To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model.

5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10⁶ and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls.

All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings.

The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested.

Aims

Assessment of bone mineral density (BMD) with dual-energy X-ray absorptiometry (DXA) is a well-established clinical technique, but it is not available in the acute trauma setting. Thus, it cannot provide a preoperative estimation of BMD to help guide the technique of fracture fixation. Alternative methods that have been suggested for assessing BMD include: 1) cortical measures, such as cortical ratios and combined cortical scores; and 2) aluminium grading systems from preoperative digital radiographs. However, limited research has been performed in this area to validate the different methods. The aim of this study was to investigate the evaluation of BMD from digital radiographs by comparing various methods against DXA scanning.

Abstract

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.

Trabecular bone is a multiscale hierarchical composite material that is known to display time-dependant properties. However, most biomechanical models treat this material as time independent. Time-dependant properties, such as creep and relaxation, are thought to play an important role in many clinically relevant orthopaedic issues: implant loosening, vertebral collapse, and non-traumatic fractures. In this study compressive multiple-load-creep-unload-recovery (MLCUR) tests were applied to human trabecular bone specimens.

15 female femoral heads were harvested, with full ethical approval and patient consent, at the time of total hip replacement. Central cores were extracted and cut parallel under constant irrigation. Specimens were embedded in end caps using surgical cement, an epoxy tube was secured around the end caps and filled with phosphate buffered saline (PBS) to ensure the specimens remained hydrated throughout. Embedded samples were scanned by microCT (SkyScan 1172, Bruker) at a resolution of 17µm to determine microarchitecture. Bone volume fraction (BVF) was used to represent microarchitecture. Specimens had an effective length of 16.37mm (±1.90SD) with diameter of 8.08mm (±0.05SD), and BVF of 19.22% (±5.61SD).

The compressive MLCUR tests were conducted at 5 strain levels, 2000µε, 4000µε, 6000µε, 8000µε and 10000µε. At each strain level, the load required to maintain each strain was held for 200s (creep) then unloaded to 1N for 600s (recovery). The instantaneous, creep, unloading and recovered strains can be easily obtained from the strain-time curves. Stress-strain plots revealed the Young's modulus. Data was modelled using line of best fit with appropriate curve fitting. R2 values were used to indicate association. Mechanical testing demonstrated the expected time independent relationship between BVF and stiffness: higher stiffness was found for specimen with higher BVF and this was consistent for all strain levels.

Creep strain was found to depend on instantaneous strain and BVF. At low levels of instantaneous strain, there was a greater amount of creep strain in low BVF samples (R2 = 0.524). This relationship was no longer apparent at higher strain levels (R2 = 0.058). Residual strain also depended on the applied instantaneous strain and BVF: at low levels of strain, residual strain was similar with all BVF (R2 = 0.108) and at high levels of strain, residual strain was greater in low BVF samples (R2 = 0.319). The amount of instantaneous strain applied to each sample is constant, variations in stiffness result in different applied loads. In low BVF bone, the stiffness is also low, therefore the stress required to reach designed strain is also lower: yet, there is more creep and less recovery.

We have demonstrated that even at loads below recognised yield levels, time-dependence affects the mechanical response and residual strain is present. In cases of low BVF, deflection due to creep, and increased irrecoverable strain could have clinically relevant consequences, such as implant loosening and vertebral collapse. The role of time-dependant properties of bone is seldom considered. This data could be developed into a constitutive model allowing these time-dependant behaviours to be incorporated in finite element modelling, leading to better predictions of implant loosening, especially for lower quality bone.

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.

Femoral shaft fractures are potentially devastating injuries. Despite this, clinical studies of the biomechanics of this injury are lacking. We aimed to clinically evaluate bone behaviour under high and low energy trauma in paediatric, adult and older patients. Single-centre retrospective study identifying all diaphyseal femoral fractures between Feb 2015-Feb 2017. Peri-prosthetic and pathological fractures were excluded. Patients were subdivided into groups 1 (paediatric, <16yo), 2 (adult, 17–55yo) and 3 (older, >55yo) to reflect immature, peak bone age and osteoporotic bone respectively. Chi-Squared analysis assessed significance of bone age to degree of comminution and fracture pattern. A p-value <0.05 was significant. A total 4130 radiographs were analysed with 206 femoral shaft fractures identified. Forty-three patients were excluded with 163 remaining. Group 1, 2 and 3 included 38, 37 and 88 patients respectively. Mean age 50.8 (SD 32.8) with male-to-female ratio of 1:1.2. Groups 1 and 3 included majority simple fractures (35/38 and 62/88 respectively). Group 2 included more comminuted injuries (33/37). Bone age to degree of comminution proved significant (p<0.05) with a bimodal distribution of simple fractures noted in groups 1 and 3. Energy to fracture was significant in group 2, where a high energy injury was associated with comminution (p<0.05). This study is the first to demonstrate an association between fracture comminution and age. Simple femoral shaft fractures showed a bimodal age distribution in paediatric and older patients regardless of mechanism energy. High energy mechanism trauma was directly related to fracture comminution at peak bone age.

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.

Kirschner wires are commonly used in paediatric fractures, however, the requirement for removal and the possibility of pin site infection provides opportunity for the development of new techniques that eliminate these drawbacks. Bioabsorbable pins that remain in situ and allow definitive closure of skin at the time of insertion could provide such advantages.

Three concurrent studies were performed to assess the viability of bioabsorbable pins across the growth plate. (1) An epidemiological study to identify Kirschner wire infection rates. (2) A mechanical assessment of a bioabsorbable pin compared to Kirschner wires in a simulated supracondylar fracture. (3) The insertion of the implants across the physis of sheep to assess effects of the bioabsorbable implant on the growth plate via macroscopic, pathohistological and micro-CT analysis.

An infection rate of 8.4% was found, with a deep infection rate of 0.4%. Mechanically the pins demonstrated comparable resistance to extension forces (p=) but slightly inferior resistance to rotation (p=). The in vivo component showed that at 6 months: there was no leg length discrepancy (p=0.6), with micro-CT evidence of normal physeal growth without tethering, and comparable physeal width (p=0.3).

These studies combine to suggest that bioabsorbable pins do not represent a threat to the growth plate and may be considered for physeal fracture fixation.

Aim

To investigate the effects of strain rate and mineral level on the stress at failure, stiffness and toughness of whole bones.

Introduction

Bending tests are commonly used to evaluate the mechanical behaviour of small animal bones. To test whole bones, it is normal that soft tissue should be removed before testing. However, cleaning the specimens might disturb the callus, interfering with the mechanical properties. This study compares mechanical properties of rat tibia between specimen with and without muscle cleaning

Aims

The aim of this study was to compare biomechanical properties of pre-contoured plate fixation using different screw fixation modes in a mid-shaft clavicle fracture model.

There is an established link between bone quality and fracture risk. It has been suggested that reduced bone quality will also reduce the toughening mechanisms displayed during loading at a high strain rate. We hypothesised that partially decalcified bone will not demonstrate an increase in force required to cause failure when comparing low and high strain rate loading.

Mechanical properties were defined by the maximum force at failure. Bone quality was defined by the mineral content. This was altered by subjecting the bones to ultrasonically assisted decalcification in 10M EDTA to achieve an average 18% mineral reduction (A 70 yr old woman has approx 18% of her peak bone mass). 20 pairs of sheep femurs were harvested and split into four equal groups: normal bone quality, fast strain rate (NF); normal bone quality, slow strain rate (NS); low bone quality, fast strain rate (LF) and low bone quality, slow strain rate (LS). All mechanical testing was carried out by means of 3-point bending. Load representing the slow strain rate was applied by a mechanical testing machine (Zwick) at a rate resulting in a deflection of 1mm/s. The dynamic loading was applied by a custom designed pneumatic ram at a mean rate of deflection between the specimens of 2983 mm/s (±SD 1155), this equates to strain rates experienced in a road traffic accident.

The following results for force at failure were found (mean ± SD). NF: Force 5503N (± 1012); NS: Force 3969N (± 572); LF: Force 3485N (± 772); LS: Force 3165N (± 605). Groups were compared using a Mann-Whitney U test. Significant results were found between the following groups: Normal bone quality, strain rate compared (NF-NS) p<0.002; Fast strain rate, bone quality compared (NF-LF) p=0.008; Slow strain rate, bone quality compared (NS-LS) p=0.02. No statistical significance was found when comparing low bone quality, strain rate compared (LF-LS) p=0.47.

These results show that normal healthy bone has an ability to withstand higher strain rates which protects it against fracture. This ability to withstand high strain rates is lost in decalcified bone making it more susceptible to fracture. The results of this study indicate the importance of strain rate reduction as well as energy absorption in the design of hip protectors and in environmental modifications.

Fractures repair by two mechanisms; direct fracture healing and indirect fracture healing via callus formation. Research concerning the effects of bisphosphonate on fracture repair has solely assessed indirect fracture healing. Patients with osteoporosis on bisphosphonates continue to sustain fragility fractures. A proportion of osteoporotic fractures require plate fixation. Bisphosphonates impair osteoclast activity and therefore, may adversely affect direct fracture healing that predominates with plate fixation.

Five skeletally mature Sprague-Dawley rats received daily subcutaneous injections of 1mg/kg Ibandronate (IBAN). Similarly, five control rats received saline (CONTROL). Three weeks following commencement of injections a tibial osteotomy was rigidly fixed with compression plating similar to that seen in routine clinical practice. Fracture healing was monitored with radiographs. Six weeks post plate fixation, animals were sacrificed. Radiographs were performed of the extricated tibiae following plate removal. The visibility of the osteotomy site was scored as totally visible, partially visible or absent as previously described. Mechanical testing was conducted on the healing osteotomies via 4-point bending.

Fractures healed without visible external callus. In the IBAN group three animals had totally visible osteotomy lines and two had partially visible osteotomy lines. The CONTROL group had three animals with absent osteotomy lines and two with partially visible osteotomy lines. The mean (±SD) stress at failure for the healing tibial osteotomies at 6 weeks was 28.8 (±23.97)MPa in the IBAN group and 37.4(±29.20) MPa in the CONTROL group (p=0.62)

Our results indicate that Ibandronate adversely affected direct fracture repair as demonstrated by the radiographic density of the fracture line. The strength of the repair was reduced but this did not reach statistical significance. Our results suggest that a sample size of 220 animals is required to detect a 15% difference (alpha 0.05, beta 0.2) which suggests the effect of bisphosphonates on direct fracture repair may be small.

The best management for acute rupture of the Achilles tendon remains controversial. An unacceptably high re-rupture rate following conservative management has been quoted as a reason for surgical management, however, many of these studies do not stand up to critical scrutiny. Since 1989 I have personally treated over 1600 consecutive patients with acute rupture of the tendo-achilles using a conservative functional management protocol. This protocol was developed in the light of experience over a number of years. I will present an independent review of 1044 consecutive patients presenting to my tendo-achilles clinic between 1996 and 2008. After examination, 975 patients were found to have an acute tendon rupture, the rest being gastrocnemius muscle tears, painful Achilles tendonopathy or mis-referrals. Of these 975 patients 29 were late presenters in whom the tendon ends did not approximate well on plantar flexion. These patients were therefore treated surgically. One patient was too obese to be included in the standard protocol, although he was still treated conservatively. With a minimum follow up of two years, the overall re-rupture rate was 2.9%. The re-rupture rate using this management protocol is similar to or better than the published operative re-rupture rates. The protocol, complications and outcome will be discussed and I recommend a well defined and tested non operative functional management protocol for treatment of acute tendo-achilles rupture. This protocol is suitable for all ages including those with significant sporting demands. It is essential that patients have assessment and follow up carried out by a senior and experienced surgeon.

The effect of bisphosphonates on the mechanical properties of the uninjured contra-lateral cortical bone during fracture healing is poorly reported. There remains conflicting evidence with regards the effect of bisphosphonate therapy on cortical bone strength. We assessed the effect of nine weeks of Ibandronate therapy, in a dose known to preserve cancellous bone BMD and strength, on the mechanical properties of the uninjured rat tibial diaphyses using a standardised model of tibial osteotomy and plate fixation. Skeletally mature ex-breeder rats were used. Stress at failure of the tibial diaphyses was measured by a four-point bending test using a custom made jig for rat tibiae. The mechanical strength was compared with radiographic measurements of bone density. Animals received daily subcutaneous injections. 11 rats received 1μg/kg Ibandronate (IBAN) daily and 17 rats received 1ml 0.9% Sodium Chloride (CONTROL) daily.

The IBAN group had a statistically significant, p=0.024, higher stress at failure 212.7 (±42.04) MPa compared to the CONTROL group 171.7 (±46.13)MPa. There was a positive correlation between the mechanical strength of bone and the radiological measure of bone density.

Osteopenia is known to occur following a fracture even in the contra-lateral limb. This study demonstrates that ibandronate therapy has no detrimental effect and may even increase the strength of uninjured cortical bone during the fracture healing process. The longer term effect of ibandronate on cortical bone especially in relation to the accumulation of mico-damage requires further study. Bisphosphonate effect on the uninjured limb needs to be considered when reporting proportional strength of fracture repair compared to the uninjured limb.

The suture properties associated with a successful tendon repair are: high tensile strength, little tissue response, good handling characteristics and minimal plastic deformation. Plastic deformation contributes to gap formation at a tendon repair site. Gaps greater than 4mm are prone to failing. This study investigates whether the plastic deformation demonstrated by two commonly used suture materials can be reduced by manual pre-tensioning.

Twenty sutures of both Prolene 3/0 (Ethicon, UK) and Ethibond 3/0 (Excel, Johnson and Johnson, UK) were tested. Half of the sutures in each group were manually pre-tensioned prior to knot tying and half were knotted without pre-tensioning. All knots were standard surgical knots with six throws. The suture lengths were measured before and after a standardised cyclical loading regime on an Instron tensile tester. The regime was designed to represent the finger flexion forces produced in a typical rehabilitation programme. All sutures were subsequently tested to their ultimate tensile strength.

After cyclical loading the pre-tensioned sutures demonstrated a mean increase in suture length of 0.7% (range 0.1-1.9%). The sutures not pre-tensioned showed a mean increase of 5.4% (range 3.3-7%). This equates to 87% less plastic deformation (p <0.05 Students' T-test) upon pre-tensioning. There were no differences with Ethibond. Pre-tensioning had no effect on ultimate tensile strength for either group.

Manual pre-tensioning reduces plastic deformation in Prolene 3/0 sutures without affecting the ultimate tensile strength. This simple technique could theoretically diminish gap formation at the site of a tendon repair.

Purpose: There is no consensus whether the posterior cruciate ligament (PCL)should be preserved (CR) or sacrificed (CS) during primary total knee replacement (TKR). The purpose of this study was to compare the greater than 10 year survivorship and health related outcomes of CR and CS TKRs using a single implant system.

Method: Between 1996 and 2000, 478 Genesis II Primary TKRs were inserted in 414 patients. Excluding those with a primary diagnosis other than osteoarthritis, body mass index greater then 40, history of prior patellectomy, fusion or osteotomy, 358 cases in 310 patients were included. 134 (37%) had a PCL preserving (CR) and 224 (63%) had a PCL sacrificing implant (CS). The two patient cohorts were compared for Kaplan-Meier survivorship, health-related outcomes (Knee Society scores, WOMAC, SF-12), range of motion (ROM) and radiographic loosening or wear.

Results: Mean follow-up was 11.87±1.04 years for CR and 10.96±0.87 years for CS (p=0.001). Four cases were revised for infection. No significant differences were noted between the CR and CS Genesis II cohorts at 10 year Kaplan-Meier survivorship excluding infections (CR 0.984±0.011, CS 0.986±0.008, p=0.30). Overall revisions were two for CR (1.5%, no infections) and seven for CS (1.7%, four for infection; 1.3% excluding infections). Revision rates were not significantly different between groups including or excluding infections (p=0.493 and p=1.00 respectively). CS had significantly greater postoperative ROM than CR (CS=114.20±13.60, CR=111.35±12.38, p=0.024). At 10 years, no differences were observed in satisfaction, health-related outcomes or radiographic wear/loosening. Crepitus was reported more frequently in CS design.

Conclusion: Most studies comparing PCL sacrificing (CS) versus retaining (CR) TKRs are short term. In this large, long term, single implant CR versus CS study, no differences were found in Kaplan-Meier survivorship, health-related outcomes or patient satisfaction. The CS design had more range of motion, but also a higher incidence of peripatellar crepitus than the CR design. We conclude that both CR and CS TKR designs can yield excellent long term clinical outcomes.