Recellularization of auricular cartilage via elastase-generated channels.

Decellularized tissue matrices are promising substrates for tissue generation by stem cells to replace poorly regenerating tissues such as cartilage. However, the dense matrix of decellularized cartilage impedes colonisation by stem cells. Here, we show that digestion of elastin fibre bundles traversing auricular cartilage creates channels through which cells can migrate into the matrix. Human chondrocytes and bone marrow-derived mesenchymal stromal cells efficiently colonise elastin-treated scaffolds through these channels, restoring a glycosaminoglycan-rich matrix and improving mechanical properties while maintaining size and shape of the restored tissue. The scaffolds are also rapidly colonised by endogenous cartilage-forming cells in a subcutaneously implanted osteochondral biopsy model. Creating channels for cells in tissue matrices may be a broadly applicable strategy for recellularization and restoration of tissue function.

An automated algorithm for the detection of cortical interruptions and its underlying loss of trabecular bone; a reproducibility study.

BACKGROUND: We developed a semi-automated algorithm that detects cortical interruptions in finger joints using high-resolution peripheral quantitative computed tomography (HR-pQCT), and extended it with trabecular void volume measurement. In this study we tested the reproducibility of the algorithm using scan/re-scan data. METHODS: Second and third metacarpophalangeal joints of 21 subjects (mean age 49 (SD 11) years, 17 early rheumatoid arthritis and 4 undifferentiated arthritis, all diagnosed < 1 year ago) were imaged twice by HR-pQCT on the same day with repositioning between scans. The images were analyzed twice by one operator (OP1) and once by an additional operator (OP2), who independently corrected the bone contours when necessary. The number, surface and volume of interruptions per joint were obtained. Intra- and inter-operator reliability and intra-operator reproducibility were determined by intra-class correlation coefficients (ICC). Intra-operator reproducibility errors were determined as the least significant change (LSCSD). RESULTS: Per joint, the mean number of interruptions was 3.1 (SD 3.6), mean interruption surface 4.2 (SD 7.2) mm2, and mean interruption volume 3.5 (SD 10.6) mm3 for OP1. Intra- and inter-operator reliability was excellent for the cortical interruption parameters (ICC ≥0.91), except good for the inter-operator reliability of the interruption surface (ICC = 0.70). The LSCSD per joint was 4.2 for the number of interruptions, 5.8 mm2 for interruption surface, and 3.2 mm3 for interruption volume. CONCLUSIONS: The algorithm was highly reproducible in the detection of cortical interruptions and their volume. Based on the LSC findings, the potential value of this algorithm for monitoring structural damage in the joints in early arthritis patients needs to be tested in clinical studies.

Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.

AIMS: Combining mesenchymal stem cells (MSCs) and chondrocytes has great potential for cell-based cartilage repair. However, there is much debate regarding the mechanisms behind this concept. We aimed to clarify the mechanisms that lead to chondrogenesis (chondrocyte driven MSC-differentiation versus MSC driven chondroinduction) and whether their effect was dependent on MSC-origin. Therefore, chondrogenesis of human adipose-tissue-derived MSCs (hAMSCs) and bone-marrow-derived MSCs (hBMSCs) combined with bovine articular chondrocytes (bACs) was compared. METHODS: hAMSCs or hBMSCs were combined with bACs in alginate and cultured in vitro or implanted subcutaneously in mice. Cartilage formation was evaluated with biochemical, histological and biomechanical analyses. To further investigate the interactions between bACs and hMSCs, (1) co-culture, (2) pellet, (3) Transwell® and (4) conditioned media studies were conducted. RESULTS: The presence of hMSCs-either hAMSCs or hBMSCs-increased chondrogenesis in culture; deposition of GAG was most evidently enhanced in hBMSC/bACs. This effect was similar when hMSCs and bAC were combined in pellet culture, in alginate culture or when conditioned media of hMSCs were used on bAC. Species-specific gene-expression analyses demonstrated that aggrecan was expressed by bACs only, indicating a predominantly trophic role for hMSCs. Collagen-10-gene expression of bACs was not affected by hBMSCs, but slightly enhanced by hAMSCs. After in-vivo implantation, hAMSC/bACs and hBMSC/bACs had similar cartilage matrix production, both appeared stable and did not calcify. CONCLUSIONS: This study demonstrates that replacing 80% of bACs by either hAMSCs or hBMSCs does not influence cartilage matrix production or stability. The remaining chondrocytes produce more matrix due to trophic factors produced by hMSCs.

Quantitative morphometric patterns in cartilage and bone from the humeral heads of end-stage osteoarthritis patients.

OBJECTIVE: The purpose of this work is to investigate in a quantitative manner, the gross and regional structural patterns in cartilage and bone from the humeral head of end-stage OA patients, with the goal of identifying patterns of disease. Since the prevalence of primary OA of the shoulder is increasing as the population ages and the non-traumatic degenerative changes leading to this disease are poorly understood, a site-specific morphometric analysis speaks to the structure-function remodelling relationship of the pathological anatomy. METHODS: Humeral heads were harvested from twenty-one patients undergoing shoulder arthroplasty for end-stage primary OA. The samples were scanned with micro-computed tomography and magnetic resonance imaging (MRI), and registered to provide reconstructed 3D datasets of the cartilage, cortical and trabecular bone tissues. Gross visual examination of the datasets allowed samples to be classified as OA-like, osteoporosis (OP)-like or OA/OP-like. RESULTS: Volumes of interest (VOI) separating the OA-like samples into five distinct regions showed positive correlations between bone and cartilage morphometric parameters; specifically in areas where more cartilage has been lost, the underlying subchondral cortical bone was more porous and thicker, while the subchondral trabecular bone was more dense, including more connections and trabeculae. These differences were site-specific, where the central humeral head saw the greatest destruction of cartilage and bone sclerosis, followed by the anterior aspects. CONCLUSION: The ability to correlate bone and cartilage changes is valuable, as these structural cues may allow a more targeted diagnostic approach and a better classification of the disease, leading to improved therapies.

The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage.

Cartilage has limited self-regenerative capacity. Tissue engineering can offer promising solutions for reconstruction of missing or damaged cartilage. A major challenge herein is to define an appropriate cell source that is capable of generating a stable and functional matrix. This study evaluated the performance of culture-expanded human chondrocytes from ear (EC), nose (NC) and articular joint (AC), as well as bone-marrow-derived and adipose-tissue-derived mesenchymal stem cells both in vitro and in vivo. All cells (≥ 3 donors per source) were culture-expanded, encapsulated in alginate and cultured for 5 weeks. Subsequently, constructs were implanted subcutaneously for 8 additional weeks. Before and after implantation, glycosaminoglycan (GAG) and collagen content were measured using biochemical assays. Mechanical properties were determined using stress-strain-indentation tests. Hypertrophic differentiation was evaluated with qRT-PCR and subsequent endochondral ossification with histology. ACs had higher chondrogenic potential in vitro than the other cell sources, as assessed by gene expression and GAG content (p < 0.001). However, after implantation, ACs did not further increase their matrix. In contrast, ECs and NCs continued producing matrix in vivo leading to higher GAG content (p < 0.001) and elastic modulus. For NC-constructs, matrix-deposition was associated with the elastic modulus (R² = 0.477, p = 0.039). Although all cells--except ACs--expressed markers for hypertrophic differentiation in vitro, there was no bone formed in vivo. Our work shows that cartilage formation and functionality depends on the cell source used. ACs possess the highest chondrogenic capacity in vitro, while ECs and NCs are most potent in vivo, making them attractive cell sources for cartilage repair.

Multimodal imaging demonstrates concomitant changes in bone and cartilage after destabilisation of the medial meniscus and increased joint laxity.

OBJECTIVE: Alterations in joint mechanics can cause osteoarthritis, which results in degeneration of both cartilage and bone tissue. The objective of this work is to measure changes in the laxity of the mouse knee joint after destabilisation of the medial meniscus (DMM) and to visualise and quantify the resulting three-dimensional changes in the bone and cartilage. METHODS: Skeletally mature C57Bl6 male mice underwent DMM surgery in the right leg. Animals were sacrificed immediately 0 weeks (n=15), 4 weeks (n=11) or 8 weeks (n=12) after surgery. For the 0-week group, the anterior-posterior (AP) and varus-valgus laxity of the DMM limb were compared to the contralateral limb. For 4 and 8-week groups, tibiae were scanned with micro-computed tomography (µCT) to quantify and visualise bone changes and with confocal scanning laser microscopy (CSLM) to measure changes in cartilage. RESULTS: Laxity testing measured an increase in AP range of motion, particularly in the anterior direction. The DMM limbs showed a decrease in epiphyseal trabecular bone at 8 weeks and a decrease in cartilage volume, primarily on the posterior medial plateau, compared to the contralateral limb. Significant bone remodelling was observed at the periphery of the joint and in severe cases, osteolysis extended through the growth plate. CONCLUSION: Multimodal imaging allowed quantifiable 3D assessment of bone and cartilage and indicated extensive changes in the tissues. The increase in AP laxity suggests that DMM surgery redistributes loading posteriorly on the medial plateau, resulting in bone and cartilage loss primarily on the posterior portion of the medial plateau.

Conceptual fracture parameters for articular cartilage.

BACKGROUND: Superficial cracks can occur in articular cartilage due to trauma or wear and tear. Our understanding of the behaviour of such cracks in a loaded matrix is limited. A notable study investigated the growth of cracks induced in the bottom layer of the matrix. This paper extends existing studies, characterizing the propagation of superficial cracks and matrix resistance under tension at varying rates of loading. METHODS: Cartilage strips with artificially induced superficial cracks were subjected to tensile loading at different loading velocities using a miniature tensile testing device. Load-displacement data, video and still images were recorded for analysis. FINDINGS: The propagation of superficial cracks in articular cartilage does not follow the classical crack tip advance that is characteristic of most engineering materials. Instead, the crack tip exhibited a negligible movement while the side edges of the crack rotated about it, accompanied by matrix stretching and an upward pull (necking) of the bottom layer of the sample. As loading progresses, the crack edges stretch and rotate to assume a position parallel to the articular surface, followed by the final fracture of the matrix at a point just below the crack tip. Using the recorded mechanical data and images, an analogous poroelastic fracture toughness, Kp(Ic)=1.83 MPa.square root mm (SD 0.8) is introduced. INTERPRETATION: It is extremely difficult for a superficial crack to propagate through articular cartilage. This may be because of the energy dissipation from the crack due to the movement and exudation of water, and large stretching of the matrix.

A qualitative analysis of crack propagation in articular cartilage at varying rates of tensile loading.

A custom-built miniature tensile testing apparatus was used to study the propagation of cracks through the articular cartilage matrix at various loading rates and initial crack lengths. The crack propagation mechanism was observed to be significantly dissimilar to that normally seen in traditional fracture mechanics opening mode, where fracture propagates through the thickness of samples or perpendicularly to the applied load. Instead, an artificially initiated microcrack in the surface layer of an articular cartilage sample grew laterally in the direction of the applied load, stretching about the crack tip, whose initial position remained unchanged throughout the fracture process. A progressive upward pull of the bottom layer toward the surface, which resulted in necking of the specimen, was observed. Our analysis revealed that the rate of necking was the same as that of the lateral stretch of the growing crack. We hypothesize that necking is due to the response of the collagen meshwork especially in the deep zones of the matrix to the tensile load. Our samples exhibited unstable fracture growth immediately after each microcrack grew to the base of the articular surface layer, with very fast crack propagation to failure, thereby indicating that the fracture toughness of the articular cartilage matrix is significantly determined by the toughness of its articular surface.