injected material. Observation revealed no blue stain could be detected over the skin, X -rays revealed that the radiopaque dye remained around the tibia with no escape of the material into the superficial layers or onto the skin surface. Therefore, the number of cells delivered and maintained at a target site could be increased by the Z-track method and therefore, the therapeutic benefit of stem cell injections could be optimised with this simple technique.
Appropriate in vivo models can be used to understand atrophic non-union pathophysiology. In these models, X-ray assessment is essential and a reliable good quality images are vital in order to detect any hidden callus formation or deficiency. However, the radiographic results are often variable and highly dependent on rotation and positioning from the detector/film. Therefore, standardised A-P and lateral x-ray views are essential for providing a full radiological picture and for reliably assessing the degree of fracture union. We established and evaluated a method for standardised imaging of the lower limb and for reliably obtaining two perpendicular views (e.g. true A-P and true lateral views). The normal position of fibula in murine models is posterolateral to the tibia, therefore, a proper technique must show fibula in both views. In order to obtain the correct position, the knee joint and ankle joints were flexed to 90 degrees and the foot was placed in a perpendicular direction with the x-ray film. To achieve this, a leg holder was made and used to hold the foot and the knee while the body was in the supine position. Lateral views were obtained by putting the foot parallel to the x-ray film. Adult Wister rat cadavers were used and serial x-rays were taken. A-P view in supine position showed the upper part of the fibula clearly, however, there was an unavoidable degree of external rotation in the whole lower limb, and the lower part of the fibula appeared behind the tibia. Therefore, a true A-P view whilst the body was in the supine position was difficult. To overcome this, a P-A view of the leg was performed with the body prone position, this allowed both upper and lower parts of the fibula to appear clearly in both views. This method provides two true perpendicular views (P-A and lateral) and helped to optimise radiological assessment.
There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4). Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for
We treated 15 patients with atrophic nonunion of a diaphyseal fracture of the humerus with an associated bony defect using an autogenous cancellous bone graft and a plate to bridge the defect. There were nine men and six women with a mean age of 48 years. The mean length of the bony defect was 3 cm. At a mean follow-up of 30 months only one fracture failed to unite. This suggests that, in the presence of a well-vascularised envelope of muscle, the application of an autogenous cancellous bone graft in conjunction with a bridging plate represents a good alternative to more demanding surgical techniques.
Over 1 million fractures occur each year in the UK. Approximately 5-10% of these fractures have problems with healing. The treatments used for these patients often have a poor outcome and are associated with increased morbidity and disability. Application of synthetic peptides such as thrombin degradation peptide (TP508) has been shown to accelerate fracture repair in a closed rat femoral fracture model. Controlled release of TP508 using microspheres has been shown to enhance repair of articular cartilage defects and stimulate bone formation in segmental defects in rabbits. The aim of this study was to determine whether TP508 could bring about healing in an established fracture non-union model. A validated rat model of fracture non-union was used. The model was created and left for 8 weeks in order to represent a clinically equivalent model of a non union of a fracture. Rats were randomised into two treatment groups receiving 10microg and 1microg doses of TP508 diluted in 50microL of microspheres and delivered directly to the non union site using percutaneous injection 8 weeks after surgery. The control group received no treatment. At 16 weeks post-surgery, osseous bridging was assessed both radiographically and histologically. Radiographically there was no difference between the control and two treatment groups. However, histomor-phometric analysis demonstrated that bone formation increased by 43.9% in animals that received high dose of TP508 compared to the control animals. The analysis also indicated that administration of the low dose of TP508 increased the amount of bone formation compared to the control by 9.9 %. Administration of TP508 has been shown to enhance healing of segmental defects in both critically and noncritically sized defects. However, in our model which is an established fracture non-union model, TP508 did not manage to achieve full osseous union. It has been suggested that the action of this peptide is concentration and environment dependent possibly indicating that TP508 might be less effective when administered in a chronic situation such as that associated with the established non-union fracture. However, even in this sub-optimal situation an increased amount of bone formation was observed.
This review is aimed at clinicians appraising
preclinical trauma studies and researchers investigating compromised bone
healing or novel treatments for fractures. It categorises the clinical
scenarios of poor healing of fractures and attempts to match them
with the appropriate animal models in the literature. We performed an extensive literature search of animal models
of long bone fracture repair/nonunion and grouped the resulting
studies according to the clinical scenario they were attempting
to reflect; we then scrutinised them for their reliability and accuracy
in reproducing that clinical scenario. Models for normal fracture repair (primary and secondary), delayed
union, nonunion (atrophic and hypertrophic), segmental defects and
fractures at risk of impaired healing were identified. Their accuracy
in reflecting the clinical scenario ranged greatly and the reliability
of reproducing the scenario ranged from 100% to 40%. It is vital to know the limitations and success of each model
when considering its application.
The aim of this study was to establish an atrophic non-union model in the rat femur under well defined biomechanical conditions and with minimised interactions between the processes in the healing zone and the implant by using external fixation.
There is a growing trend towards using pre-clinical models of atrophic non-union. This study investigated different fixation devices, by comparing the mechanical stability at the fracture site of tibia bone fixed by either intramedullary nail, compression plate or external fixator. 40 tibias from adult male Wistar rats' cadavers were osteotomised at the mid-shaft and a gap of 1 mm was created and maintained at the fracture site to simulate criteria of atrophic non-union model. These were divided into five groups (n=8 in each): the first group was fixed with 20G intramedullary nail, the second group with 18G nail, the third group with 4-hole plate, the fourth group with 6-hole plate, and the fifth group with external fixator. Tibia was harvested by leg disarticulation from the knee and ankle joints, the soft tissues were carefully removed from the leg, and tibias were kept hydrated throughout the experiment. Each group was then subdivided into two subgroups for mechanical testing: one for axial loading (n=4) and one for 4-point bending (n=4). Statistical analysis was carried out by ANOVA with a fisher post-hoc comparison between groups. A p-value less than 0.05 was considered statistically significant. Axial load to failure data and stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices, however there was no statistically significant difference axially between the nail thicknesses. In bending, load to failure revealed that 18G nails are significantly stronger than 20G. We concluded that 18G nail is superior to the other fixation devices, therefore it has been used for in-vivo experiments to create a novel model of atrophic non-union with stable fixation.
We present our clinical experience in treating atrophic non-union of long bones by injecting, percutaneously, autologous bone marrow aspirate concentrated as a source of progenitors stem cells Bone marrow aspirated from the iliac crest contains progenitor cells that can be used to obtain bone-healing of non-union. However, its efficacy appears to be related to the number and concentration of progenitors in the graft. The last three-year period, 11 patients (8 men-3 women) with established atrophic non-union were treated in our department. In all cases, the gap between the fragments was smaller than 5 mm. A constant volume of 60+60 ml of marrow were aspirated from both iliac crests and centrifuged for 15 minutes aiming at the increase of concentration of progenitor-mononucleotide cells. An average volume of 20 ml (+/− 2) concentrated bone marrow was injected percutaneously, under C-arm, at the site of non-union. The graft contained an average of 272.64 x 10(6)/ ml mononucleotide cells. The evaluation of treatment was based on the clinical and radiological findings after 3, 6, 9 and 12 months. However, prior to administration of bone marrow stem cells, there was no case with evidence of ongoing deep sepsis. Bone union was obtained in 10 out of 11 patients (full weight bearing, callus formation in 3 out of 4 cortices). In one case a second operation was needed due to impaired indications of treatment. However, in all cases, there were no signs of local or systematic complications. Percutaneous concentrated bone marrow grafting is an efficient and safe method, for treating atrophic non-unions, with a minimal invasion technique. Contraindications for the above technique are a gap larger than 5 mm and a preexisting angular and axial deformity.
We retrospectively reviewed eight children with idiopathic chondrolysis (IC) of the hip and nine with atrophic tuberculosis (TB) of the hip treated over the 10 years 1990 to 1999. Both conditions present with a stiff hip and radiographic joint space narrowing. Our aim was to delineate clinical, radiological and histological differences between the two conditions, thereby obviating the need for biopsy in IC, which could worsen the prognosis. In the IC group all patients were girls. Their mean age was 12 years (11.5 to 13). They presented with a flexion abduction and external rotation deformity of the hip. Chest radiographs were normal in all patients, and all except one had an ESR below 20. The Mantoux was negative in six of the eight. Radiographs showed joint space narrowing and osteopoenia, but the subchondral bony line remained present. Four of the eight had a synovial biopsy, which showed non-specific chronic synovitis. The cartilage looked pale and lustreless. In one hip the cartilage was biopsied and showed cartilage necrosis. In the TB group, five of the nine patients were boys. The mean age was 7 years (5 to 13.5). The only constant hip deformity was flexion. Chest radiographs were normal in all patients. In all patients the ESR was below 20 and the Mantoux was positive. Hip radiographs showed osteopoenia with loss of the subchondral bony line. Peri-articular lytic lesions were present in all patients except one. Histology of synovial biopsy showed caseous necrosis in all hips, and seven of the nine had a positive culture for TB. Macroscopically the cartilage looked normal, and in one hip the cartilage biopsy was histologically normal. We confirmed that in IC the joint space narrowing is due to cartilage necrosis. We postulate that in atrophic TB the loss of subchondral bone due to subchondral erosion gives the impression of joint space narrowing. We also concluded that IC was a diagnoses per se and not by exclusion, and that biopsy was not required.
During fracture repair, a number of growth factors and cytokines are present at elevated levels at the fracture site such as Transforming Growth Factor Beta (TGF-), Fibroblast Growth Factor (FGF) and Platelet Derived Growth Factor (PDGF). The aim of the study was to investigate the presence of these growth factors in healing fractures and fracture non-unions, in order to test the hypothesis that atrophic non-unions express a lower level of growth factors than hypertrophic non-unions and healing fractures. Biopsies were taken from the fracture site of 23 patients (mean age 46) with uninfected non-unions, 12 patients with hypertrophic (mean 13.8 months after fracture) and 11 patients with atrophic (mean 16.5 months after fracture). A comparison group of biopsies from early fracture callus (one to four weeks after fracture) in five patients with healing fractures was also included. Five-micron paraffin sections were immunohistochemically stained for TGF-, FGF-II and PDGF. Growth factors were then assessed in six different cell types. Fibroblasts, endothelial cells and macrophages were found to express TGF-, FGF-II and PDGF in all three-fracture groups. Osteoblasts, osteoclasts and chondrocytes were not present in the healing fracture group. The growth factor expression in osteoblasts, osteoclasts and chondrocytes in the non-union groups were found to be variable, however, the expression of these growth factors appeared to be less in the atrophic non-unions than hypertrophic non-unions. The expression of these growth factors was found to be less in the atrophic non-union group than the hypertrophic non-union group in osteoblasts, osteoclasts and chondrocytes. These results may have relevance for new therapies that can be aimed at delivering growth factors to treat fracture non-unions. By further investigation of the differential expression of these growth factors it may be possible to determine which factors are likely to stimulate fracture healing.