Pulsed ultrasound for bone regeneration - outcomes and hurdles in the clinical application: a systematic review.

Impaired bone-fracture healing is associated with long-term musculoskeletal disability, pain and psychological distress. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive and side-effect-free treatment option for fresh, delayed- and non-union bone fractures, which has been used in patients since the early 1990s. Several clinical studies, however, have questioned the usefulness of the LIPUS treatment for the regeneration of long bones, including those with a compromised healing. This systematic review addresses the hurdles that the clinical application of LIPUS encounters. Low patient compliance might disguise the effects of the LIPUS therapy, as observed in several studies. Furthermore, large discrepancies in results, showing profound LIPUS effects in regeneration of small-animal bones in comparison to the clinical studies, could be caused by the suboptimal parameters of the clinical set-up. This raises the question of whether the so-called "acoustic dose" requires a thorough characterisation to reveal the mechanisms of the therapy. The adequate definition of the acoustic dose is especially important in the elderly population and patients with underlying medical conditions, where distinct biological signatures lead to a delayed regeneration. Non-industry-funded, randomised, double-blind, placebo-controlled clinical trials of the LIPUS application alone and as an adjuvant treatment for bones with complicated healing, where consistent control of patient compliance is ensured, are required.

Ultrasound palpation for fast in-situ quantification of articular cartilage stiffness, thickness and relaxation capacity.

Most current cartilage testing devices require the preparation of excised samples and therefore do not allow intra-operative application for diagnostic purposes. The gold standard during open or arthroscopic surgery is still the subjective perception of manual palpation. This work presents a new diagnostic method of ultrasound palpation (USP) to acquire applied stress and strain data during manual palpation of articular cartilage. With the proposed method, we obtain cartilage thickness and stiffness. Moreover, repeated palpations allow the quantification of relaxation effects. USP measurements on elastomer phantoms demonstrated very good repeatability for both, stage-guided (97.2%) and handheld (96.0%) applications. The USP measurements were compared with conventional indentation experiments and revealed very good agreement on elastomer phantoms ([Formula: see text]) and good agreement on porcine cartilage samples ([Formula: see text]). Artificially degenerated cartilage samples showed reduced stiffness, weak capacity to relax after palpation and an increase of stiffness of approximately 50% with each single palpation. Intact cartilage was measured by USP directly at the patella (in situ) and after excision and removal of the subchondral bone (ex situ), leading to stiffness values of [Formula: see text] and [Formula: see text] ([Formula: see text]), respectively. The results demonstrate the potential of the USP system for cartilage testing, its sensitivity to degenerative changes and as a method for quantifying relaxation processes by means of repeated palpations. Furthermore, the differences in the results of in-situ and ex-situ measurements are of general interest, since such comparison has not been reported previously. We point out the limited comparability of ex-situ cartilage with its in-situ biomechanical behavior.

Can a continuous mineral foam explain the stiffening of aged bone tissue? A micromechanical approach to mineral fusion in musculoskeletal tissues.

UNLABELLED: Recent experimental data revealed a stiffening of aged cortical bone tissue, which could not be explained by common multiscale elastic material models. We explain this data by incorporating the role of mineral fusion via a new hierarchical modeling approach exploiting the asymptotic (periodic) homogenization (AH) technique for three-dimensional linear elastic composites. We quantify for the first time the stiffening that is obtained by considering a fused mineral structure in a softer matrix in comparison with a composite having non-fused cubic mineral inclusions. We integrate the AH approach in the Eshelby-based hierarchical mineralized turkey leg tendon model (Tiburtius et al 2014 Biomech. MODEL: Mechanobiol. 13 1003-23), which can be considered as a base for musculoskeletal mineralized tissue modeling. We model the finest scale compartments, i.e. the extrafibrillar space and the mineralized collagen fibril, by replacing the self-consistent scheme with our AH approach. This way, we perform a parametric analysis at increasing mineral volume fraction, by varying the amount of mineral that is fusing in the axial and transverse tissue directions in both compartments. Our effective stiffness results are in good agreement with those reported for aged human radius and support the argument that the axial stiffening in aged bone tissue is caused by the formation of a continuous mineral foam. Moreover, the proposed theoretical and computational approach supports the design of biomimetic materials which require an overall composite stiffening without increasing the amount of the reinforcing material.

3D ultrasound biomicroscopy for assessment of cartilage repair tissue: volumetric characterisation and correlation to established classification systems.

Objective and sensitive assessment of cartilage repair outcomes lacks suitable methods. This study investigated the feasibility of 3D ultrasound biomicroscopy (UBM) to quantify cartilage repair outcomes volumetrically and their correlation with established classification systems. 32 sheep underwent bilateral treatment of a focal cartilage defect. One or two years post-operatively the repair outcomes were assessed and scored macroscopically (Outerbridge, ICRS-CRA), by magnetic resonance imaging (MRI, MOCART), and histopathology (O'Driscoll, ICRS-I and ICRS-II). The UBM data were acquired after MRI and used to reconstruct the shape of the initial cartilage layer, enabling the estimation of the initial cartilage thickness and defect volume as well as volumetric parameters for defect filling, repair tissue, bone loss and bone overgrowth. The quantification of the repair outcomes revealed high variations in the initial thickness of the cartilage layer, indicating the need for cartilage thickness estimation before creating a defect. Furthermore, highly significant correlations were found for the defect filling estimated from UBM to the established classification systems. 3D visualisation of the repair regions showed highly variable morphology within single samples. This raises the question as to whether macroscopic, MRI and histopathological scoring provide sufficient reliability. The biases of the individual methods will be discussed within this context. UBM was shown to be a feasible tool to evaluate cartilage repair outcomes, whereby the most important objective parameter is the defect filling. Translation of UBM into arthroscopic or transcutaneous ultrasound examinations would allow non-destructive and objective follow-up of individual patients and better comparison between the results of clinical trials.

Articular cartilage degeneration classification by means of high-frequency ultrasound.

CONTEXT: To date only single ultrasound parameters were regarded in statistical analyses to characterize osteoarthritic changes in articular cartilage and the potential benefit of using parameter combinations for characterization remains unclear. OBJECTIVE: Therefore, the aim of this work was to utilize feature selection and classification of a Mankin subset score (i.e., cartilage surface and cell sub-scores) using ultrasound-based parameter pairs and investigate both classification accuracy and the sensitivity towards different degeneration stages. DESIGN: 40 punch biopsies of human cartilage were previously scanned ex vivo with a 40-MHz transducer. Ultrasound-based surface parameters, as well as backscatter and envelope statistics parameters were available. Logistic regression was performed with each unique US parameter pair as predictor and different degeneration stages as response variables. The best ultrasound-based parameter pair for each Mankin subset score value was assessed by highest classification accuracy and utilized in receiver operating characteristics (ROC) analysis. RESULTS: The classifications discriminating between early degenerations yielded area under the ROC curve (AUC) values of 0.94-0.99 (mean ± SD: 0.97 ± 0.03). In contrast, classifications among higher Mankin subset scores resulted in lower AUC values: 0.75-0.91 (mean ± SD: 0.84 ± 0.08). Variable sensitivities of the different ultrasound features were observed with respect to different degeneration stages. CONCLUSIONS: Our results strongly suggest that combinations of high-frequency ultrasound-based parameters exhibit potential to characterize different, particularly very early, degeneration stages of hyaline cartilage. Variable sensitivities towards different degeneration stages suggest that a concurrent estimation of multiple ultrasound-based parameters is diagnostically valuable. In-vivo application of the present findings is conceivable in both minimally invasive arthroscopic ultrasound and high-frequency transcutaneous ultrasound.

Ultrasound biomicroscopy (UBM) and scanning acoustic microscopy (SAM) for the assessment of hernia mesh integration: a comparison to standard histology in an experimental model.

BACKGROUND: Mesh integration is a key parameter for reliable and safe hernia repair. So far, its assessment is based on histology obtained from rare second-look operations or experimental research. Therefore, non-invasive high-resolution imaging techniques would be of great value. Ultrasound biomicroscopy (UBM) and scanning acoustic microscopy (SAM) have shown potential in the imaging of hard and soft tissues. This experimental study compared the detection of mesh integration, foreign body reaction and scar formation in UBM/SAM with standard histology. MATERIALS AND METHODS: Ten titanized polypropylene meshes were implanted in rats in a model of onlay repair. 17 days postoperative animals were killed and samples were paraffin embedded for histology (H&E, Cresyl violet) or processed for postmortem UBM/SAM. The observation period was uneventful and meshes appeared well integrated. RESULTS: Relocation of neighboring cross-sectional levels could easily be achieved with the 40-MHz UBM and granulation tissue could be distinguished from adjacent muscle tissue layers. The spatial resolution of approximately 8 µm of the 200-MHz UBM system images was comparable to standard histology (2.5-5× magnification) and allowed a clear identification of mesh fibers and different tissue types, e.g., scar, fat, granulation, and muscle tissues, as well as vessels, abscedations, and foreign body giant cell clusters. CONCLUSION: This pilot study demonstrates the potential of high-frequency ultrasound to assess hernia mesh integration non-invasively. Although the methods lack cell-specific information, tissue integration could reliably be assessed. The possibility of conducting UBM in vivo advocates this method as a guidance tool for the indication of second-look operations and subsequent elaborate histological analyses.

3-d high-frequency ultrasound improves the estimation of surface properties in degenerated cartilage.

High-frequency ultrasound (US) surface parameters are well known to be sensitive to degenerative changes in cartilage tissue, but estimates deteriorate if the sample is inclined. We propose 3-D US to precisely estimate the local surface and inclination. For this purpose, the most common ultrasonic surface parameters ultrasound roughness index and integrated reflection coefficient were extended to 2-D surface measurements. Tissue-mimicking phantoms and human cartilage samples with varying degrees of degeneration were measured using a 40-MHz transducer. Characteristic inclination dependencies of the parameters aided in the distinction between specular reflected or backscattered signal origins and allowed a restriction to suitable local inclinations. In the application to cartilage, comparisons with histologic grading (structural Mankin-score) depicted a statistically significant (p < 0.05) increase of US roughness index for scores larger than 0 and decrease of integrated reflection coefficient for scores larger than 1. The presented findings will increase the reliability of ultrasonic surface parameters and can in principal be applied in vivo.

Longitudinal elastic properties and porosity of cortical bone tissue vary with age in human proximal femur.

Tissue level structural and mechanical properties are important determinants of bone strength. As an individual ages, microstructural changes occur in bone, e.g., trabeculae and cortex become thinner and porosity increases. However, it is not known how the elastic properties of bone change during aging. Bone tissue may lose its elasticity and become more brittle and prone to fractures as it ages. In the present study the age-dependent variation in the spatial distributions of microstructural and microelastic properties of the human femoral neck and shaft were evaluated by using acoustic microscopy. Although these properties may not be directly measured in vivo, there is a major interest to investigate their relationships with the linear elastic measurements obtained by diagnostic ultrasound at the most severe fracture sites, e.g., the femoral neck. However, before the validity of novel in vivo techniques can be established, it is essential to understand the age-dependent variation in tissue elastic properties and porosity at different skeletal sites. A total of 42 transverse cross-sectional bone samples were obtained from the femoral neck (Fn) and proximal femoral shaft (Ps) of 21 men (mean±SD age 47.1±17.8, range 17-82years). Samples were quantitatively imaged using a scanning acoustic microscope (SAM) equipped with a 50MHz ultrasound transducer. Distributions of the elastic coefficient (c33) of cortical (Ct) and trabecular (Tr) tissues and microstructure of cortex (cortical thickness Ct.Th and porosity Ct.Po) were determined. Variations in c33 were observed with respect to tissue type (c33Trc33(Ct.Fn)=35.3GPa>c33(Tr.Ps)=33.8GPa>c33(Tr.Fn)=31.9GPa), and cadaver age (R(2)=0.28-0.46, p<0.05). Regional variations in porosity were found in the neck (superior 13.1%; inferior 6.1%; anterior 10.1%; posterior 8.6%) and in the shaft (medial 9.5%; lateral 7.7%; anterior 8.6%; posterior 12.0%). In conclusion, significant variations in elastic coefficients were detected between femoral neck and shaft as well as between the quadrants of the cross-sections of neck and shaft. Moreover, an age-related increase in cortical porosity and a stiffening of the bone tissue were observed. These findings may explain in part the increase in susceptibility to suffer low energy fractures during aging and highlight the potential of ultrasound in clinical osteoporosis diagnostics.

Prediction of the intramuscular fat content in loin muscle of pig carcasses by quantitative time-resolved ultrasound.

A novel method for non-destructive intramuscular fat (IMF) estimation via spectral ultrasound backscatter analysis of signals obtained from pig carcasses early post mortem is described. A commercial hand-held ultrasound device (center frequency: 2.7 MHz) was modified to focus the sound beam to the longissimus muscle at the 2nd/3rd last rib. Time-resolved ultrasound backscatter signals of loin muscle were recorded 45 min p.m. on 82 pig carcass sides. Backfat width (d(BF)=18.9±3.8 mm) and muscle attenuation (α(muscle)=.77±.15 dB MHz(-1) cm(-1)) were assessed from the measured pulse echo data. Other propagation properties of skin, backfat and muscle tissue obtained in a previous investigation were incorporated into the signal pre-processing to minimize parameter estimation artifacts. Spectral and cepstral parameters were derived from time-gated backscattered signals measured in the central muscle region. The range of intramuscular fat (IMF) determined by ether extraction was representative for German pig populations (.7%≤IMF(chem)≤3.6%, coefficient of variation CV(IMF(chem))=44.8%). Variations of IMF were associated with variations of backfat width (CV(d(BF))=20.2%), muscle attenuation (CV(α(muscle))=19.3%), and slope of the backscattered amplitude spectrum (CV(m)=28.8%). A full cross validated multiple linear regression model using these parameters resulted in good predictability of IMF(chem) (R(2)=.76, RMSEP=.34%). Among all tested carcasses, 73% could be correctly classified into one of three IMF classes (LOW: <1%, MID: 1-2%, HIGH: >2%). Using a single threshold (2% IMF), about 92% of all carcasses were correctly classified. With respect to the inherent variability of IMF within a single muscle and the different tissue volumes used for the chemical and ultrasound based IMF estimations the remaining prediction errors are acceptable. Compared to previous ultrasound based studies, the number of acoustic parameters used for the IMF prediction could be reduced. Moreover, the used parameters are based on time-of-flight and spectral slope estimations, which are i) more robust with respect to measurement artifacts and ii) have a causal link to structural variations associated with IMF variations in pork loin.

Quantitative ultrasound biomicroscopy for the analysis of healthy and repair cartilage tissue.

The increasing spectrum of different cartilage repair strategies requires the introduction of adequate non-destructive methods to analyse their outcome in-vivo, i.e. arthroscopically. The validity of non-destructive quantitative ultrasound biomicroscopy (UBM) was investigated in knee joints of five miniature pigs. After 12 weeks, six 5-mm defects, treated with different cartilage repair approaches, provided tissues with different structural qualities. Healthy articular cartilage from each contralateral unoperated knee joint served as a control. The reflected and backscattered ultrasound signals were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB) parameters. The cartilage repair tissues were additionally assessed biomechanically by cyclic indentation, histomorphologically and immunohistochemically. UBM allowed high-resolution visualisation of the structure of the joint surface and subchondral bone plate, as well as determination of the cartilage thickness and demonstrated distinct differences between healthy cartilage and the different repair cartilage tissues with significant higher IRC values and a steeper negative slope of the depth-dependent backscatter amplitude AIBslope for healthy cartilage. Multimodal analyses revealed associations between IRC and the indentation stiffness. Furthermore, AIBslope and AIB at the cartilage-bone boundary (AIBdC) were associated with the quality of the repair matrices and the subchondral bone plate, respectively. This ex-vivo pilot study confirms that UBM can provide detailed imaging of articular cartilage and the subchondral bone interface also in repaired cartilage defects, and furthermore, contributes in certain aspects to a basal functional characterization of various forms of cartilage repair tissues. UBM could be further established to be applied arthroscopically in-vivo.

Microelastic imaging of bone.

Several high-frequency ultrasound techniques have been developed during the last decade with the intention of assessing elastic properties of bone at the tissue level. The basic measurement principles can be divided into: 1) measurement of the compressional wave velocity in thin tissue sections; 2) measurement of surface acoustic wave velocities in thick sections; and 3) derivation of the acoustic impedance from the confocal reflection amplitude in thick sections. In this paper, the 3 principles are described with example measurements given in the frequency range from 50 MHz to 1.2 GHz. The measurements were made with 2 microscopes operating in the pulse-echo mode, either with frequencies up to 200 MHz and time-resolved detection or between 100 MHz and 2 GHz and amplitude detection. The methods are compared and their application potentials and limitations are discussed with respect to the hierarchical structure of cortical bone. Mapping of the confocal reflection amplitude has superior capabilities for deriving quantitative elastic and structural parameters in the heterogeneous bone material. Even at low frequencies (50 MHz), the mineralized tissue matrix can be separated from the larger pores (Haversian canals), and the elastic coefficient in the probing direction can be measured in 2 dimensions. Depending on the type of sample surface preparation (flat or cylindrically shaped), local distribution of a single elastic coefficient or the average transverse isotropic stiffness tensor can be derived. With frequencies in the GHz range, the lamellar bone structure can be analyzed. However, at one GHz, the acoustic wavelength is still one order of magnitude larger than the individual mineralized collagen fibrils. Although the thickness of a lamellar unit can easily be assessed from the acoustic image, the derivation of the anisotropic elastic properties of the mineralized collagen fibrils as well as the detailed structure of a lamella can only be accomplished with further model assumptions.

Spatial distribution of anisotropic acoustic impedance assessed by time-resolved 50-MHz scanning acoustic microscopy and its relation to porosity in human cortical bone.

We used quantitative scanning acoustic microscopy (SAM) to assess tissue acoustic impedance and microstructure of cortical bone of human radii with the aim to provide data on regional distribution of acoustic impedance along the circumferential and across the radial directions in the entire cross-section of the radius diaphysis as well as to determine the range of impedance values in transverse (perpendicular to bone axis) and longitudinal (parallel to bone axis) cross-sections. Several microstructural features related to cortical porosity were analyzed in order to determine whether these features differ in different parts of the cortex and to assess the relationship between the microstructure and tissue acoustic impedance. Fifteen fresh bone specimens (human radius) were investigated using a SAM (center frequency of 50 MHz and -6 dB lateral resolution of approximately 23 microm). The sample acoustic impedance was obtained by means of a calibration curve correlating the reflected signal amplitude of reference materials with their corresponding well-known acoustic impedance. Tissue acoustic impedance and microstructural features were derived from the morphometric analysis of the segmented impedance images. A higher porosity was found in the inner cortical layer (mean+/-SD=8.9+/-2.3%) compared to the peripheral layer (2.7+/-1.5%) (paired t-test, p<10(-5)). ANOVA showed that most of the variance can be explained by the regional effect across the radial direction with a minor contribution due to between-sample variability. Similar to porosity, the number and diameter of pores were greater in the inner layer. In contrast to porosity, ANOVA showed that impedance variability can mostly be explained by between-specimen variability. Two-way ANOVA revealed that after compensation for the between-sample variability the variation in acoustic impedance across the radial direction was much larger than that along the circumferential direction. In addition to the significant difference between the inner cortical layer (8.25+/-0.4 Mrayl) and peripheral layer (8.0+/-0.5 Mrayl) (unilateral paired t-test, p<10(-4)), the values in the anterior region (8.2+/-0.5 Mrayl) were found to be significantly higher than those of the posterior region (7.9+/-0.6 Mrayl). Impedance mean value of longitudinal sections was lower than mean value measured in transverse cross-sections, resulting in an impedance acoustic anisotropy ratio of 1.17+/-0.03 in the inner cortical layer and 1.19+/-0.02 in the peripheral layer. SAM is a valuable tool to provide data on the spatial distribution of microstructural and microelastic bone properties that is useful to improve our understanding of the impact of bone microstructure on tissue material properties.

Site-matched assessment of structural and tissue properties of cortical bone using scanning acoustic microscopy and synchrotron radiation muCT.

200 MHz scanning acoustic microscopy (SAM) and synchrotron radiation muCT (SR-muCT) were used to assess microstructural parameters and tissue properties in site-matched regions of interest in cortical bone. Anterior and postero-lateral regions of ten cross sections from human cortical radius were explored. Structural parameters, including diameter and number of Haversian canals per cortical area (Ca.Dm, N.Ca/Ar) and porosity Po were assessed with both methods using a custom-developed image fusion and analysis software. Acoustic impedance Z and degree of mineralization of bone DMB were extracted separately for osteonal and interstitial tissues from the fused images. Structural parameter estimations obtained from radiographic and acoustic images were almost identical. DMB and impedance values were in the range between 0.77 and 1.28 g cm(-3) and 5.13 and 12.1 Mrayl, respectively. Interindividual and regional variations were observed, whereas the strongest difference was found between osteonal and interstitial tissues (Z: 7.2 +/- 1.1 Mrayl versus 9.3 +/- 1.0 Mrayl, DMB: 1.06 +/- 0.07 g cm(-3) versus 1.16 +/- 0.05 g cm(-3), paired t-test, p < 0.05). Weak, but significant correlations between DMB and Z were obtained for the osteonal (R(2) = 0.174, p < 10(-4)) and for the pooled (osteonal and interstitial) data. The regression of the pooled osteonal and interstitial tissue data follows a second-order polynomial (R(2) = 0.39, p < 10(-4)). Both modalities fulfil the requirement for a simultaneous evaluation of cortical bone microstructure and material properties at the tissue level. While SAM inspection is limited to the evaluation of carefully prepared sample surfaces, SR-muCT provides volumetric information on the tissue without substantial preparation requirements. However, SAM provides a quantitative estimate of elastic properties at the tissue level that cannot be captured by SR-muCT.

Channel defect detection in food packages using integrated backscatter ultrasound imaging.

Hermetically-sealed flexible food packages require very effective seal integrity testing to extend the shelf stability of thermally processed food. The initial goal of this study was to estimate the detection limits of laboratory-generated channels which simulate defects in food packages using pulse-echo ultrasonic imaging techniques. Packages with well characterized (via transmission optical microscopy) laboratory-generated channels that simulate defects with diameters between 9 and 325 microm in the seal plane traversing the major axis of the heat seal were generated in heat-sealed microwavable retort-pouch plastic film (trilaminate). Pulse-echo techniques in the 13-17 MHz center frequency range were investigated. The samples were examined with a conventional B-mode imaging technique, which was found to be inadequate for subwavelength imaging of the types of typical channel defects found in shelf-stable food packages. Based on conventional B-mode image features, a new goal of this study was established to develop and evaluate an imaging technique which would exhibit subwavelength imaging capabilities. The new imaging technique called backscattered amplitude integral (BAI) is introduced here. It was observed that BAI-mode imaging has the ability for subwavelength detection of channel defects, e.g., detection of a 10-microm diameter channel defect at a center frequency of 13.1 MHz (lambda=182 microm).