Effects of ultrasound frequency, temporal sampling frequency, and spatial sampling step on the quantitative ultrasound parameters of articular cartilage

Quantitative ultrasound imaging may provide a technique for diagnosing initial signs of osteoarthritis (OA), such as surface fibrillation of articular cartilage. Because subchondral sclerosis and osteophyte formation occur in OA as well, ultrasonic analysis of subchondral bone could yield useful diagnostic information. In this study, we investigated whether low-frequency (5 MHz) ultrasound, typically used in bone diagnostics, would be feasible for evaluating the integrity of the surface of the cartilage. The reflection parameters in the time and frequency domains, the ultrasound roughness index, and the wavelet-based parameters were evaluated using ultrasound transducers operating at 5, 10, and 50 MHz frequencies. The effects of variable size of spatial sampling steps and of temporal sampling frequencies were also investigated. Custom-made phantoms and cartilage samples with various surface characteristics were analyzed. The reflection parameters detected the surface degradation with all ultrasound frequencies. The roughness of the surface could only be evaluated reliably with the 50 MHz-focused transducer. In conclusion, simultaneous analysis of the reflection parameters of the cartilage and the subchondral bone is feasible at low (5 MHz) ultrasound frequencies. However, reliable evaluation of the microtopography of the cartilage requires use of a higher ultrasound frequency.     

Effects of non-optimal focusing on dual-frequency ultrasound measurements of bone

In pulse-echo (PE) ultrasound measurements, the use of focused transducers is desirable for quantitative assessment of bone characteristics because of the attenuation in the overlying soft tissues. However, the variable thickness and composition of the soft tissue overlying bone affect the focal depth of the ultrasound beam and induce errors into the measurements. To compensate for the attenuation-related effects caused by the interfering soft tissue (i.e., fat and lean tissue), a dual-frequency ultrasound (DFUS) technique was recently introduced. The aim of this study was to investigate the effect of non-optimal focal depth of the ultrasound beam on the determination of the integrated reflection coefficient (IRC) of bone when overlaid by an interfering layer composed of oil and water. The feasibility of the DFUS-based correction of the IRC was evaluated through numerical simulations and experimental measurements. Even when the interfering layer-bone interface was out of focus, the total thickness of the interfering layer could be accurately determined with the technique. However, based on the simulations, the errors in the determination of the composition of the interfering layer increased (0.004 to 113.8%) with the increase in distance between the interfering layer-bone interface and the focus of the ultrasound beam. Attenuation compensation, based on the true composition of the interfering layer, resulted in an average relative error of 22.3% in the IRC values calculated from the simulations. Interestingly, the attenuation compensation with the interfering layer composition estimated using the DFUS technique resulted in a smaller average relative error of 14.9% in the IRC values. The simulations suggest that DFUS can reduce the errors induced by soft tissue in bone PE ultrasound measurements. The experimental measurements indicate that the accuracy of the IRC measurements is rather similar when using DFUS correction or correction based on the true composition of the interfering layer. However, the results suggest that accurate determination of soft tissue composition may be difficult without optimal focusing of the ultrasound beam on the soft tissue-bone interface.     

Characterization of the repair tissue in articular cartilage defects using silver-enhanced colloidal gold immunostaining

The newly developed silver-enhanced colloidal gold staining method was used in a rabbit model to characterize the repair tissue in large articular cartilage defects filled with a heterocyclic methacrylate polymer. By 6 weeks the resurfacing tissue consisted of highly organized hyaline-like articular cartilage, fully integrated with the adjacent normal cartilage. Immuno-histochemistry detected collagen type ll, keratan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate in the matrix of the neocartilage. The level to which the polymer plug was recessed apeared to be critical to the overall quality of the repair tissue. Optimum results were obtained when the top surface of the biomaterial was at the level of the subchondral bone, below the level of the surrounding articular cartilage. Other technical aspects of implantation, that also affect the repair, are also discussed.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.     

Zone-dependent mechanical properties of human articular cartilage obtained by indentation measurements

Emerging 3D printing technology permits innovative approaches to manufacture cartilage scaffolds associated with layer-by-layer mechanical property adaptation. However, information about gradients of mechanical properties in human articular cartilage is limited. In this study, we quantified a zone-dependent change of local elastic modulus of human femoral condyle cartilage by using an instrumented indentation technique. From the cartilage superficial zone towards the calcified layer, a gradient of elastic modulus values between 0.020?±?0.003?MPa and 6.44?±?1.02?MPa was measured. To validate the tissue quality, the histological tissue composition was visualized by glycosaminoglycan and collagen staining. This work aims to introduce a new protocol to investigate the zone-dependent mechanical properties of graded structures, such as human articular cartilage. From this knowledge, better cartilage repair strategies could be tailored in the future.     

An evaluation of human articular cartilage on femoral head

In this study, we investigated stress relaxation behavior of the human articular cartilage on femoral head. Articular cartilage is a white dense connective tissue that covers the bone ends within diarthrodial joints and works as a weight-transmitting and energy-absorbing material. Human articular cartilage on femoral head was used as test material. Relaxation tests were carried out by using the indentation technique via Instron Universal Testing Machine. Test materials were investigated in an isotonic salt solution at 37 °C. To keep the temperature constant, two vessels being in each other were utilized. Thus, hot water was circulated in the outer vessel and isotonic salt solution was kept in the inner vessel. Experimental results showed that there is a remarkable difference between normal and degenerated cartilage for the same age and sex. It was observed that the relaxation percent of normal cartilage as a function of relaxation time is much higher than that of degenerated cartilage.     

实时超声膨胀测量系统在关节软骨中的实验研究

关节软骨是覆盖关节表面的一层承载生物重量的组织。关节软骨在正常状态下，软骨蛋白多糖的弹性和胶原纤维的张力保持平衡。这种平衡的微小变化会引起关节软骨的退化。关节软骨特别是其表层区域的膨胀是和骨关节炎的退化相联系的，定量的测量关节软骨的膨胀可以表征骨关节炎的退化变化。本文的主要目的是介绍一种新的实时超声膨胀测量系统，并把该系统应用到关节软骨的研究中。该系统使用50MHz的超声来实时动态观测用胰岛素和0．15M生理盐水溶液分别浸泡处理的牛膝盖关节软骨的非均匀瞬时深度依赖膨胀行为。实验表明，实时超声检测的方法为关节软骨的退化研究提供了独特的工具。这项技术结合关节内窥镜检查，具有潜在的早期诊断体内关节退化的价值。     

The regeneration of articular cartilage using a new polymer system

A polymer system based on room temperature polymerising poly (ethylmethacrylate) polymer powder and tetrahydrofurfuryl monomer has been investigated as a biomaterial for encouraging articular cartilage repair. This heterocyclic methacrylate polymer system swells slightly in situ and thus provides a good interface with subchondral bone resulting in mechanical stability with favourable uptake kinetics. Another feature of this polymer system is that it exhibits high water uptake which leads to absorption of the surrounding tissue fluid and matrix proteins, including growth factors; this may encourage the formation of new cartilage. Three weeks after implantation the tissue overgrowth contained cartilage components: chondrocytes, collagen type II, chondroitin 4-sulphate and chondroitin 6-sulphate. In addition numerous chondrocyte clones were observed at the edge of the defect and in the newly repaired tissue. By six weeks a superficial articulating surface was continuous with the normal articular cartilage with underlying tissue which showed some evidence of endochondral ossification. By nine weeks the surface covering of new cartilage had a widened and an irregular zone of calcified cartilage with thickened subchondral bone was present. At eight months the resurfaced cartilage remained intact above a remodelled subchondral bone end plate.     

Long term results after implantation of tissue engineered cartilage for the treatment of osteochondral lesions in a minipig model

In present study we determined the long term in vivo integration and histological modeling of an in vitro engineered cartilage construct. Tissue engineered autologous cartilagenous tissue was cultured on calcium phosphate cylinders and implanted into osteochondral defects into the femoral condyles in minipigs. Radiological follow-up was performed at 2, 8, 26 and 52 weeks, condyles were harvested 26 and 52 weeks post-implantation. Thickness of cultivated tissue (1.10 ± 0.55 mm) was comparable to in situ cartilage and cells produced in vitro cartilage specific proteins. In vivo, 26 and 52 weeks post-implantation defects were resurfaced with hyaline-like tissue, the implants were well integrated with no gap at the interface between the engineered neocartilage and the adjacent articular cartilage. Synthesis of type II collagen was detected 26 and 52 weeks after implantation. The modified ICRS score increased from 26 to 52 weeks. Histomorphometric evaluation revealed a decrease in cellularity in tissue engineered cartilage from 2.2-fold of native cartilage after 26 weeks to 1.5-fold after 52 weeks. In conclusion, these findings demonstrate the integration and maturation of tissue engineered cartilage pellets attached on a bone substitute carrier implanted in osteochondral defects over a long time. J. P. Petersen, P. Ueblacker, C. Goepfert have contributed equally to this study.     

Fourier transform infrared spectral analysis of degenerative cartilage: an infrared fiber optic probe and imaging study

A preliminary investigation into the diagnostic potential of an infrared fiber optic probe (IFOP) for evaluating degenerative human articular cartilage is described. Twelve arthritic human tibial plateaus obtained during arthroplasty were analyzed using the IFOP. Infrared spectra were obtained from IFOP contact with articular surface sites visually graded normal or degraded (Collins Scale grade 1 and grade 3, respectively). Comparisons of infrared spectral parameters (peak heights and areas) were made to elucidate spectral indicators of surface degeneration. IFOP spectral analysis revealed subtle but consistent changes between grades 1 and 3 sites. Infrared absorbance bands arising from type II collagen were observed to change with degradation. More degraded tissues exhibited increased amide II (1590-1480 cm(-1))/1338 cm(-1) area ratio (p=0.034) and decreased 1238/1227 cm(-1) peak ratio (p = 0.017); similar changes were seen with Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Grades 1 and 3 cartilage showed consistent spectral differences in the amide II, III, and 1338 cm(-1) regions that are likely related to type II collagen degradation that accompanies cartilage degeneration. These results suggest that it may be possible to monitor subtle changes related to early cartilage degeneration, allowing for IFOP use during arthroscopy for in situ determination of cartilage integrity.     

超声弹性显微镜成像系统开发与应用的初步研究

传统的超声弹性成像技术一般使用1MHz～10MHz的超声波,这一频段超声波的空间分辨率在毫米量级,它不能满足对生物组织中微细结构(如皮肤层,关节软骨等)的研究。文中介绍一套新开发的超声弹性显微镜成像系统,并将其初步应用于对关节软骨和老鼠表皮的成像研究中。该系统由加压系统和背向散射超声显微镜系统两部分组成,其中超声探头频率为50MHz。通过对关节软骨和老鼠表皮的成像实验表明,该系统可以清晰的对生物组织中微细结构成像,研究它们的机械特性。     

Matrix generation within a macroporous non-degradable implant for osteochondral defects is not enhanced with partial enzymatic digestion of the surrounding tissue: evaluation in an in vivo rabbit model

Articular cartilage defects are a significant source of pain, have limited ability to heal, and can lead to the development of osteoarthritis. However, a surgical solution is not available. To tackle this clinical problem, non-degradable implants capable of carrying mechanical load immediately after implantation and for the duration of implantation, while integrating with the host tissue, may be viable option. But integration between articular cartilage and non-degradable implants is not well studied. Our objective was to assess the in vivo performance of a novel macroporous, nondegradable, polyvinyl alcohol construct. We hypothesized that matrix generation within the implant would be enhanced with partial digestion of the edges of articular cartilage. Our hypothesis was tested by randomizing an osteochondral defect created in the trochlea of 14 New Zealand white rabbits to treatment with: (i) collagenase or (ii) saline, prior to insertion of the implant. At 1 and 3-month post-operatively, the gross morphology and histologic appearance of the implants and the surrounding tissue were assessed. At 3 months, the mechanical properties of the implant were also quantified. Overall, the hydrogel implants performed favorably; at all time-points and in all groups the implants remained well fixed, did not cause inflammation or synovitis, and did not cause extensive damage to the opposing articular cartilage. Regardless of treatment with saline or collagenase, at 1 month post-operatively implants from both groups had a contiguous interface with adjacent cartilage and were populated with chondrocyte-like cells. At 3 months fibrous encapsulation of all implants was evident, there was no difference between area of aggrecan staining in the collagenase versus saline groups, and implant modulus was similar in both groups; leading us to reject our hypothesis. In summary, a porous PVA osteochondral implant remained well fixed in a short term in vivo osteochondral defect model; however, matrix generation within the implant was not enhanced with partial digestion of adjacent articular cartilage.     

An analytical solution of the rebound indentation problem for an isotropic linear viscoelastic layer loaded with a spherical punch

A unilateral frictionless axisymmetric contact problem for an isotropic viscoelastic layer attached to a rigid substrate and loaded with a spherical indenter is considered. It is assumed that the indentation protocol is composed of two stages. In the indentation phase, the layer is subjected to displacement loading, while at the end of the first stage, the load is immediately removed and the second stage, called the recovery phase, lasts for a theoretically indefinite time. Under the assumption of time-independent Poisson’s ratio, we derive closed-form analytical expressions for the contact force (in the indentation phase) and for the indentation displacement (in the the recovery phase). The obtained closed-form analytical solution is valid for the indentation phase with an arbitrary monotonic loading displacement and can be used for evaluation of the rebound indentation test for soft biological tissues and originally suggested for assessment of articular cartilage viability.     

可降解水凝胶作为关节软骨修复材料的研究进展

可降解水凝胶因其良好的生物相容性和生物降解性被广泛用于关节软骨的修复和再生。本文以可降解水凝胶在软骨组织工程中的三类应用策略为主线,概述了用于原位成型可注射水凝胶的蛋白多糖类材料及纳米复合类材料;系统总结了传统工艺制造组织工程支架的优缺点及多种工艺结合的制备方法;重点归纳了近年来3D打印组织工程支架从纯软骨到骨/软骨一体化、从单层到多层的研究进展;最后分析了可降解水凝胶作为关节软骨支架材料在微观定向结构和生物活性功能化方面的局限性,并作出展望：未来开展多材料、多尺度、多诱导的高仿生梯度支架是关节软骨组织工程的一个重要研究方向。     

A simple method of cartilage regeneration using a new polymerizing system: ultrastructural characteristics of the repair tissue

The ultrastructural characteristics of the repair tissue in large articular cartilage defects, filled with a heterocyclic polymerizing system were studied using transmission electron microscopy (TEM) and energy dispersive micro-analysis (EDMA). By six weeks post-implantation, the defects were resurfaced with predominantly hyaline-like articular cartilage. Chondrocytes in both the superficial and deep zones of the repair tissue were highly productive, secreting large amounts of proteoglycans, into a well-organized, rich in collagen fibrils, extracellular matrix. By contrast, in the repair tissue of the defects treated without the biomaterial, proteoglycan synthesis was less and the structure of the matrix was inferior. We conclude that the polymer enhances both chondrocyte metabolism and matrix organization, thus improving the quality of the repair tissue in articular cartilage defects.     

Experimental investigation of the contact mechanics of rough fractal surfaces

The nonstationary character of roughness is a widely recognized property of surface morphology and suggests modeling several solid surfaces by fractal geometry. In the field of contact mechanics, this demands novel investigations attempting to clarify the role of multiscale roughness during physical contact. Here we review the results we recently obtained in the characterization of the contact mechanics of fractal surfaces by depth-sensing indentation. One class of experiments was conducted on organic thin films, load-displacement curves being acquired by atomic force microscopy using custom-designed tips. Another class of experiments focused on well-defined crystalline and mechanically polished ceramic substrates probed by a traditional nanoindenter. We observed the first-loading cycle to be considerably affected by surface roughness. Plastic failure was found to dominate incipient contact while contact stiffness increased on decreasing fractal dimension and roughness. Our findings suggest fractal parameters to drive contact mechanics whenever the penetration depth is kept below the interface width.     

Markov models of specular and diffuse scattering in restoration ofmedical ultrasound images

Observed medical ultrasound images are degraded representations of the true tissue reflectance. The specular reflections at boundaries between regions of different tissue types are blurred, and the diffuse scattering within such regions also contains speckle. This reduces the diagnostic value of such images. In order to remove both blur and speckle, the authors develop a maximum a posteriori deconvolution algorithm for two-dimensional (2-D) ultrasound radio frequency (RF) images based on a new Markov random field image model incorporating spatial smoothness constraints and physical models for specular reflections and diffuse scattering. During stochastic relaxation, the algorithm alternates steps of restoration and segmentation, and includes estimation of reflectance parameters. The smoothness constraints regularize the overall procedure, and the algorithm uses the specular reflection model to locate region boundaries. The resulting restorations of some simulated and real RF images are significantly better than those produced by Wiener filtering     

Estimation of fixed charge density and diffusivity profiles in cartilage using contrast enhanced computer tomography

Contrast enhanced computer tomography (CT) imaging of articular cartilage has been proposed for diagnostics of cartilage degeneration, that is, osteoarthritis. Previous studies also indicate that acute cartilage damage can be detected by measuring diffusion of contrast agent into cartilage using CT. However, currently, there is no reliable method to measure spatial diffusion rates within cartilage tissue, and only average bulk values have been reported. In this paper, we develop a method to determine depthwise diffusivity of contrast agents in cartilage tissue using contrast enhanced CT. The triphasic mechano‐electrochemical theory of cartilage is modified to include diffusion of contrast agents. By applying statistical inversion theory and Bayesian approximation error approach, the method allows us to estimate a fixed charge density distribution in the cartilage tissue, an important determinant for mechanical competence of articular cartilage. The method is tested by using a one‐dimensional simulation study. Preliminary tests with experimental data on diffusion of anionic iodine contrast agent in bovine articular cartilage indicate that the method can provide realistic estimates for depth dependent fixed charge density. Thereby, the present study can improve our understanding on the feasibility of contrast enhanced CT for cartilage diagnostics. Copyright © 2014 John Wiley & Sons, Ltd.     

Enhanced mechanical properties of thermosensitive chitosan hydrogel by silk fibers for cartilage tissue engineering

Articular cartilage has limited repair capability following traumatic injuries and current methods of treatment remain inefficient. Reconstructing cartilage provides a new way for cartilage repair and natural polymers are often used as scaffold because of their biocompatibility and biofunctionality. In this study, we added degummed chopped silk fibers and electrospun silk fibers to the thermosensitive chitosan/glycerophosphate hydrogels to reinforce two hydrogel constructs which were used as scaffold for hyaline cartilage regeneration. The gelation temperature and gelation time of hydrogel were analyzed by the rheometer and vial tilting method. Mechanical characterization was measured by uniaxial compression, indentation and dynamic mechanical analysis assay. Chondrocytes were then harvested from the knee joint of the New Zealand white rabbits and cultured in constructs. The cell proliferation, viability, production of glycosaminoglycans and collagen type II were assessed. The results showed that mechanical properties of the hydrogel were significantly enhanced when a hybrid with two layers of electrospun silk fibers was made. The results of GAG and collagen type II in cell-seeded scaffolds indicate support of the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fiber–hydrogel composite for GAG content and in two-layer electrospun fiber–hydrogel composite for Col II. It was concluded that these two modified scaffolds could be employed for cartilage tissue engineering.     

Engineering cartilage tissue interfaces using a natural glycosaminoglycan hydrogel matrix — An in vitro study

Hydrogels are suitable matrices for cartilage tissue engineering on account of their resemblance to native extracellular matrix of articular cartilage and also considering its ease of application, they can be delivered to the defect site in a minimally invasive manner. In this study, we evaluate the suitability of a fast gelling natural biopolymer hydrogel matrix for articular cartilage tissue engineering. A hydrogel based on two natural polymers, chitosan and hyaluronic acid derivative was prepared and physicochemically characterized. Chondrocytes were then encapsulated within the hydrogel and cultured over a period of one month. Cartilage regeneration was assessed by histological, biochemical and gene expression studies. Chondrocytes maintained typical round morphology throughout the course of this investigation, indicating preservation of their phenotype with sufficient production of extracellular matrix and expression of typical chondrogenic markers Collagen type 2 and aggrecan. The results suggest that the natural polymer hydrogel matrix can be used as an efficient matrix for articular cartilage tissue engineering.