Virpi Tiitu

Detection of mechanical injury of articular cartilage using contrast enhanced computed tomography.

OBJECTIVE Osteoarthritic degeneration may be initiated by mechanical overloading of articular cartilage. Mechanical injury increases the permeability of tissue, thereby probably affecting the diffusion of contrast agents in articular cartilage. We investigated whether it is possible to detect acute cartilage injury by measuring contrast agent diffusion into articular cartilage using contrast enhanced computed tomography (CECT). METHODS Osteochondral plugs (Ø=6.0 mm, n=36) were prepared from intact bovine patellae (n=9). Two of the adjacent samples were injured by impact loading, using a drop tower, while the others served as paired controls. The samples were imaged before immersion in contrast agent solution [ioxaglate (Hexabrix™) or sodium iodide (NaI)] and 1, 3, 5, 7, 10, 15, 20 and 25 h after immersion using a MicroCT-instrument. Contrast agent content, diffusion coefficient and diffusion flux were determined for each sample. RESULTS Already after 1 h the penetration of contrast agents into cartilage was significantly (P<0.05) greater in the injured samples. The diffusion coefficient was not altered by the injury, which suggests that reaching the diffusion equilibrium takes the same time in injured and intact cartilage. However, the diffusion flux of ioxaglate through the articular surface was significantly higher in injured samples at 30-60 min after immersion. CONCLUSIONS To conclude, CECT could diagnose articular cartilage injuries, and determination of the diffusion flux of ioxaglate helped to detect tissue injury without waiting for the diffusion equilibrium. These results are encouraging, however, in vivo application of CECT is challenging and systematic further studies are needed to reveal its clinical potential.

Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.

OBJECTIVE The effect of threose-induced collagen cross-linking on the mechanical and diffusive properties of cartilage was investigated in vitro. In particular, we investigated the potential of Contrast Enhanced Computed Tomography (CECT) to detect changes in articular cartilage after increased collagen cross-linking, which is an age-related phenomenon. METHODS Osteochondral plugs (Ø=6.0 mm, n=28) were prepared from intact bovine patellae (n=7). Two of the four adjacent samples, prepared from each patella, were treated with threose to increase the collagen cross-linking, while the other two specimen served as paired controls. One sample pair was mechanically tested and then mechanically injured using a material testing device. Contrast agent [ioxaglate (Hexabrix™)] diffusion was imaged in the other specimen pair for 25 h using CECT. Water fraction, collagen and proteoglycan content, collagen network architecture and the amount of cross-links [hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent)] of the samples were also determined. RESULTS Cartilage collagen cross-linking, both Pent and LP, were significantly (P<0.001) increased due to threose treatment. CECT could detect the increased cross-links as the contrast agent penetration and the diffusion flux were significantly (P<0.05) lower in the threose treated than in untreated samples. The equilibrium modulus (+164%, P<0.05) and strain dependent dynamic modulus (+47%, P<0.05) were both significantly greater in the threose treated samples than in reference samples, but there was no association between the initial dynamic modulus and the threose treatment. The water fraction, proteoglycan and collagen contents, as well as collagen architecture, were not significantly altered by the threose treatment. CONCLUSIONS To conclude, the CECT technique was found to be sensitive at detecting changes in cartilage tissue due to increased collagen cross-linking. This is important since increased cross-linking has been proposed to be related to the increased injury susceptibility of tissue.

Quantitative evaluation of spontaneously and surgically repaired rabbit articular cartilage using intra-articular ultrasound method in situ.

During the last decade, a major effort has been devoted to developing surgical methods for repairing localized articular cartilage lesions. Despite some promising results no ultimate breakthrough in surgical cartilage repair has been achieved. Improvements in repair techniques would benefit from more sensitive and quantitative methods for long-term follow-up of cartilage healing. In this study, the potential of a new ultrasound technique for detecting the compositional and structural changes in articular cartilage after surgery, using recombinant human type II collagen gel and spontaneous repair was, investigated. Rabbit knee joints containing intact (n = 13) and surgically (n = 8) or spontaneously (n = 5) repaired tissue were imaged in situ at 6 months after the operation using a clinical intravascular high-frequency (40 MHz) ultrasound device. Based on the ultrasound raw data, ultrasound reflection coefficient (R), integrated ultrasound reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI) were determined for each sample. URI was significantly higher in both repair groups than in intact cartilage (p < 0.05). The reflection parameters (R and IRC) were significantly lower in surgically repaired cartilage (p < 0.05) than in intact cartilage. Furthermore, AIB was significantly higher in surgically repaired cartilage than in intact tissue (p < 0.05). To conclude, the integrity of the rabbit articular cartilage repair could be quantitatively evaluated with the nondestructive ultrasound approach. In addition, clinically valuable qualitative information on the changes in cartilage integration, structure and composition could be extracted from the ultrasound images. In the present study, the structure and properties of repaired tissue were inferior to native tissue at 6 months after the operation. The applied ultrasound device and probes are FDA approved and, thus, applicable for the quantitative in vivo evaluation of human articular cartilage.

Diffusion of Gd-DTPA2− into articular cartilage

OBJECTIVES The delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC) technique is a method proposed for non-invasive measurement of cartilage glycosaminoglycan (GAG) content. In this method, gadopentetate (Gd-DTPA²⁻) is assumed to distribute in cartilage in inverse relation to the GAG distribution, thus allowing quantification of the GAG content. For accurate GAG quantification, the kinetics of Gd-DTPA²⁻ in articular cartilage is of critical importance. However, the diffusion of Gd-DTPA²⁻ has not been systematically studied over long time periods using MRI-feasible gadopentetate concentrations. Thus, the present study aims to investigate the diffusion of gadopentetate into cartilage in vitro in intact and enzymatically degraded cartilage. METHODS The diffusion of gadopentetate into bovine articular cartilage was investigated at 9.4 T over 18-h time period using repeated T(1) measurements in two models, (1) comparing intact and trypsin-treated tissue and (2) assessing the effect of penetration direction. The diffusion process was further assessed by determining the gadopentetate flux and diffusivity. The results were compared with histological and biochemical reference methods. RESULTS AND CONCLUSIONS The results revealed that passive diffusion of Gd-DTPA²⁻ was significantly slower than previously assumed, leading to overestimation of the GAG content at equilibrating times of few hours. Moreover, Gd-DTPA²⁻ distribution was found to depend not only on GAG content, but also on collagen content and diffusion direction. Interestingly, the dGEMRIC technique was found to be most sensitive to cartilage degradation in the early stages of diffusion process, suggesting that full equilibrium between gadopentetate and cartilage may not be required in order to detect cartilage degeneration.

Comparison of ultrasound and optical coherence tomography techniques for evaluation of integrity of spontaneously repaired horse cartilage

The aim of this study was to compare sensitivity of ultrasound and optical coherence tomography (OCT) techniques for the evaluation of the integrity of spontaneously repaired horse cartilage. Articular surfaces of horse intercarpal joints, featuring both intact tissue and spontaneously healed chondral or osteochondral defects, were imaged ex vivo with arthroscopic ultrasound and laboratory OCT devices. Quantitative ultrasound (integrated reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI)) and optical parameters (optical reflection coefficient (ORC), optical roughness index (ORI) and optical backscattering (OBS)) were determined and compared with histological integrity and mechanical properties of the tissue. Spontaneously healed tissue could be quantitatively discerned from the intact tissue with ultrasound and OCT techniques. Furthermore, several significant correlations (p < 0.05) were detected between ultrasound and OCT parameters. Superior resolution of OCT provided a more accurate measurement of cartilage surface roughness, while the ultrasound backscattering from the inner structures of the cartilage matched better with the histological findings. Since the techniques were found to be complementary to each other, dual modality imaging techniques could provide a useful tool for the arthroscopic evaluation of the integrity of articular cartilage.

Engineering of cartilage in recombinant human type II collagen gel in nude mouse model in vivo

OBJECTIVE Our goal was to test the recombinant human type II collagen (rhCII) material as a gel-like scaffold for chondrocytes in a nude mouse model in vivo. DESIGN Isolated bovine chondrocytes (6x10(6)) were seeded into rhCII gels (rhCII-cell) and injected subcutaneously into the backs of nude mice. For comparison, chondrocytes (6x10(6)) in culture medium (Med-cell) and cell-free rhCII gels (rhCII-gel) were similarly injected (n=24 animals, total of three injections/animal). After 6 weeks, the tissue constructs were harvested and analyzed. RESULTS Chondrocytes with or without rhCII-gel produced white resilient tissue, which in histological sections had chondrocytes in lacunae-like structures. Extracellular matrix stained heavily with toluidine blue stain and had strongly positive collagen type II immunostaining. The tissue did not show any evidence of vascular invasion or mineralization. The cell-free rhCII-gel constructs showed no signs of cartilage tissue formation. Cartilage tissue produced by Med-cell was thin and macroscopically uneven, while the rhCII-cell construct was smooth and rounded piece of neotissue. RhCII-cell constructs were statistically thicker than Med-cell ones. However, no statistical differences were found between the groups in terms of glycosaminoglycan (GAG) content or biomechanical properties. CONCLUSIONS These results show that rhCII-gel provides good expansion and mechanical support for the formation of cartilage neotissue. RhCII material may allow favorable conditions in the repair of chondral lesions.

Multiparametric MRI assessment of human articular cartilage degeneration: Correlation with quantitative histology and mechanical properties

To evaluate the sensitivity of quantitative MRI techniques (T1, T1,Gd, T2, continous wave (CW) T1ρ dispersion, adiabatic T1ρ, adiabatic T2ρ, RAFF and inversion‐prepared magnetization transfer (MT)) for assessment of human articular cartilage with varying degrees of natural degeneration.

Ultrasound evaluation of mechanical injury of bovine knee articular cartilage under arthroscopic control

A local cartilage injury can trigger development of posttraumatic osteoarthritis (OA). Surgical methods have been developed for repairing cartilage injuries. Objective and sensitive methods are needed for planning an optimal surgery as well as for monitoring the surgical outcome. In this laboratory study, the feasibility of an arthroscopic ultrasound technique for diagnosing cartilage injuries was investigated. In bovine knees (n = 7) articular cartilage in the central patella and femoral sulcus was mechanically degraded with a steel brush modified for use under arthroscopic control. Subsequently, mechanically degraded and intact adjacent tissue was imaged with a high frequency (40 MHz) intravascular ultrasound device operated under arthroscopic guidance. After opening the knee joint, mechanical indentation measurements were also conducted with an arthroscopic device at each predefined anatomical site. Finally, cylindrical osteochondral samples were extracted from the measurement sites and prepared for histological analysis. Quantitative parameters, i.e., reflection coefficient (R), integrated reflection coefficient (IRC), apparent integrated backscattering (AIB), and ultrasound roughness index (URI) were calculated from the ultrasound signals. The reproducibilities (sCV %) of the measurements of ultrasound parameters were variable (3.7% to 26.1%). Reflection and roughness parameters were significantly different between mechanically degraded and adjacent intact tissue (p <; 0.05). Surface fibrillation of mechanically degraded tissue could be visualized in ultrasound images. Furthermore, R and IRC correlated significantly with the indentation stiffness. The present results are encouraging; however, further technical development of the arthroscopic ultrasound technique is needed for evaluation of the integrity of human articular cartilage in vivo.

Surface characterization and in vitro biocompatibility assessment of photosensitive polyimide films

Polyimide (PI) is a commonly used polymer in microelectronics. Recently, numerous PI-based flexible neural interfaces have been developed for reducing mechanical mismatch between rigid implant and soft neural tissue. Most approaches employ non-photosensitive PI, which has been proven earlier to be biocompatible. However, photosensitive polyimide (PSPI) would simplify device fabrication remarkably, but its biocompatibility has been only sparsely reported. In this study, cytotoxicity of spin-coated PSPI (HD Microsystems PI-2771) and conventional PI (HD Microsystems PI-2525) films were evaluated in vitro using BHK-21 fibroblasts according to the ISO-10993-5 standard. PSPIs were tested as cured at a temperature of 200 degrees C (PI-2771-200) and 350 degrees C (PI-2771-350). The PI film surfaces were characterized in terms of their roughness, energy and zeta potential which are hypothesized to affect cell-material interactions. The values of the total surface free energy (SFE), and its polar and dispersive component, were significantly (p<0.001) greater for the PI-2525 film (SFE: 47.3 mJ/m2) than for the PI-2771-200 (25.6 mJ/m2) or PI-2771-350 films (26.2 mJ/m2). The curing temperature of the PI-2771 had a significant effect on the zeta potential values (p<0.001), but not on surface energy (p=0.091) or roughness (p=0.717). The results from the MTS proliferation assays and live/dead staining revealed that PSPI is almost as non-cytotoxic as conventional PI and polyethylene (negative control). The morphology and spreading of BHK-21 cells were similar on all the PI materials tested. In conclusion, PSPI seems to be a promising biocompatible material, while further studies in vitro and in vivo are needed to clarify the long-term effects.

Improved adherence and spreading of Saos-2 cells on polypropylene surfaces achieved by surface texturing and carbon nitride coating

The adhesion and contact guidance of human primary osteogenic sarcoma cells (Saos-2) were characterized on smooth, microstructured (MST) and micro- and nano-structured (MNST) polypropylene (PP) and on the same samples with a silicon-doped carbon nitride (C3N4-Si) coating. Injection molding was used to pattern the PP surfaces and the coating was obtained by using ultra-short pulsed laser deposition (USPLD). Surfaces were characterized using atomic force microscopy and surface energy components were calculated according to the Owens-Wendt model. The results showed C3N4-Si coated surfaces to be significantly more hydrophilic than uncoated ones. In addition, there were 86% more cells in the smooth C3N4-Si coated PP compared to smooth uncoated PP and 551%/476% more cells with MST/MNST C3N4-Si coated PP than could be obtained with MST/MNST uncoated PP. Thus the adhesion, spreading and contact guidance of osteoblast-like cells was effectively improved by combining surface texturing and deposition of osteocompatible C3N4-Si coating.