Introduction

The fracture healing outcome is often evaluated via ex vivo testing of the fracture callus. However, there is only a small time window, where the callus stiffness is significantly different, i.e. a delayed fracture healing might be undetected if the time point of sacrifice is improper. The aim of this study was to develop an in vivo monitoring concept, which allows determining the fracture callus stiffness in vivo over the whole healing time in rats.

A multicenter retrospective analysis of patients treated for tibial fracture was conducted to develop a score that correlates with fracture healing time and, ultimately, to identify the risk gradient of delayed healing.

The clinical records of 93 patients treated for tibial fracture at three orthopaedic centers were evaluated. Patients were considered healed when full weight bearing was allowed and no further controls were scheduled. For the purpose of our analysis, we separated patients healed within or after 180 days.

Patient's risk factors known to be associated to delay healing, as well as fracture morphology and orthopaedic treatment were recorded in an electronic Case Report Form (e-CRF). Information available in the literature was used to weight the relative risk (RR) associated to each risk factor; values were combined to calculate a score to be correlated to the fracture healing time: L-ARRCO (Literature-Algoritmo Rischio Ritardo Consolidazione Ossea). Among all information collected in e-CRFs, we identified other risk factors, associated to delayed healing, that were used to calculate a new score: ARRCO. Univariate logistic analysis was used to determine a correlation between the score and healing time. Analysis by receiver operating characteristic (ROC) and calculation of the area under the curve (AUC) were used for sensitivity and specificity.

Complete information was available for 53 patients. The mean value of the L-ARRCO score among patients healed within 180 days was 5.78 ± 1.59 and 7.05 ± 2.46 among those healed afterwards, p=0.044. The mean value of the ARRCO score of patients healed within 180 days was 5.92 ± 1.78 and 9.03 ± 2.79 among those healed afterwards, p<0.0001. The ROC curve shows an AUC of 0.62±0.09 for L-ARRCO and an AUC of 0.82±0.07 for ARRCO, (p<0.0001).

We have shown that the ARRCO score value is significantly correlated to fracture healing time. The score may be used to identify fractures at risk of delayed healing, thus allowing surgeon's early intervention to stimulate osteogenesis.

Introduction

The hematoma occurring at a fracture site is known to play an important role in fracture healing. Previously, we demonstrated that fracture hematoma contained multilineage mesenchymal progenitor cells. On the other hand, the process of fracture healing is associated by two different mechanisms, intramembranous and endochondral. However, there are no reports proving the details about cellular analysis in the process of endochondoral ossification.

In the assessment of fracture healing by monitoring stiffness with vibrational analysis or instrumented external fixators, it has been assumed that there is a workable correlation between stiffness and strength. We used four-point bending tests to study time-related changes in stiffness and strength in healing tibial fractures in sheep. We aimed to test the validity of the measurement of stiffness to assess fracture strength.

At each duration of healing examined, we found marked variations in stiffness and strength. Stiffness was shown to be load-dependent: measurements at higher loads reflected ultimate strength more accurately. There was a biphasic relationship between stiffness and strength: at first there was a strong correlation regardless of loading conditions, but in the second phase, which included the period of ‘clinical healing’, stiffness and strength were not significantly correlated.

We conclude that the monitoring of stiffness is useful primarily in assessing progress towards union but is inherently limited as an assessment of strength at the time of clinical union. Any interpretation of stiffness must take into account the load conditions.

There is evidence that fracture healing is impaired in patients with chronic immune disorders the reasons remaining unclear so far. To further elucidate the role of the immune system in bone healing, this study investigated the hypothesis that fracture healing would be considerably disturbed in a mouse model with severe defects of the innate as well as adaptive immune system.

Immune deficient Nod-scidIL2Rγ^null and immune competent BALBcByJ mice were used (12 weeks, male, each n=24). The mice received a femur osteotomy stabilized by an external fixator and were sacrificed at d 21, 28, and 35. The calli were evaluated by three-point-bending testing, μCT and histomorphometry.

The flexural rigidity of the callus did not significantly differ between both genotypes after 21 and 28 days but was significantly lower in Nod-scidIL2Rγ^null mice after 35 days (31%). The maximum moment of inertia was significantly increased after 21 days (by 34%), and the callus cross section area after 21, 28 and 35 days in Nod-scidIL2Rγ^null mice. BV/TV of the callus of Nod-scidIL2Rγ^null mice was significantly decreased after 28 and 35 days (by 32% and 41%). The histological evaluation showed a significantly enhanced amount of cartilage in the fracture gap of Nod-scidIL2Rγ^null mice.

These data indicate an only moderate delay in fracture healing in Nod-scidIL2Rγ^null mice suffering on severe defects in innate and adaptive immune response.

Since osteoimmunology is gaining increasingly interest and evidence for involvement of complement in bone biology was found, the role of complement in bone biology and fracture healing was evaluated.

After characterizing the bone phenotype, a fracture healing experiment with C3- and C5- deficient mice was performed. After osteotomy of the right femur and external fixation, healing was analyzed after 1, 3, 7 and 21 days. Bone characterization revealed a reduced number of osteoclasts in C5-deficient animals with a significantly reduced resorption activity. While bone mineral density was significantly higher in complement-deficient strains, stiffness was significantly reduced. After 21 days of fracture healing, C5-deficient animals showed reduced stiffness and a smaller callus volume compared to controls. Interestingly, C3- more than C5-deficient animals showed reduced bone formation. Altogether, bone phenotype of complement-deficient animals resembles a mild form of osteopetrosis.

This might be due to the resorption defect seen in C5-deficient mice. A reason for the minor involvement of C3-deficient mice compared to the C5-deficient animals could be the cross-talk between the coagulation cascade with side activation of complement factor C5 by thrombin.

These results indicate for the first time an essential role of complement in bone biology and fracture healing. Future studies should focus on the molecular basis of complement involvement and the osteoclastic resorption defect.

There is evidence that fracture healing is delayed in severely injured patients. We recently demonstrated that a blunt chest trauma, which induced posttraumatic systemic inflammation, considerably impaired fracture healing in rats. Because the complement anaphylatoxin C5a is an important trigger of systemic inflammation, we tested the hypothesis, whether the impairment of fracture healing observed after a severe trauma resulted from systemically activated complement.

16 male Wistar rats received a thoracic trauma and a femur osteotomy stabilized by an external fixator. Immediately and 12 h after the trauma, half of the animals received a C5aR-antagonist to prevent the C5a-dependent systemic inflammation. Control rats received a nonsense peptide, which does not provoke any biological effect. The animals were killed after 35 days and the calli were analyzed by three point bending testing, μCT and histomorphometry. Statistics: Mann-Whitney U test, level of significance to p<0.05.

The treatment with the C5aR-antagonist increased flexural rigidity significantly by 55%, improved bony bridging of the fracture gap and led to a slightly larger and qualitatively improved callus as evaluated by μCT and histological measurements.

This study shows, that the immunomodulation by a C5aR-antagonist significantly reduced the deleterious effects of a thoracic trauma on fracture healing. C5a could possibly represent a target to prevent delayed bone healing in patients with severe trauma.

Introduction

Failures in fracture healing are mainly caused by a lack of neovascularization. We have previously demonstrated that G-CSF-mobilized peripheral blood (GM-PB) CD34+ cells, an endothelial progenitor enriched cell population, contributed to fracture healing via vasculogenesis and osteogenesis. We postulated the hypothesis that local transplantation of culture expanded bone marrow (cEx-BM) CD34+ cells could exhibit therapeutic potential for fracture healing.

Introduction

Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown.

Clinical evidence that patients with type 2 diabetes mellitus (T2DM) have increased risk of fractures is reported. Furthermore, thiazolidinediones, used to treat T2DM increases the risk of secondary osteoporosis & subsequent fractures. The osteogenic potency of metformin is reported in vitro, few studies have investigated the effects of metformin on bone mass and fracture healing in vivo. We aimed to investigate the effects of metformin on fracture healing in vivo.

In polytrauma patients invasive surgeries can potentiate the posttraumatic systemic inflammation thus increasing the risk of multi organ dysfunction. Therefore, fractures are initially treated by external fixators, which later are replaced by intramedullary nails. We showed that a severe trauma impaired the healing of fractures stabilized by external fixation. Here we studied, whether the conversion to an intramedullary nail increases posttraumatic inflammation and leads to further impairment of healing.

44 rats received a femur osteotomy stabilized by an external fixator (FixEx). Half of the rats underwent a thoracic trauma (TXT) at the same time. After 4 days the fixator was replaced by an intramedullary nail (IMN) in half of the rats of each group. The rats were killed after 40 and 47 days. C5a serum levels were measured 0, 6, 24, and 72h after the 1st as well as the 2nd surgery. The calli were evaluated by three-point-bending test, μCT and histomorphometry.

The TXT significantly increased serum C5a levels after the 2nd surgical intervention. After 40 days the switch from FixEx to IMN significantly decreased bending stiffness in rats with and without TXT. After 47 days flexural rigidity in rats subjected to conversion was significantly decreased compared to rats treated only with a FixEx, particularly in combination with TXT.

This study showed that after a severe trauma the conversion of the fixation could provoke a second hit and contribute to delayed fracture healing.

In fractures electrical currents generated by piezoelectric and junctional diode effects initiate and augment healing. Conductive fixators may interfere with these currents causing delayed/nonunion which can be avoided by non conductive fixators, facilitating osteosynthesis. Null hypothesis of no difference and two tailed alternate hypothesis of any could be better was used. Impugn change in Electrical properties for demarcating union rate.

Patients of Gustillo's grade I and II tibia fractures were randomised in conductive and non conductive fixator groups in a blinded manner. Electrical and clinico-radiological properties were compared every two weeks for 20 weeks, recoding magnitude and significant difference. Capacitance(p=0.03), Impedance(p=0.002), Inductance(p=0.01) and Reactance(p=0.02) are the electrical parameters which not only demarcated union rate but orchestrated diagnosis of fracture healing. In Non-conducting group, after removal of fixator at week 10, Local Tenderness was consistently absent, Rust Score was higher at week 18 and 20(p=0.01), absence of abnormal mobility was 58% higher and 100% at week 12(p < 0.05), Presence of weight bearing was higher from week 16(OR=15, p=0.03), presence of transmitted movement was 2.4 times higher at week 10(95% CI=0.17–34.93, p=0.52) and was 100% at week 14.

Fractures heal at a faster rate if fixed by insulated non-conducting external fixators. Electrical parameters can be used to demarcate and monitor fracture healing.

Introduction

Regenerative medicine is a rapidly expanding discipline. However due to a lack of validated outcome measures, clinical trials have been far few. This study aims to assess the validity, inter-observer reliability and intra-observer reproducibility of experimental fracture healing assessment on plain radiographies. This technique involves implantation of mesenchymal stem cell (MSC) seeded constructs on only one side of the fracture after randomisation.

Secondary fracture healing processes are strongly influenced by interfragmentary motion. Shear movement is assumed to be more critical than axial movement, however experimental results are controversial. Numerical fracture healing models allow to simulate the fracture healing process with variation of single input parameters and under comparable normalized mechanical conditions. Therefore, a direct comparison of different in vivo scenarios is possible. The aim of this study was to simulate fracture healing under several axial and shear movement scenarios and compare their respective time to heal. We hypothesize that shear movement is always more critical than axial loading. For the presented study, we used a corroborated numerical model for fracture healing in sheep. Numerous variations of the movement amplitude, the fracture gap size and the musculoskeletal loads were simulated for comparable axial compressive and shear load cases. In all simulated cases, axial compressive load had less inhibitory influences on the healing process than shear load. Therefore, shear loading is more critical for the fracture healing outcome in general. Thus, our findings suggest osteosynthesis implants to be optimized to limit shear movements under musculoskeletal loading.