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.

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 in-vivo experiments to create a novel model of atrophic non-union with stable 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.

Objectives

A successful outcome following treatment of nonunion requires the correct identification of all of the underlying cause(s) and addressing them appropriately. The aim of this study was to assess the distribution and frequency of causative factors in a consecutive cohort of nonunion patients in order to optimise the management strategy for individual patients presenting with nonunion.

Objectives

To explore the therapeutic potential of combining bone marrow-derived mesenchymal stem cells (BM-MSCs) and hydroxyapatite (HA) granules to treat nonunion of the long bone.

Previous classification systems of failure of limb salvage focused primarily on endoprosthetic failures and lacked sufficient depth for the effective study of the causes of failure. In order to address these inadequacies, the International Society of Limb Salvage (ISOLS) formed a committee to recommend revisions of the previous systems. The purpose of this study was to report on their recommendations. The modifications were prepared using an earlier, evidence-based model with subclassification based on the existing medical literature. Subclassification for all five primary types of failure of limb salvage following endoprosthetic reconstruction were formulated and a complementary system was derived for the failure of biological reconstruction. An additional classification of failure in paediatric patients was also described.

Limb salvage surgery presents a complex array of potential mechanisms of failure, and a complete and precise classification of types of failure is required. Earlier classification systems lacked specificity, and the evidence-based system outlined here is designed to correct these weaknesses and to provide a means of reporting failures of limb salvage in order to allow the interpretation of outcome following reconstructive surgery.

Cite this article: Bone Joint J 2014;96-B:1436–40.

Objectives

Nonunion is one of the most troublesome complications to treat in orthopaedics. Former authors believed that atrophic nonunion occurred as a result of lack of mesenchymal stem cells (MSCs). We evaluated the number and viability of MSCs in site of atrophic nonunion compared with those in iliac crest.

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. Atrophic nonunion was defined as a lack of continuity and atrophy of both defect ends radiologically and histologically at eight weeks. Results. In the 1 mm defect group, all defects had healed. In the 2 mm group, one-fifth remained atrophic nonunion. In the 3 mm group, three-fifths had atrophic nonunion, and all of the defects of groups of 4 mm and over were atrophic nonunion. Conclusion. This study showed that we were able to predictively produce an atrophic nonunion animal model by creating defects of at least 4 mm in the Fischer 344 rat femur

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.

Nonunion of the tibia associated with bone loss, previous infection, obliteration of the intramedullary canal or located in the distal metaphysis poses a challenge to the surgeon and significant morbidity to patients. We retrospectively reviewed the records of 24 patients who were treated by central bone grafting and compared them to those of 20 who were treated with a traditional posterolateral graft. Central bone grafting entails a lateral approach, anterior to the fibula and interosseous membrane which is used to create a central space filled with cancellous iliac crest autograft. Upon consolidation, a tibiofibular synostosis is formed that is strong enough for weight-bearing. This procedure has advantages over other methods of treatment for selected nonunions.

Of the 24 patients with central bone grafting, 23 went on to radiographic and clinical union without further intervention. All healed within a mean of 20 weeks (10 to 48). No further bone grafts were required, and few complications were encountered. These results were comparable to those of the 20 patients who underwent posterolateral bone grafting who united at a mean of 31.3 weeks (16 to 60) but one of whom required below-knee amputation for intractable sepsis.

Central bone grafting is a safe and effective treatment for difficult nonunions of the tibia.

Introduction: Atrophic nonunion is a well recognised complication of long bone fractures. Clinical trials show that BMP-2 accelerates healing and reduces nonunion in open tibial fractures. We are interested in a natural small molecule that has been previously demonstrated to stimulate angiogenesis in vivo. Our aim is to assess the two treatments in the prevention of nonunion. The small animal model we used is a non-critical size defect of the tibia deprived it of its blood supply by surgical stripping of the periosteum and curetting of the local endosteum thus closely reflecting the clinical situation. The outcomes were measured by radiographic assessment and histology. Methods: Wistar rats were treated with either the angiogenic molecule (0.1% or 0.003%), BMP-2 or vehicle alone (PBS) soaked in a type I collagen sponge. All animals underwent a 2mm osteotomy, stripping of the periosteum and endosteum proximally and distally for the length of the diameter of the tibia. Fluorescent markers were injected at 2 weekly intervals. The rats were sacrificed at 8 weeks. Both tibiae were disarticulated; fixator and soft tissues were removed and AP and lateral X-rays were taken. Subjective assessment of the healing on X-ray was carried out in two ways; using a radiographic scoring system and by grey scale analysis. The samples were embedded, sectioned and stained for new bone formation. Results: Bridging or potential to bridge was seen in a number of animals on x-ray. Bridging or potential to bridge was judged to be present in 72.22% of the BMP-2 group and 66.67% of the high dose group compared to 22.22% of the control group. Histological analysis is being performed to confirm these findings. Discussion: Atrophic nonunion is a serious clinical complication, unfortunately BMP-2 is a highly costly treatment option and therefore alternative molecular therapies are much sought after. We describe here an angiogenic molecule has some potential in preventing formation of nonunion

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting

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.