Harri T. Kokkonen

Preparation and characterization of collagen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds for cartilage tissue engineering

In this study, three-dimensional (3D) porous scaffolds were developed for the repair of articular cartilage defects. Novel collagen/polylactide (PLA), chitosan/PLA, and collagen/chitosan/PLA hybrid scaffolds were fabricated by combining freeze-dried natural components and synthetic PLA mesh, where the 3D PLA mesh gives mechanical strength, and the natural polymers, collagen and/or chitosan, mimic the natural cartilage tissue environment of chondrocytes. In total, eight scaffold types were studied: four hybrid structures containing collagen and/or chitosan with PLA, and four parallel plain scaffolds with only collagen and/or chitosan. The potential of these types of scaffolds for cartilage tissue engineering applications were determined by the analysis of the microstructure, water uptake, mechanical strength, and the viability and attachment of adult bovine chondrocytes to the scaffolds. The manufacturing method used was found to be applicable for the manufacturing of hybrid scaffolds with highly porous 3D structures. All the hybrid scaffolds showed a highly porous structure with open pores throughout the scaffold. Collagen was found to bind water inside the structure in all collagen-containing scaffolds better than the chitosan-containing scaffolds, and the plain collagen scaffolds had the highest water absorption. The stiffness of the scaffold was improved by the hybrid structure compared to plain scaffolds. The cell viability and attachment was good in all scaffolds, however, the collagen hybrid scaffolds showed the best penetration of cells into the scaffold. Our results show that from the studied scaffolds the collagen/PLA hybrids are the most promising scaffolds from this group for cartilage tissue engineering.

Diffusion and near-equilibrium distribution of MRI and CT contrast agents in articular cartilage

Charged contrast agents have been used both in vitro and in vivo for estimation of the fixed charge density (FCD) in articular cartilage. In the present study, the effects of molecular size and charge on the diffusion and equilibrium distribution of several magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents were investigated. Full thickness cartilage disks (Ø = 4.0 mm, n = 64) were prepared from fresh bovine patellae. Contrast agent (gadopentetate: Magnevist((R)), gadodiamide: Omniscan, ioxaglate: Hexabrix or sodium iodide: NaI) diffusion was allowed either through the articular surface or through the deep cartilage. CT imaging of the samples was conducted before contrast agent administration and after 1, 5, 9, 16, 25 and 29 h (and with three samples after 2, 3, 4 and 5 days) diffusion using a clinical peripheral quantitative computed tomography (pQCT) instrument. With all contrast agents, the diffusion through the deep cartilage was slower when compared to the diffusion through the articular surface. With ioxaglate, gadopentetate and gadodiamide it took over 29 h for diffusion to reach the near-equilibrium state. The slow diffusion of the contrast agents raise concerns regarding the validity of techniques for FCD estimation, as these contrast agents may not reach the equilibrium state that is assumed. However, since cartilage composition, i.e. deep versus superficial, had a significant effect on diffusion, imaging of the nonequilibrium diffusion process might enable more accurate assessment of cartilage integrity.

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.

In vivo diagnostics of human knee cartilage lesions using delayed CBCT arthrography

The aim of this study was to investigate the feasibility of delayed cone beam (CBCT) arthrography for clinical diagnostics of knee cartilage lesions. Knee joints with cartilage lesions were imaged using native radiography, MRI, and delayed CBCT arthrography techniques in vivo. The joints were imaged three times with CBCT, just before, immediately after (arthrography) and 45 min after the intra‐articular injection of contrast agent. The arthrographic images enabled sensitive detection of the cartilage lesions. Use of arthrographic and delayed images together with their subtraction image enabled also detection of cartilage with inferior integrity. The contrast agent partition in intact cartilage (ICRS grade 0) was lower (p < 0.05) than that of cartilage surrounding the ICRS grade I–IV lesions. Delayed CBCT arthrography provides a novel method for diagnostics of cartilage lesions. Potentially, it can also be used in diagnostics of cartilage degeneration. Due to shorter imaging times, higher resolution, and lower costs of CT over MRI, this technique could provide an alternative for diagnostics of knee pathologies. However, for comprehensive evaluation of the clinical potential of the technique a further clinical study with a large pool of patients having a wide range of cartilage pathologies needs to be conducted.

Delayed Computed Tomography Arthrography of Human Knee Cartilage In Vivo

Objective: We investigated the feasibility of delayed computed tomography (CT) arthrography for evaluation of human knee cartilage in vivo. Especially, the diffusion of contrast agent out of the joint space and the optimal time points for imaging were determined. Design: Two patients were imaged using delayed CT arthrography and delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) techniques. Results: Two hours after injection, the concentration of contrast agent in the joint space was still high enough (20% to 24.5% of the initial concentration at 0 minutes) to allow delayed CT arthrography. The half-life of the contrast agent in the joint space varied from 30 to 60 minutes. The contrast agent concentration in patellar and femoral cartilage reached the maximum after 30 and 60 minutes, respectively. According to dGEMRIC, there were no differences between patients. However, in delayed CT arthrography, the penetration of the contrast agent was higher in the osteoarthritic knee cartilage. Conclusions: Contrast agent remained in the joint space long enough to enable delayed CT arthrography of cartilage. After 30 minutes, the normalized contrast agent concentration was higher in the cartilage of the osteoarthritic knee in comparison with the healthy knee. To conclude, delayed CT arthrography exhibited potential for use in the clinical evaluation of cartilage integrity.

Diffusion of ionic and non-ionic contrast agents in articular cartilage with increased cross-linking : contribution of steric and electrostatic effects

OBJECTIVE To investigate the effect of threose-induced collagen cross-linking on diffusion of ionic and non-ionic contrast agents in articular cartilage. DESIGN Osteochondral plugs (Ø=6mm) were prepared from bovine patellae and divided into two groups according to the contrast agent to be used in contrast enhanced computed tomography (CECT) imaging: (I) anionic ioxaglate and (II) non-ionic iodixanol. The groups I and II contained 7 and 6 sample pairs, respectively. One of the paired samples served as a reference while the other was treated with threose to induce collagen cross-linking. The equilibrium partitioning of the contrast agents was imaged after 24h of immersion. Fixed charge density (FCD), water content, contents of proteoglycans, total collagen, hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP) and pentosidine (Pent) cross-links were determined as a reference. RESULTS The equilibrium partitioning of ioxaglate (group I) was significantly (p=0.018) lower (-23.4%) in threose-treated than control samples while the equilibrium partitioning of iodixanol (group II) was unaffected by the threose-treatment. FCD in the middle and deep zones of the cartilage (p<0.05) and contents of Pent and LP (p=0.001) increased significantly due to the treatment. However, the proteoglycan concentration was not systematically altered after the treatment. Water content was significantly (-3.5%, p=0.007) lower after the treatment. CONCLUSIONS Since non-ionic iodixanol showed no changes in partition after cross-linking, in contrast to anionic ioxaglate, we conclude that the cross-linking induced changes in charge distribution have greater effect on diffusion compared to the cross-linking induced changes in steric hindrance.

Quantitative Evaluation of Knee Subchondral Bone Mineral Density Using Cone Beam Computed Tomography

Contrast agent enhanced cone beam computed tomography (CE-CBCT), a technique capable of high-resolution in vivo imaging with small radiation dose, has been applied successfully for clinical diagnostics of cartilage degeneration, i.e., osteoarthritis (OA). As an X-ray technique, CE-CBCT may also detect changes in mineral density of subchondral bone (volumetric bone mineral density, vBMD), known to be characteristic for OA. However, its feasibility for density measurements is not clear due to limited signal-to-noise ratio and contrast of CBCT images. In the present study, we created clinically applicable hydroxyapatite phantoms and determined vBMDs of cortical bone, trabecular bone, subchondral trabecular bone and subchondral plate of 10 cadaver (ex vivo) and 10 volunteer (in vivo) distal femora using a clinical CBCT scanner, and for reference, also using a conventional CT scanner. Our results indicated strong linear correlations between the vBMD values measured with the CT and CBCT scanners , however, absolute vBMD values were dependent on the scanner in use. Further, the differences between the vBMDs of cortical bone, trabecular bone and subchondral bone were similar and independent of the scanner. The present results indicate that vBMD values might not be directly comparable between different instruments. However, based on our present and previous results, we propose that, for OA diagnostics, clinical CBCT enables not only quantitative analysis of articular cartilage but also subchondral bone vBMD. Quantitative information on both cartilage and subchondral bone could be beneficial in OA diagnostics.

Contrast-Enhanced Computed Tomography Enables Quantitative Evaluation of Tissue Properties at Intrajoint Regions in Cadaveric Knee Cartilage:

Objective The aim of this study was to investigate whether the concentration of the anionic contrast agent ioxaglate, as quantitated by contrast-enhanced computed tomography (CECT) using a clinical cone-beam CT (CBCT) instrument, reflects biochemical, histological, and biomechanical characteristics of articular cartilage imaged in an ex vivo, intact human knee joint. Design An osteoarthritic human cadaveric knee joint (91 years old) was injected with ioxaglate (36 mg I/mL) and imaged using CBCT over 61 hours of ioxaglate diffusion into cartilage. Following imaging, the joint surfaces were excised, rinsed to remove contrast agent, and compressive stiffness (equilibrium and instantaneous compressive moduli) was measured via indentation testing (n = 17 sites). Each site was sectioned for histology and assessed for glycosaminoglycan content using digital densitometry of Safranin-O stained sections, Fourier transform infrared spectroscopy for collagen content, and morphology using both the Mankin and OARSI semiquantitative scoring systems. Water content was determined using mass change after lyophilization. Results CECT attenuation at all imaging time points, including those <1 hour of ioxaglate exposure, correlated significantly (P < 0.05) with cartilage water and glycosaminoglycan contents, Mankin score, and both equilibrium and instantaneous compressive moduli. Early time points (<30 minutes) also correlated (P < 0.05) with collagen content and OARSI score. Differences in cartilage quality between intrajoint regions were distinguishable at diffusion equilibrium and after brief ioxaglate exposure. Conclusions CECT with ioxaglate affords biochemical and biomechanical measurements of cartilage health and performance even after short, clinically relevant exposure times, and may be useful in the clinic as a means for detecting early signs of cartilage pathology.

Nondestructive fluorescence-based quantification of threose-induced collagen cross-linking in bovine articular cartilage

Abstract. Extensive collagen cross-linking affects the mechanical competence of articular cartilage: it can make the cartilage stiffer and more brittle. The concentrations of the best known cross-links, pyridinoline and pentosidine, can be accurately determined by destructive high-performance liquid chromatography (HPLC). We explore a nondestructive evaluation of cross-linking by using the intrinsic fluorescence of the intact cartilage. Articular cartilage samples from bovine knee joints were incubated in threose solution for 40 and 100 h to increase the collagen cross-linking. Control samples without threose were also prepared. Excitation-emission matrices at wavelengths of 220 to 950 nm were acquired from the samples, and the pentosidine and pyridinoline cross-links and the collagen concentrations were determined using HPLC. After the threose treatment, pentosidine and lysyl pyridinole (LP) concentrations increased. The intrinsic fluorescence, excited below 350 nm, decreased and was related to pentosidine [r=−0.90, 240/325  nm (excitation/emission)] or LP (r=−0.85, 235/285  nm) concentrations. Due to overlapping, the changes in emission could not be linked specifically to the recorded cross-links. However, the fluorescence signal enabled a nondestructive optical estimate of changes in the pentosidine and LP cross-linking of intact articular cartilage.