Electron microscopic observation of human auricular chondrocytes transplanted into peritoneal cavity of nude mice for cartilage regeneration

Restoration of damaged cartilage tissue has been deemed futile with current treatments. Although there have been many studies on cartilage regeneration thus far, there is no report that chondrocytes were completely re-differentiated in vitro. The clarification of cellular composition and matrix production during cartilage regeneration must be elucidated to fabricate viable mature cartilage in vitro. In order to achieve this aim, the chondrocytes cultured on coverslips were transplanted into the peritoneal cavities of mice. At different time points post-transplantation, the cartilage maturation progression and cells composing the regeneration were examined. Cartilage regeneration was confirmed by hematoxylin & eosin (HE) and toluidine blue staining. The maturation progression was carefully examined further by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). At the first and second week time points, various cell shapes were observed using SEM. Chronologically, by the third week, the number of fibers increased, suggesting the progression of extracellular matrix (ECM) maturation. Observation through TEM revealed the chondrocytes located in close proximity to various cells including macrophage-like cells. On the second week, infiltration of lymphocytes and capillary vessels were observed, and after the third week, the chondrocytes had matured and were abundantly releasing extracellular matrix. Chronological observation of transplanted chondrocytes by electron microscopy revealed maturation of chondrocytes and accumulation of matrix during the re-differentiation process. Emerging patterns of host-derived cells such as macrophage-like cells and subsequent appearance of lymphocytes-like cells and angiogenesis were documented, providing crucial context for the identification of the cells responsible for in vivo cartilage regeneration. Keywords: Chondrocytes, Macrophage, Lymphocyte, Endothelial cells, Peritoneal cavity, Electron microscop

Improving chondrocyte harvests with poly(2-hydroxyethyl methacrylate) coated materials in the preparation for cartilage tissue engineering

Remarkable advances have been made in cartilage regenerative medicine to cure congenital anomalies including microtia, tissue defects caused by craniofacial injuries, and geriatric diseases such as osteoarthritis. However, those procedures require a substantial quantity of chondrocytes for tissue engineering. Previous studies have required several passages to obtain sufficient cell numbers for three-dimensional and monolayer cultures. Thus, our objective was to improve the quantity of chondrocytes that can be obtained by examining an anti-fouling polyhydrophilic chemical called poly(2-hydroxyethyl methacrylate) (pHEMA). To determine the effectiveness of the chemical, pHEMA solution was applied via dip-coating to centrifuge tubes, serological pipettes, and pipette tips. The cell quantity obtained during standard cell culturing and passaging procedures was measured alongside non-coated materials as a control. A significant 2.2-fold increase of chondrocyte yield was observed after 2 passages when pHEMA was applied to the tubes compared to when non-coated tubes were utilized. The 3-dimensional chondrocyte pellets prepared from the respective cell populations and transplanted into nude mice were histologically and biochemically analyzed. No evidence of difference in matrix production for in vitro and in vivo cultures was found as well as similar proliferation rates and colony formation abilities. The use of pHEMA provides a powerful alternative method for expanding the quantity of chondrocytes harvested and handled during cell isolation and passaging to enhance cartilage tissue engineering

Perilipin 2 downregulation in β cells impairs insulin secretion under nutritional stress and damages mitochondria

Perilipin 2 (PLIN2) is a lipid droplet (LD) protein in β cells that increases under nutritional stress. Downregulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was downregulated in mouse β cells, INS1 cells, and human islet cells. β Cell–specific deletion of PLIN2 in mice on a high-fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Downregulation of PLIN2 in INS1 cells blunted GSIS after 24-hour incubation with 0.2 mM palmitic acid. Downregulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Downregulation of PLIN2 decreased specific OXPHOS proteins in all 3 models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2-deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress, as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells has an important role in preserving insulin secretion, β cell metabolism, and mitochondrial function under nutritional stress