A number of anti-retroviral therapies (ART) have been implicated in potentially contributing to HIV-associated bone disease. The aim of this study was to evaluate the effect of combination ART on the fracture healing process. A total of 16 adult male Wistar rats were randomly divided into two groups (n = eight each): Group 1 was given a combination of Tenfovir 30 mg, Lamivudine 30 mg, and Efavirenz 60 mg per day orally, whereas Group 2 was used as a control. After one week of medication preload, all rats underwent a standardized surgical procedure of mid-shaft tibial osteotomy fixed by intramedullary nail with no gap at the fracture site. Progress in fracture healing was monitored regularly for eight weeks. Further evaluations were carried out after euthanasia by micro-CT, mechanically and histologically. Two blinded orthopaedic surgeons used the Radiological Union Scoring system for the Tibia (RUST) to determine fracture healing.Aims
Methods
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.
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. Atrophic non-union is usually developed following high energy trauma leading to periosteal stripping. The most recent reliable model with these aspects involves creating a non-critical gap of 1mm with periosteal and endosteal stripping. However, this method uses an external fixator for fracture fixation, whereas intramedullary nailing is the standard fixation device for long bone fractures. To establish a clinically relevant model of atrophic non-union using intramedullary nail and (1) ex vivo and in vivo validation and characterization of this model, (2) establishing a standardized method for leg positioning for a reliable x-ray imaging. Ex vivo evaluation: 40 rat's cadavers (adult male 5–6 months old), 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 an external fixator. Tibiae were harvested by leg disarticulation from the knee and ankle joints. 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) using Zwick/Roell® machine. 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. To maintain the non-critical gap, a spacer was inserted in the gap, the design was refined to minimize the effect on the healing surface area. In vivo evaluation was done to validate and characterize the model. Here, a 1 mm gap was created with periosteal and endosteal stripping to induce non-union. The fracture was then fixed by a hypodermic needle. A proper x-ray technique must show fibula in both views. Therefore, a leg holder was used to hold the knee and ankle joints in 90º flexion and the foot was placed in a perpendicular direction with the x-ray film. Lateral view was taken with the foot parallel to the x-ray film. Ex vivo: axial load stiffness data revealed that intramedullary nails are significantly stronger and stiffer than other devices. Bending load to failure showed that 18G nails are significantly stronger than 20G, thus it is used for the in vivo experiments. In vivo: final iteration revealed 3/3 non-union, and in controls with the periosteum and endosteum intact but with the 1mm non-critical gap, it progressed to 3/3 union. X-ray positioning: A-P view in supine position, there was an unavoidable degree of external rotation in the lower limb, thus the lower part of the fibula appeared behind the tibia. To overcome this, a P-A view of the leg was performed with the body in prone rather, this arrangement allowed both upper and lower parts of the fibula to appear clearly in both views. We report a novel model of atrophic non-union, the surgical procedure is relatively simple and the model is reproducible.OBJECTIVES
