Nondestructive Assessment of Glycosaminoglycans in Engineered Cartilages Using Hexabrix-Enhanced Micro-Computed Tomography

It is very useful to evaluate the content and 3D distribution of extracellular matrix non-destructively in tissue engineering. This study evaluated the feasibility of using micro-computed tomography (µCT) with Hexabrix to measure quantitatively sulfated glycosaminoglycans (GAGs) of engineered cartilage. Rabbit chondrocytes at passage 2 were used to produce artificial cartilages in polyglycolic acid scaffolds in vitro. Engineered cartilages were incubated with Hexabrix 320 for 20 min and analyzed via µCT scanning. The number of voxels in the 2D and 3D scanning images were counted to estimate the amount of sulfated GAGs. The optimal threshold value for quantification was determined by regression analysis. The 2D µCT images of an engineered cartilage showed positive correlation with the histological image of Safranin-O staining. Quantitative data obtained with the 3D µCT images of 14 engineered cartilages showed strong correlation with sulfated GAGs contents obtained by biochemical analysis (R² = 0.883, p < 0.001). Repeated exposure of engineered cartilages to Hexabrix 320 and µCT scanning did not significantly affect cell viability, total DNA content, or the total content of sulfated GAGs. We conclude that µCT imaging using Hexabrix 320 provides high spatial resolution and sensitivity to assess the content and 3D distribution of sulfated GAGs in engineered cartilages. It is expected to be a valuable tool to evaluate the quality of engineered cartilage for commercial development in the future.

Repair of Partial Thickness Cartilage Defects Using Cartilage Extracellular Matrix Membrane-Based Chondrocyte Delivery System in Human Ex Vivo Model

Treatment options for partial thickness cartilage defects are limited. The purpose of this study was to evaluate the efficacy of the chondrocyte-seeded cartilage extracellular matrix membrane in repairing partial thickness cartilage defects. First, the potential of the membrane as an effective cell carrier was investigated. Secondly, we have applied the chondrocyte-seeded membrane in an ex vivo, partial thickness defect model to analyze its repair potential. After culture of chondrocytes on the membrane in vitro, cell viability assay, cell seeding yield calculation and cell transfer assay were done. Cell carrying ability of the membrane was also tested by seeding different densities of cells. Partial defects were created on human cartilage tissue explants. Cell-seeded membranes were applied using a modified autologous chondrocyte implantation technique on the defects and implanted subcutaneously in nude mice for 2 and 4 weeks. In vitro data showed cell viability and seeding yield comparable to standard culture dishes. Time dependent cell transfer from the membrane was observed. Membranes supported various densities of cells. Ex vivo data showed hyaline-like cartilage tissue repair, integrated on the defect by 4 weeks. Overall, chondrocyte-seeded cartilage extracellular membranes may be an effective and feasible treatment strategy for the repair of partial thickness cartilage defects.

Cartilage Repair Using Mesenchymal Stem Cells

Articular cartilage defect rarely heals spontaneously due to its avascularity and low cellularity. Even small articular cartilage defects can develop into osteoarthritis, and subsequently, its management has been a major clinical concern. Although there are several treatment options for cartilage defect, no treatment has been established as a gold standard procedure. Bone marrow stimulation techniques which is equivalent to microfracture these days has been adapted as first line treatment, attributed to their technical easiness and minimal invasiveness to patients. However, this procedure has limitation in reproducing hyaline cartilage, so recent cell-based therapies using autologous chondrocytes or mesenchymal stem cells have drawn particular attention. MSCs regardless of its origin have shown significant potential for chondrogenesis. Novel approaches using MSCs as an alternative cell source for patient derived chondrocytes are currently on trial. In this review, stem cells from various origins considered as cell sources and potential application of mesenchymal stem cells to promote cartilage repair will be discussed. While differentiation of stem cell can be well controlled in vitro, it is not easy to predict the course of differentiation when the stem cell is transplanted. Some novel methods using physical stimulation and material based techniques for differentiation control are introduced in this context. Such differentiation control will be beneficial when it is adapted before transplantation. We call it preconditioning.

A Study on the Potential of Hydroxyapatite Based Bioactive Bone Cement / 대한정형외과연구학회지

Study on the Potential of Hydroxapatite Based Bioactive Bone Cement PURPOSE: The purpose of this study is to propose a new bioactive bone cement (BBC) composed of bone powder (hydroxyapatite; HA), chitosan powder, and currently available polymethylmethacrylate (PMMA) bone cement for use in orthopaedic surgeries such as vertebroplasty or bone filler. MATERIALS AND METHODS: Three types of proposed BBCs and a currently available commercial PMMA were tested. In vitro studies the surface morphology, chemical composition, changes in pH value along the time, exothermic temperatures, intrusion and cellular responses were investigated. SEM, radiological and histological examinations were performed in animal studies. RESULTS: The major components of BBCs were C, O, Ca, P, Cl, Si, S, Ba and Mg. The pH values in BBCs decreased after 1 day, however they eventually reached 7.2-7.4. The water absorbency, weight loss, and porosity in BBCs increased more than PMMA more than during degradation (p<0.05). However, the compressive Young's moduli and ultimate compressive strength (UCS) of BBCs were lower than those of PMMA (<0.05). The exothermic temperatures of the BBCs were considerably lower than that of PMMA (p<0.05). In view of setting time, it takes relatively longer for BBCII and III to be solidified than PMMA (p<0.05). The intrusion tests showed that the BBCs were more intrusive than PMMA (p<0.05). The cell proliferation test on BBCII showed that the BBCII was more preferable than the PMMA. No cytotoxic characteristics were found in all BBCs. In the animal test, BBC II was more biocompatible as well as osteoconductible than the PMMA. CONCLUSION: The results of in vitro and animal studies indicated that the proposed BBCs have a potential of clinical application as replacement of the current PMMA bone cements.

The Biomechanical Responses of Intervertebral Disc due to Degenerative Change in Porcine Lumbar Spine / 대한정형외과연구학회지

PURPOSE: The biomechanical responses of degenerative porcine intervertebral disc were compared with those MATERIALS AND METHODS: Two groups were set; Group A (44.0+/-2.8 months old, female) and Group B (6.2 +/-1.3 months old, female). Histological (H&E stain) observations were carried out to see the degeneration for both groups. Then biomechanical responses were investigated by measuring height changes in disc, intradiscal pressure values and relaxation time for each specimen under axial compressive loads. RESULTS: Degenerative changes were confirmed through H&E staining in Group A. The ratios of the nucleus pulposus area to total area were 14.7+/-4.5% and 29.2+/-6.0% in Group A and B, respectively (p=0.000). The decrease rates in disc height were 12.1+/-3.3% and 21.6+/-7.6%, in Group A and B, respectively under the axial compression of 740 N (p=0.000). No significant difference in intradiscal pressure measured in anterior zone between-groups except at axial load of 740N (p> 0.05). However, significant difference in pressure was found in posterolateral zone when the load was 542 N and higher (542 N: p=0.015, 740 N: p=0.010). The average relaxation time for Group A was significantly longer than that for Group B at 740N, i.e., at maximum load (anterior: p=0.010, posterolateral: p=0.014). CONCLUSION: Different biomechanical responses in degenerative disc were confirmed. They are 1) less flexible, 2) slower in energy relaxation under axial loading, and 3) larger portion of the external load were taken up at posterior part of annulus fibrous, especially in degenerative disc.