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.

Infrared microspectroscopic determination of collagen cross-links in articular cartilage.

Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples ( n = 27 ) were treated with threose to increase the collagen cross-linking while the other half ( n = 27 ) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r = 0.84 ( p < 0.001 ), r = 0.87 ( p < 0.001 ) and r = 0.92 ( p < 0.001 ) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

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.

Correlation of Subchondral Bone Density and Structure from Plain Radiographs with Micro Computed Tomography Ex Vivo.

Osteoarthritis causes changes in the subchondral bone structure and composition. Plain radiography is a cheap, fast, and widely available imaging method. Bone tissue can be well seen from plain radiograph, which however is only a 2D projection of the actual 3D structure. Therefore, the aim was to investigate the relationship between bone density- and structure-related parameters from 2D plain radiograph and 3D bone parameters assessed from micro computed tomography (µCT) ex vivo. Right tibiae from eleven cadavers without any diagnosed joint disease were imaged using radiography and with µCT. Bone density- and structure-related parameters were calculated from four different locations from the radiographs of proximal tibia and compared with the volumetric bone microarchitecture from the corresponding regions. Bone density from the plain radiograph was significantly related with the bone volume fraction (r = 0.86; n = 44; p < 0.01). Mean homogeneity index for orientation of local binary patterns (HI(angle,mean)) and fractal dimension of vertical structures (FD(Ver)) were related (p < 0.01) with connectivity density (HI(angle,mean): r = -0.73, FD(Ver): r = 0.69) and trabecular separation (HI(angle,mean): r = 0.73, FD(Ver): r = -0.70) when all ROIs were pooled together (n = 44). Bone density and structure in tibia from standard clinically available 2D radiographs are significantly correlated with true 3D microstructure of bone.

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.

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.

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

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.

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.