an-efficient-reproducible-protocol-for-distraction-osteogenesis-rat

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Aix Marseille Univ

APHM

As a determinant of skeletal fragility, the organic matrix is responsible for the post-yield and creep behavior of bone and for its toughness, while the mineral apatite acts on stiffness. Specific to the fibula and ulna in children, greenstick fractures show a plastic in vivo mechanical behavior before bone fracture. During growth, the immature form of collagen enzymatic cross-links gradually decreases, to be replaced by the mature form until adolescence, subsequently remaining constant throughout adult life. However, the link between the cortical bone organic matrix and greenstick fractures in children remains to be explored. Here, we sought to determine: 1) whether plastic bending fractures can occur in vitro, by testing cortical bone samples from children's fibula and 2) whether the post-yield behavior (ωp plastic energy) of cortical bone before fracture is related to total quantity of the collagen matrix, or to the quantity of mature and immature enzymatic cross-links and the quantity of non-enzymatic cross-links. We used a two-step approach; first, a 3-point microbending device tested 22 fibula machined bone samples from 7 children and 3 elderly adults until fracture. Second, biochemical analysis by HPLC was performed on the sample fragments. When pooling two groups of donors, children and elderly adults, results show a rank correlation between total energy dissipated before fracture and age and a linear correlation between plastic energy dissipated before fracture and ratio of immature/mature cross-links. A collagen matrix with more immature cross-links (i.e. a higher immature/mature cross-link ratio) is more likely to plastically deform before fracture. We conclude that this ratio in the sub-nanostructure of the organic matrix in cortical bone from the fibula may go some way towards explaining the variance in post-yield behavior. From a clinical point of view, therefore, our results provide a potential explanation of the presence of greenstick fractures in children.

Ratio between mature and immature enzymatic cross-links correlates with post-yield cortical bone behavior: An insight into greenstick fractures of the child fibula.

Various mathematical models represent the effects of local mechanical environment on the regulation of skeletal regeneration. Their relevance relies on an accurate description of the evolving mechanical properties of the regenerating tissue. The object of this study was to develop an experimental model which made it possible to characterize the temporal evolution of the structural and mechanical properties during unloaded enchondral osteogenesis in the New Zealand rabbit, a standard animal model for studies of osteogenesis and chondrogenesis. A 25 mm segment of tibial diaphysis was removed sub-periosteally from rabbits. The defect was repaired by the preserved periosteum. An external fixator was applied to prevent mechanical loading during osteogenesis. The regenerated skeletal tissues were studied by CT scan, histology and mechanical tests. The traction tests between 7 and 21 days post-surgery were done on formaldehyde-fixated tissue allowing to obtain force/displacement curves. The viscoelastic properties of the regenerating skeletal tissues were visualized throughout the repair process.

Temporal evolution of mechanical properties of skeletal tissue regeneration in rabbits: an experimental study.

The objective here was to experimentally characterize the temporal evolution of the structural and mechanical properties of large volume immature regenerated tissues. We studied these evolving tissues from their genesis in controlled mechanical conditions. We developed an animal model based on the periosteal properties leading to unloaded regenerated skeletal tissue. To characterize the temporal evolution of mechanical properties, we carried out indentation tests coupled with macroscopic examinations and histological studies. This combined methodology yielded a range of information on osteogenesis at different scales: macroscopic by simple observation, mesoscopic by indentation test and microscopic by histological study. Results allowed us to identify different periods, providing a link between biological changes and material property evolution in bone tissue regeneration. The regenerated tissue evolves from a viscous, homogeneous, soft material to a heterogeneous stiffer material endowed with a lower viscosity. From a biological point of view, cell organization progresses from a proliferated cell clot to a mature structure closer to that of the bone. During the first 7 days, mechanical and biological results revealed the same evolution: first, the regenerated tissue grew, then, differentiated into an osteochondral tissue and finally calcification began. While our biological results confirm those of other studies, our mechanical results provide the first experimental mechanical characterization by reduced Young's modulus of such tissue.

Temporal evolution of skeletal regenerated tissue: what can mechanical investigation add to biological?

Adolescent idiopathic scoliosis (AIS) causes a spine and rib cage three-dimensional (3D) deformity previously treated by bracing. Whatever the manufacturing process, this rigid system acts biomechanically on the patient through the "three-point bending" mechanical principle. It applies corrective forces to a limited area and acts especially in the frontal plane. It seemed to us that a nonrigid system, called "Cbrace," with 3D action allowing distribution of forces could increase compliance and provide better long-term correction prospects.

Beyond the classic correction system: a numerical nonrigid approach to the scoliosis brace.

Fracture of the proximal femur is a major public health problem in elderly persons. It has recently been suggested that combining texture analysis and bone mineral density measurement improves the failure load prediction in human femurs. In this study, we aimed to compare bone texture analysis with three-dimensional (3D) microarchitecture and mechanical properties of trabecular bone in osteoporotic femurs. Eight femoral heads from osteoporotic patients who fractured their femoral neck provided 31 bone cores. Bone samples were studied using a new high-resolution digital X-ray device (BMA™, D3A Medical Systems) allowing for texture analysis with fractal parameter H (mean), and were examined using micro-computed tomography (microCT) for 3D microarchitecture. Finally, uniaxial compression tests to failure were performed to estimate failure load and apparent modulus of bone samples. The fractal parameter H (mean) was strongly correlated with bone volume fraction (BV/TV) (r = 0.84) and trabecular thickness (Tb.Th) (r = 0.91) (p < 0.01). H (mean) was also markedly correlated with failure load (r = 0.84) and apparent modulus (r = 0.71) of core samples (p < 0.01). Bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) demonstrated significant correlations with failure load (r = 0.85 and 0.72, respectively) and apparent modulus (r = 0.72 and 0.64, respectively) (p < 0.01). Overall, the best predictors of failure load were H (mean), bone volume fraction, and trabecular thickness, with r (2) coefficients of 0.83, 0.76, and 0.80 respectively. This study shows that the fractal parameter H (mean) is correlated with 3D microCT parameters and mechanical properties of femoral head bone samples, which suggests that radiographic texture analysis is a suitable approach for trabecular bone microarchitecture assessment in osteoporotic femurs.

Bone texture analysis is correlated with three-dimensional microarchitecture and mechanical properties of trabecular bone in osteoporotic femurs.

Compression biomechanical tests using fresh cadaveric thoracolumbar motion segments.

Radiographical texture analysis improves the prediction of vertebral fracture: an ex vivo biomechanical study.

The assessment of elastic properties in children's cortical bone is a major challenge for biomechanical engineering community, more widely for health care professionals. Even with classical clinical modalities such as X-ray tomography, MRI, and/or echography, inappropriate diagnosis can result from the lack of reference values for children bone. This study provides values for elastic properties of cortical bone in children using ultrasonic and mechanical measurements, and compares them with adult values. 18 fibula samples from 8 children (5-16 years old, mean age 10.6 years old ±4.4) were compared to 16 fibula samples from 3 elderly adults (more than 65 years old). First, the dynamic modulus of elasticity (Edyn) and Poisson's ratio (ν) are evaluated via an ultrasonic method. Second, the static modulus of elasticity (Esta) is estimated from a 3-point microbending test. The mean values of longitudinal and transverse wave velocities measured at 10 MHz for the children's samples are respectively 3.2mm/μs (±0.5) and 1.8mm/μs (±0.1); for the elderly adults' samples, velocities are respectively 3.5mm/μs (±0.2) and 1.9 mm/μs (±0.09). The mean Edyn and the mean Esta for the children's samples are respectively 15.5 GPa (±3.4) and 9.1 GPa (±3.5); for the elderly adults' samples, they are respectively 16.7 GPa (±1.9) and 5.8 GPa (±2.1). Edyn, ν and Esta are in the same range for children's and elderly adults' bone without any parametric statistical difference; a ranking correlation between Edyn and Esta is shown for the first time.

In vitro ultrasonic and mechanic characterization of the modulus of elasticity of children cortical bone.

Most acetabular cups implanted today are press-fit impacted cementless. Anchorage begins with the primary stability given by insertion of a slightly oversized cup. This primary stability is key to obtaining bone ingrowth and secondary stability. We tested the hypothesis that primary stability of the cup is related to surface roughness of the implant, using both an experimental and a numerical models to analyze how three levels of surface roughness (micro, macro and combined) affect the primary stability of the cup. We also investigated the effect of differences in diameter between the cup and its substrate, and of insertion force, on the cups' primary stability. The results of our study show that primary stability depends on the surface roughness of the cup. The presence of macro-roughness on the peripheral ring is found to decrease primary stability; there was excessive abrasion of the substrate, damaging it and leading to poor primary stability. Numerical modeling indicates that oversizing the cup compared to its substrate has an impact on primary stability, as has insertion force.

Does surface roughness influence the primary stability of acetabular cups? A numerical and experimental biomechanical evaluation.

Child cortical bone tissue is rarely studied because of the difficulty of obtaining samples. Yet the preparation and ultrasonic characterization of the small samples available, while challenging, is one of the most promising ways of obtaining information on the mechanical behavior of non-pathological children׳s bone. We investigated children׳s cortical bone obtained from chirurgical waste. 22 fibula or femur samples from 21 children (1-18 years old, mean age: 9.7±5.8 years old) were compared to 16 fibula samples from 16 elderly patients (50-95 years old, mean age: 76.2±13.5 years old). Stiffness coefficients were evaluated via an ultrasonic method and anisotropy ratios were calculated as the ratio of C33/C11, C33/C22 and C11/C22. Stiffness coefficients were highly correlated with age in children (R>0.56, p<0.01). No significant difference was found between C11 and C22 for either adult or child bone (p>0.5), nor between C44 and C55 (p>0.5). We observe a transverse isotropy with C33>C22=C11>C44C55>C66. For both groups, we found no correlation between age and anisotropy ratios. This study offers the first complete analysis of stiffness coefficients in the three orthogonal bone axes in children, giving some indication of how bone anisotropy is related to age. Future perspectives include studying the effect of the structure and composition of bone on its mechanical behavior.

Analyzing the anisotropic Hooke's law for children's cortical bone.

When assessing ultrasonic measurements of material parameters, the signal processing is an important part of the inverse problem. Measurements of thickness, ultrasonic wave velocity and mass density are required for such assessments. This study investigates the feasibility and the robustness of a wavelet-based processing (WBP) method based on a Jaffard-Meyer algorithm for calculating these parameters simultaneously and independently, using one single ultrasonic signal in the reflection mode. The appropriate transmitted incident wave, correlated with the mathematical properties of the wavelet decomposition, was determined using a adapted identification procedure to build a mathematically equivalent model for the electro-acoustic system. The method was tested on three groups of samples (polyurethane resin, bone and wood) using one 1-MHz transducer. For thickness and velocity measurements, the WBP method gave a relative error lower than 1.5%. The relative errors in the mass density measurements ranged between 0.70% and 2.59%. Despite discrepancies between manufactured and biological samples, the results obtained on the three groups of samples using the WBP method in the reflection mode were remarkably consistent, indicating that it is a reliable and efficient means of simultaneously assessing the thickness and the velocity of the ultrasonic wave propagating in the medium, and the apparent mass density of material.

Measuring mass density and ultrasonic wave velocity: A wavelet-based method applied in ultrasonic reflection mode.

Cortical porosity is a major determinant of bone strength. Haversian and Volkmann׳s canals are׳seen' as pores in 2D cross-section but fashion a dynamic network of interconnected channels in 3D, a quantifiable footprint of intracortical remodeling. Given the changes in bone remodeling across life, we hypothesized that the 3D microarchitecture of the cortical pore network influences its stiffness during growth and ageing. Cubes of cortical bone of 2 mm side-length were harvested in the distal 1/3 of the fibula in 13 growing children (mean age±SD: 13±4 yrs) and 16 adults (age: 75±13 yrs). The cubes were imaged using desktop micro-CT (8.14µm isotropic voxel size). Pores were segmented as a solid to assess pore volume fraction, number, diameter, separation, connectivity and structure model index. Elastic coefficients were derived from measurements of ultrasonic bulk compression and shear wave velocities and apparent mass density. The pore volume fraction did not significantly differ between children and adults but originates from different microarchitectural patterns. Compared to children, adults had 42% (p=0.033) higher pore number that were more connected (Connective Density: +205%, p=0.001) with a 18% (p=0.007) lower pore separation. After accounting for the contribution of pore volume fraction, axial elasticity in traction-compression mode was significantly correlated with better connectivity in growing children and with pore separation among adults. The changes in intracortical remodeling across life alter the distribution, size and connectedness of the channels from which cortical void fraction originates. These alterations in pore network microarchitecture participate in changes in compressive and shear mechanical behavior, partly in a porosity-independent manner. The assessment of pore volume fraction (i.e., porosity) provides only a limited understanding of the role of cortical void volume fraction in its mechanical properties.

Pore network microarchitecture influences human cortical bone elasticity during growth and aging.

High-resolution imaging and biomechanical investigation of ex-vivo vertebrae.

Correlative Analysis of Vertebral Trabecular Bone Microarchitecture and Mechanical Properties: A Combined Ultra-high Field (7 Tesla) MRI and Biomechanical Investigation.

Article	Year
Ratio between mature and immature enzymatic cross-links correlates with post-yield cortical bone behavior: An insight into greenstick fractures of the child fibula. Bone\| PubMed ID: 26079997	2015
Temporal evolution of mechanical properties of skeletal tissue regeneration in rabbits: an experimental study. Medical & biological engineering & computing\| PubMed ID: 17694341	2007
Temporal evolution of skeletal regenerated tissue: what can mechanical investigation add to biological? Medical & biological engineering & computing\| PubMed ID: 20517709	2010
Beyond the classic correction system: a numerical nonrigid approach to the scoliosis brace. The spine journal : official journal of the North American Spine Society\| PubMed ID: 21402500	2011
Bone texture analysis is correlated with three-dimensional microarchitecture and mechanical properties of trabecular bone in osteoporotic femurs. Journal of bone and mineral metabolism\| PubMed ID: 22886379	2013
Radiographical texture analysis improves the prediction of vertebral fracture: an ex vivo biomechanical study. Spine\| PubMed ID: 23823577	2013
In vitro ultrasonic and mechanic characterization of the modulus of elasticity of children cortical bone. Ultrasonics\| PubMed ID: 24112598	2014
Does surface roughness influence the primary stability of acetabular cups? A numerical and experimental biomechanical evaluation. Medical engineering & physics\| PubMed ID: 25080896	2014
Analyzing the anisotropic Hooke's law for children's cortical bone. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 26094248	2015
Measuring mass density and ultrasonic wave velocity: A wavelet-based method applied in ultrasonic reflection mode. Ultrasonics\| PubMed ID: 26403278	2016
Pore network microarchitecture influences human cortical bone elasticity during growth and aging. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 27389322	2016
Correlative Analysis of Vertebral Trabecular Bone Microarchitecture and Mechanical Properties: A Combined Ultra-high Field (7 Tesla) MRI and Biomechanical Investigation. Spine\| PubMed ID: 28338579	2017

Martine Pithioux

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