In vivo bone regeneration using a novel porous bioactive composite

Many commercial bone graft substitutes (BGS) and experimental bone tissue engineering scaffolds have been developed for bone repair and regeneration. This study reports the in vivo bone regeneration using a newly developed porous bioactive and resorbable composite that is composed of bioactive glass (BG), collagen (COL), hyaluronic acid (HYA) and phosphatidylserine (PS), BG-COL-HYA-PS. The composite was prepared by a combination of sol-gel and freeze-drying methods. A rabbit radius defect model was used to evaluate bone regeneration at time points of 2, 4 and 8 weeks. Techniques including radiography, histology, and micro-CT were applied to characterize the new bone formation. 8 weeks results showed that (1) nearly complete bone regeneration was achieved for the BG-COL-HYA-PS composite that was combined with a bovine bone morphogenetic protein (BMP); (2) partial bone regeneration was achieved for the BG-COL-HYA-PS composites alone; and (3) control remained empty. This study demonstrated that the novel BG-COL-HYA-PS, with or without the grafting of BMP incorporation, is a promising BGS or a tissue engineering scaffold for non-load bearing orthopaedic applications.     

Some considerations on biomaterials and bone

Osteoinduction is a property not traditionally attributed to Calcium Phosphate ceramics. Histologic, SEM and X-ray microanalyses of a biopsy of pulmonary alveolar microlithiasis allow to discredit this opinion. Bone, even lamellar type, was ectopically formed on microliths undergoing osteoclastic erosion. The SEM and X-ray microanalyses of coral granules implanted in humans indicate an osteoconductive property for both Calcium and Phosphorus. Analysis of in vitro allows to propose an enhancement of the osteocapability of coral. Lamellar bone formation in the near absence of loads undermines the opinion which sees a correlation between lamellar bone and mechanical loads. Analysis of the bone surrounding an uncemented titanium hip prosthesis highlights that both remodeled and newly formed bone have lamellae oriented parallel to prosthesis surfaces, i.e. orthogonal to loads, as opposed to that of lamellar bone of osteons which are oriented parallel to loads. Analysis of longitudinal sections of cortical bone under polarized light points out that lamellae are displaced parallel to the cement line surface both in the conic end of osteons and in Volkman's canals with thick wall, i.e. undergoing sloped load directions. In conclusion, there may be a relationship between lamellae formation and gravity.     

A method for vibrational assessment of cortical bone

Large bones from many anatomical locations of the human skeleton consist of an outer shaft (cortex) surrounding a highly porous internal region (trabecular bone) whose structure is reminiscent of a disordered cubic network. Age related degradation of cortical and trabecular bone takes different forms. Trabecular bone weakens primarily by loss of connectivity of the porous network, and recent studies have shown that vibrational response can be used to obtain reliable estimates for loss of its strength. In contrast, cortical bone degrades via the accumulation of long fractures and changes in the level of mineralization of the bone tissue. In this paper, we model cortical bone by an initially solid specimen with uniform density to which long fractures are introduced; we find that, as in the case of trabecular bone, vibrational assessment provides more reliable estimates of residual strength in cortical bone than is possible using measurements of density or porosity.     

Ultrasonic non-destructive measurements of cortical bone thickness in human cadaver femur

The knowledge of the cortical bone thickness profile in human bone has a two-fold clinical significance: to study the stress occurring in a loaded bone structure to optimize the design of prostheses; and to predict the onset of advanced bone disease such as osteoporosis. In this study, the cortical bone thickness in three embalmed human cadaver femora were measured non-destructively using an ultrasonic technique. These thickness measurements were also made using a computed tomographic (CT) scanning method. Subsequently bones were sectioned and the actual bone thicknesses in the same regions were measured using a micrometer. The correlation coefficient between the actual thickness and the ultrasonically measured thickness was 0.95 and with the CT was 0.62. Thus, these results show that, under present experimental conditions, ultrasonic thickness measurements compare well with the micrometer actual thickness results. This technique, when fully developed, can be used as a non-destructive tool for quantitative cortical bone thickness measurements. Moreover, the ultrasonic technique does not use ionizing radiation.     

Feasibility of bone assessment with leaky Lamb waves in bone phantoms and a bovine tibia

In this study, the effect of cortical thickness variation on the propagation of leaky Lamb waves is investigated by using an axial transmission technique commonly used to characterize long bones. Three Lucite plates with thicknesses of 1, 3, and 5 mm as bone phantoms and one bovine tibia with a cortical thickness of 2 mm were used at various low frequencies. Experimental measurements in bone phantoms show that the peak frequency and amplitude of excited Lamb modes strongly depend on the thickness of the Lucite plate. In the bovine tibia, the S0 and A0 Lamb modes are consistently observed in the frequency-thickness region from 0.2 to 1.0 MHz mm, and can be effectively launched at a frequency of 200 kHz, suggesting 200 kHz to be the optimal signal frequency for in vivo clinical applications. It can be also seen that both modes are affected by the frequency-thickness product, but the effect is greater for the A0 mode. Hence, the A0 Lamb mode seems more sensitive to cortical thickness change due to aging and osteoporosis. This study suggests that the use of leaky Lamb waves is feasible for ultrasonic bone assessment.     

Bone repair using mineral trioxide aggregate combined to a material carrier, associated or not with calcium hydroxide in bone defects

Mineral trioxide aggregate (MTA) is a powder aggregate containing mineral oxides with a good biological action and may facilitate the regeneration of the periodontal ligament and formation of bone. Calcium hydroxide demonstrates antibacterial properties, enhances tissue dissolution, and induces bone formation. The objective of this study was to evaluate the MTA in the bone healing process and verify if the calcium hydroxide P.A. can improve and accelerate this process. It was used forty male Wistar rats, which were divided into two groups, considering or not the use of calcium hydroxide P.A. solution before treatment. Thus, each one of these groups was divided in four groups with five animals each, according to the treatment and the defect filled by: animal's coagulum, monoolein gel, MTA in aqueous solution, and MTA combined with monoolein gel. After 10 days, the animals were perfused and the right hemimandibles removed for histological analysis. Statistical analysis of the data showed significant difference between all analyzed groups when it was made comparisons using or not calcium hydroxide P.A. (p<0.0001). There was found statistical difference between the groups that was inserted or not MTA, independently the calcium hydroxide application (p<0.05). Results showed that the MTA used was able to induce bone regeneration and had its action optimized when combined to calcium hydroxide P.A.     

Qualitative and quantitative ultrashort echo time (UTE) imaging of cortical bone

We describe the use of two-dimensional ultrashort echo time (2D UTE) sequences with minimum TEs of 8 μs to image and quantify cortical bone on a clinical 3T scanner. An adiabatic inversion pulse was used for long T(2) water and fat signal suppression. Adiabatic inversion prepared UTE acquisitions with varying TEs were used for T(2) measurement. Saturation recovery UTE acquisitions were used for T(1) measurement. Bone water concentration was measured with the aid of an external reference phantom. UTE techniques were evaluated on cadaveric specimens and healthy volunteers. A signal-to-noise ratio of around 30, contrast-to-noise ratio of around 27/20 between bone and muscle/fat were achieved in tibia in vivo with a nominal voxel size of 0.23 × 0.23 × 6.0 mm(3) in a scan time of 5 min. A mean T(1) of 223 ± 11 ms and mean T(2) of 390 ± 19 μs were found. Mean bone water concentrations of 23.3 ± 1.6% with UTE and 21.7 ± 1.3% with adiabatic inversion prepared UTE sequences were found in tibia in five normal volunteers. The results show that in vivo qualitative and quantitative evaluation of cortical bone is feasible with 2D UTE sequences.     

Multi-frequency axial transmission bone ultrasonometer

The last decade has seen a surge in the development of axial transmission QUS (Quantitative UltraSound) technologies for the assessment of long bones using various modes of acoustic waves. The condition of cortical bones and the development of osteoporosis are determined by numerous mechanical, micro-structural, and geometrical or macro-structural bone properties like hardness, porosity and cortical thickness. Such complex manifestations of osteoporosis require the evaluation of multiple parameters with different sensitivities to the various properties of bone that are affected by the disease. This objective may be achieved by using a multi-frequency ultrasonic examination The ratio of the acoustic wavelength to the cortical thickness can be changed by varying the frequency of the ultrasonic pulse propagating through the long bone that results in the change in composition of the induced wave comprised of a set of numerous modes of guided, longitudinal, and surface acoustic waves. The multi-frequency axial transmission QUS method developed at Artann Laboratories (Trenton, NJ) is implemented in the Bone Ultrasonic Scanner (BUSS). In the current version of the BUSS, a train of ultrasonic pulses with 60, 100, 400, 800, and 1200 kHz frequencies is used. The developed technology was tested on a variety of bone phantoms simulating normal, osteopenic, and osteoporotic bones. The results of this study confirm the feasibility of the multi-frequency approach for the assessment of the processes leading to osteoporosis.     

Ultrastructural observation on genesis and morphology of cortical granules in Macrobrachium nipponense (Crustacea,Caridea)

Cortical granules are secretory vesicles in oocytes that develop from the Golgi complex. In the freshwater shrimp, Macrobrachium nipponense, mitochondria participates in the formation of cortical granules. We investigated the structural changes of mitochondria and the distribution cortical granules in different stages of oocyte development. Transmission electron microscopy provided evidence for the involvement of mitochondria and a particular spiral lamellar organization and an electron-lucent area in internal cortical granules. The ooplasm provided material for the cortical granules in early oocyte development. We demonstrated that mitochondria play a role in coalescence and maturation of cortical granules in this species. Additionally, a concept of cortical granules regarded as a functional integration is put forward. The genesis of shrimp cortical granules exhibited a particular pathway of maturation. The outer shape and inner organization considering different taxa suggested general as well as specific features of the development of cortical granules.     

Two-wave behavior under various conditions of transition area from cancellous bone to cortical bone

The two-wave phenomenon, the wave separation of a single ultrasonic pulse in cancellous bone, is expected to be a useful tool for the diagnosis of osteoporosis. However, because actual bone has a complicated structure, precise studies on the effect of transition conditions between cortical and cancellous parts are required. This study investigated how the transition condition influenced the two-wave generation using three-dimensional X-ray CT images of an equine radius and a three-dimensional simulation technique. As a result, any changes in the boundary between cortical part and trabecular part, which gives the actual complex structure of bone, did not eliminate the generation of either the primary wave or the secondary wave at least in the condition of clear trabecular alignment. The results led us to the possibility of using the two-wave phenomenon in a diagnostic system for osteoporosis in cases of a complex boundary.     

The TEM characterization of the lamellar structure of osteoporotic human trabecular bone

The lamellar structure of osteoporotic human trabecular bone was characterized experimentally by means of transmission electron microscopy (TEM). More specifically, the TEM was used to determine if trabecular bone exhibits similar lamellar structural motifs as cortical bone by analyzing unmineralized, mineralized and demineralized bone, and to study the influence of the osteocyte network on the lamellar structure of osteoporotic trabecular bone. Comparison with normal trabecular bone is included. This paper summarizes partial results of a larger study, which addressed the characterization of the hierarchical structure of normal versus osteoporotic human trabecular bone [Rubin, M.A., 2001. Multiscale characterization of the ultrastructure of trabecular bone in osteoporotic and normal humans and in two inbred strains of mice. MS Thesis, Georgia Institute of Technology.] at several structural scales.     

Experimental and numerical analysis of Izod impact test of cortical bone tissue

Bones can only sustain loads until a certain limit, beyond which they fail. Usually, the reasons for bone fracture are traumatic falls, sports injuries, and engagement in transport or industrial accidents. A proper treatment of bones and prevention of their fracture can be supported by in-depth understanding of deformation and fracture behavior of this tissue in such dynamic events. In this paper, a combination of experimental and numerical analysis was carried out in order to comprehend the fracture behavior of cortical bone tissue. Experimental tests were performed to study the transient dynamic behavior of cortical bone tissue under impact bending loading. The variability of absorbed energy for different cortex positions and notch depths was studied using Izod impact tests. Also, Extended Finite-Element Method implemented into the commercial finite-element software Abaqus was used to simulate the crack initiation and growth processes in a cantilever beam of cortical bone exposed to impact loading using the Izod loading scheme. The simulation results show a good agreement with the experimental data.     

Propagation of two longitudinal waves in a cancellous bone with the closed pore boundary

Ultrasound propagation in cancellous bone (porous media) under the condition of closed pore boundaries was investigated. A cancellous bone and two plate-like cortical bones obtained from a racehorse were prepared. A water-immersion ultrasound technique in the MHz range and a three-dimensional elastic finite-difference time-domain (FDTD) method were used to investigate the waves. The experiments and simulations showed a clear separation of the incident longitudinal wave into fast and slow waves. The findings advance the evaluation of bones based on the two-wave phenomenon for in vivo assessment.     

Three-dimensional adiabatic inversion recovery prepared ultrashort echo time cones (3D IR-UTE-Cones) imaging of cortical bone in the hip

PurposeWe present three-dimensional adiabatic inversion recovery prepared ultrashort echo time Cones (3D IR-UTE-Cones) imaging of cortical bone in the hip of healthy volunteers using a clinical 3T scanner.MethodsA 3D IR-UTE-Cones sequence, based on a short pulse excitation followed by a 3D Cones trajectory, with a nominal TE of 32 μs, was employed for high contrast morphological imaging of cortical bone in the hip of heathy volunteers. Signals from soft tissues such as muscle and marrow fat were suppressed via adiabatic inversion and signal nulling. T₂^⁎ value of the cortical bone was also calculated based on 3D IR-UTE-Cones acquisitions with a series of TEs ranging from 0.032 to 0.8 ms. A total of four healthy volunteers were recruited for this study. Average T₂^⁎ values and the standard deviation for four regions of interests (ROIs) at the greater trochanter, the femoral neck, the femoral head and the lesser trochanter were calculated.ResultsThe 3D IR-UTE-Cones sequence provided efficient suppression of soft tissues with excellent image contrast for cortical bone visualization in all volunteer hips. Exponential single component decay was observed for all ROIs, with averaged T₂^⁎ values ranging from 0.33 to 0.45 ms, largely consistent with previously reported T₂^⁎ values of cortical bone in the tibial midshaft.ConclusionsThe 3D IR-UTE-Cones sequence allows in vivo volumetric imaging and quantitative T₂^⁎ measurement of cortical bone in the hip using a clinical 3T scanner.     

Water proton density in human cortical bone obtained from ultrashort echo time (UTE) MRI predicts bone microstructural properties

PurposeTo investigate the correlations between cortical bone microstructural properties and total water proton density (TWPD) obtained from three-dimensional ultrashort echo time Cones (3D-UTE-Cones) magnetic resonance imaging techniques.Materials and methods135 cortical bone samples were harvested from human tibial and femoral midshafts of 37 donors (61 ± 24 years old). Samples were scanned using 3D-UTE-Cones sequences on a clinical 3T MRI and on a high-resolution micro-computed tomography (μCT) scanner. TWPD was measured using 3D-UTE-Cones MR images. Average bone porosity, pore size, and bone mineral density (BMD) were measured from μCT images at 9 μm voxel size. Pearson's correlation coefficients between TWPD and μCT-based measures were calculated.ResultsTWPD showed significant moderate correlation with both average bone porosity (R = 0.66, p < 0.01) and pore size (R = 0.57, p < 0.01). TWPD also showed significant strong correction with BMD (R = 0.71, p < 0.01).ConclusionsThe presented 3D-UTE-Cones imaging technique allows assessment of TWPD in human cortical bone. This quick UTE-MRI-based technique was capable of predicting bone microstructure differences with significant correlations. Such correlations highlight the potential of UTE-MRI-based measurement of bone water proton density to assess bone microstructure.     

Proton magnetic relaxation in bone marrow related to age and bone mineral density: low-resolution in vitro studies.

Detailed analysis of proton spin-spin and spin-lattice relaxation behaviors of the bone marrow in the presence of trabecular bone network was performed at low-resolution (B(0) = 0.496T) on rat vertebrae specimens deprived of spinal cord. Two groups of samples, from young and old healthy animals, were investigated before cellular necrosis had started. BMD measurements were carried out to quantify the expected age-related modifications of the trabecular bone network. 1H-MR measurements were also performed on the same samples, deprived of marrow and saturated with water, in order to control the validity of a possible interpretation of the marrow 1H-MR characteristics, in terms of marrow components, and to investigate the possible employment of these samples to study the trabecular bone network properties. We pointed out that: 1) a bimodal distribution of T(2i) and T(1i) values (distinguishing "fast" and "slow" relaxations) describes satisfactorily all the 1H-MR experimental decays; 2) age-related modifications of the trabecular bone network are marked by correlate variations of the BMD value and of the proton spin-spin relaxation rates in water saturated samples; 3) age-related modifications of marrow are underlined by variations of the average value of the "fast" T(2i) and of the "slow" T(1i) relaxation time distributions, which could be attributed to the marrow components different from the fat granules of the adipose cells.Our results suggest that studies in vitro on bone tissue, by 1H-MR techniques at low-resolution, may contribute to a better bone function characterization and, therefore, to a better clinical utilization of MRI techniques.     

长骨中振动声激发超声导波的方法

为了实现一定频段内任意低频下在长骨中激励导波信号,本文提出一种采用聚焦高频(5 MHz)超声换能器在长骨皮质骨中激发低频(150 kHz)超声导波的振动声方法.首先介绍了板状超声导波理论和双声束共聚焦法与单声束调幅法激发振动声的基本原理;进而采用三维有限元仿真方法分析振动声激发低频超声导波的基本现象,然后结合牛胫骨板离体实验,验证振动声激发低频超声导波的可行性.结果均表明,双声束共焦与单声束振动超声均可在骨板中激发低频超声导波.相关研究方法有助于提高空间域长骨中超声导波测量精度,以及在一定频段内实现任意频率激励等,对发展低频超声导波在体测量长骨皮质骨的新技术具有一定的指导意义.     

Quantifying cortical bone free water using short echo time (STE-MRI) at 1.5 T

PurposeThe purpose of our study was to use Dual-TR STE-MR protocol as a clinical tool for cortical bone free water quantification at 1.5 T and validate it by comparing the obtained results (MR-derived results) with dehydration results.MethodsHuman studies were compliant with HIPPA and were approved by the institutional review board. Short Echo Time (STE) MR imaging with different Repetition Times (TRs) was used for quantification of cortical bone free water T₁ (T₁free) and concentration (ρ_free). The proposed strategy was compared with the dehydration technique in seven bovine cortical bone samples. The agreement between the two methods was quantified by using Bland and Altman analysis. Then we applied the technique on a cross-sectional population of thirty healthy volunteers (18F/12M) and examined the association of the biomarkers with age.ResultsThe mean values of ρ_free for bovine cortical bone specimens were quantified as 4.37% and 5.34% by using STE-MR and dehydration techniques, respectively. The Bland and Altman analysis showed good agreement between the two methods along with the suggestion of 0.99% bias between them. Strong correlations were also reported between ρ_free (r² = 0.62) and T_1free and age (r² = 0.8). The reproducibility of the method, evaluated in eight subjects, yielded an intra-class correlation of 0.95.ConclusionSTE-MR imaging with dual-TR strategy is a clinical solution for quantifying cortical bone ρ_free and T_1free.     

An ultrasonic method for measuring the elastic properties of human tibial cortical and cancellous bone

Ultrasonic techniques have been used to measure the elastic properties of bone. Eight human tibiae were used to determine and map the elastic properties of cortical and cancellous bone. The present study shows cortical bone to be at least orthotropic in its material symmetry. The mechanical properties of cortical bone are more homogeneous along the length than around the circumference. The variations in the properties around the quadrant of cortical bone are small, less than 10%, while differences in the properties around the circumference of cancellous bone are more apparent, approximately 5 times those of cortical bone. The elastic properties of cancellous bone exhibited inhomogeneity and some consistency pattern along both the length and the circumference.     

超声导波评价长骨骨折的仿体实验研究

定量超声评价长骨骨折已成为近年来的研究热点之一,超声导波模式转换理论可被应用于长骨骨折状况的评价。采用三维时域有限差分法分析骨折长骨中导波模式转换的基本规律,提取模式转换定量评价参数;进而结合亚克力管仿体实验,分析不同裂纹程度下导波模式的转换情况。仿真与仿体实验表明,导波模式能量参数与裂纹程度具有很好的相关性,可用于评价长骨骨折状况。