Improved chondrocyte morphology and glycogen retention in the secondary center of ossification following osmium-potassium ferrocyanide fixation

The use of osmium-potassium ferrocyanide as the secondary fixative greatly improved chondrocyte preservation and stabilized the cartilage matrix proteoglycan. The proteoglycan was similar in appearance to that seen following fixation in the presence of cationic dyes. Extensive glycogen preservation was noted in these cells, occupying the area prior to and during the formation of the secondary center of ossification. The volume and organization of the glycogen within the cell cytoplasm were greater than that following buffered osmium fixation, and the cellular vacuoles within were greatly reduced. The cells forming the secondary center prior to the onset of mineralization were of greatest interest, because other studies compared them with the primary growth plate and described them as showing signs of hypertrophy as early as 5 days postnatally, as is found in the primary growth plate. Our observations indicate that glycogen is present in these cells, and cellular enlargement was not present. The cells do not resemble the hypertrophic chondrocytes of the primary growth plate, as far as cytoplasmic content is concerned, and we suggest that they may contribute to the development of the secondary center in a different fashion.     

Autologous resurfacing

Background: There is a discrepancy between the interest in joint‐reconstructions and the current knowledge about the healing‐processes involved. Major reconstructions are performed with osteosynthesized allografts and fresh allografts for cartilage. Objectives: The main question to be answered is: what do we know about metaphyseal and epiphyseal cancellous bone healing, contact healing of the subchondral bone and its influence on cartilage healing? Can we achieve healing of all four compartments in contact? Purpose: The purpose is to systematically investigate through animal testing the healing processes of metaphyseal and epiphyseal bone, including the subchondral bone and the healing of cartilage of press‐fit‐inserted grafts, considering nondemineralized high‐resolution histology. Material and Methods: Primary cancellous‐bone healing of osteosynthesized hemi‐osteotomies was studied in 13 canine tibial heads, the contact healing was investigated in 7 dogs and 18 giant‐rabbits comparing contact‐healing of press‐fit‐inserted autologs cylindrical grafts with empty defects applying the wet‐grinding diamond‐technology. Bench‐experiments on the epiphyseal bones of swine including pullout‐tests of cylindrical grafts formed the basis for validation of that press‐fit diamond technology. Results: Primary metaphyseal and epiphyseal contact healing, including hyaline cartilage, was found in all compartments of the meta‐and epiphysis in the precisely performed experiments. The press‐fit principle, which employs cylindrical grafts and diamond instrumentation featuring a difference of 15/100 mm between graft and recipient bed, achieved high loads between 73.48 and 178.95 N (mean value 118.16 and standard deviation 32.79) in the pullout tests. Conclusion: Autologous press‐fit grafting with alignment of the bony baseplate using wet‐grinding precision has attained promising histo‐morphological results. Microsc. Res. Tech., 78:40–51, 2015. © 2014 Wiley Periodicals, Inc.     

Response of the donor and recipient cells in mesenchymal cell transplantation to cartilage defect

To facilitate the repair of articular cartilage defects, autologous mesenchymal cells from bone marrow or periosteum were transplanted in a rabbit model. Two weeks after the transplantation of the mesenchymal cells, the whole area of the original defect was occupied by cartilage. From the deep area of the reparative cartilage, which contacted with host bone, chondrocytes became hypertrophic and the invasion of bone with vasculature started, until the replacement reached the natural junction of the host cartilage and the subchondral bone about 4 weeks after transplantation. Twelve weeks after the transplantation, the repair cartilage in the defect became a little thinner than the adjacent normal cartilage, which became a little thinner 24 weeks after the transplantation (the longest observation period in the study). Large, full-thickness defects of the weight-bearing region of the articular cartilage were repaired with hyaline-like cartilage after implantation of autologous mesenchymal cells. The repair process by mesenchymal cell transplantation was explained as follows: The donor transplanted cell differentiated into cartilage and the defects were completely filled with cartilage. Then, mesenchymal cells that entered the chondrogenic lineage rapidly progressed through this lineage to the hypertrophic state, which was then the target for erosion and vascular invasion. Although this vasculature and the newly formed bone were considered to be host-derived, there was no evidence to that effect. To prove this, suitable experimental marking of these donor cells is needed. In the case of chondrocyte transplantation, the repair cartilage maintained its thickness to the full depth of the original defect; the tissue derived from the implanted chondrocytes was not invaded by vessels or replaced by subchondral bone.     

The fate of implanted autologous chondrocytes in regenerated articular cartilage

Autologous chondrocyte implantation (ACI) is used to treat some articular cartilage defects. However, the fate of the cultured chondrocytes after in-vivo transplantation and their role in cartilage regeneration remains unclear. To monitor the survival and fate of such cells in vivo, the chondrocytes were labelled with a lipophilic dye and the resultant regenerated tissue in dogs examined. It was found that, 4 weeks after implantation, the osteochondral defects were filled with regenerative tissue that resembled hyaline cartilage. Fluorescence microscopy of frozen sections of the regenerated tissue revealed that the majority of cells were derived from the DiI-labelled implanted chondrocytes. From these results, it was concluded that a large population of implanted autologous chondrocytes can survive at least 4 weeks after implantation and play a direct role in cartilage regeneration. However, it remains unknown whether other cells, such as periosteal cells or bone marrow stromal stem cells, are involved in the regeneration of cartilage after ACI.     

Cell and matrix morpho-functional analysis in chondrocyte micromasses

Micromass cultures represent a convenient means of studying chondrocyte physiology in the context of a tridimensional culture model. In this study, we present the first ultrastructural analysis of the distribution and organization of the extracellular components in micromasses in comparison with their cartilaginous counterparts. Primary chondrocytes obtained from osteoarthritis patients were pelleted in micromasses. Transmission electron microscopy and immunofluorescence were used to evaluate the distribution of major extracellular matrix proteins, i.e., aggrecan, chondroitin-4-sulfate, chondroitin-6-sulfate, and collagen I and II. Both approaches revealed a number of morphological features shared by micromass and cartilage chondrocytes. In particular, in micromasses, chondrocytes are in close contact with an organized extracellular matrix that adequately mimics that of cartilage. Cells were observed to establish specialized junctions for cell-extracellular matrix crosstalk. Noteworthy, cells seem endowed in a chondroitin sulfate-rich microenvironment, and thus possibly ensuring the immobilization of chemokines, a family of molecules emerging in osteoarthritis pathogenesis, in a haptotactic-like gradient to the chondrocytes, which facilitates the binding to their receptors. To determine the suitability of this model to investigate osteoarthritis pathogenesis, a potential apoptotic stimulus (endothelial IL-8) was used, and ultrastructural analysis assessed apoptosis induction. Micromass cultures were proved to be an experimental technique providing a large number of properly differentiated chondrocytes, and thus allowing reliable biochemical and morphological studies. They represent, therefore, a novel approach to osteoarthritis investigation that promises more thorough understanding of chondrocyte physiology in osteoarthritis.     

A new role for chondrocytes as non-professional phagocytes. An in vitro study

Chondrocytes are capable of engulfing latex particles, cell detritus, and necrotic and apoptotic remains in vitro. It is conceivable that chondrocytes might be involved in the clearance by phagocytosis of different materials within the cartilage. In fact, so far there is no evidence for the presence of "professional phagocytes" (macrophages and neutrophils) in this tissue. Chondrocyte suspensions obtained from rat knees and hips were cultured to assess phagocytosis of latex particles (1 microm), articular cartilage detritus, and necrotic and apoptotic chondrocyte remains (induced by VP-16 1 mM). We observed that chondrocytes phagocytosed latex particles as evaluated by confocal microscopy and flow cytometry. In addition, we observed that chondrocytes phagocytosed articular cartilage detritus and necrotic and apoptotic VP-16 induced-chondrocytes, as observed by bright field microscopy and transmission electron microscopy.     

Preparation of thin cryo-sections for electron probe analysis of calcifying cartilage

Conventional methods of fixation, dehydration, embedding and wet-sectioning can produce artefacts in the chemical composition of mineralizing tissues. Cryoultramicrotomy was adopted for a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Fresh rabbit epiphyseal cartilage was mounted on silver pins, frozen by immersion in liquid nitrogen, and sectioned with the specimen temperature at 133 K and the knife temperature at 273 K. Dry cryo-sections (30-70 nm in thickness) were manipulated on to coated grids and examined the same day. These cryo-sections showed good morphological and cytoplasmic detail, with large areas relatively free of ice-crystal damage. Sections stained either with osmium vapour or negatively stained with silicotungstic acid showed areas with well-preserved mitochondria with granules and endoplasmic reticulum. Unstained sections also showed dense granules (50-120 nm in diameter) in the mitochondria of chondrocytes and preliminary electron probe analysis of these has indicated a Ca/P mass ratio of approximately 1.14. In the longitudinal septa, about 2 micrometer away from the chondrocytes, matrix-vesicle-like particles could be seen with crystal needles inside them. Micro-analysis of two of these gave a Ca/P mass ratio of 1.73 and 2.68. Cryo-ultramicrotomy appears to confirm a number of conclusions derived from conventional ultrastructural study of growth cartilage and suggests for the first time how amorphous calcium phosphate and crystalline apatite can be shown to exist in different organelles in the same cryo-section of the tissue.     

Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture

Tissue engineering is a promising approach for articular cartilage repair; however, it still has proven a challenge to produce tissue from the limited number of cells that can be extracted from a single individual. Relatively few cell expansion methods exist without the problems of dedifferentiation and/or loss of potency. Previously, it has been shown that mechanical vibrations can enhance chondrocyte proliferation in monolayer culture. Thus, it was hypothesized that chondrocytes grown in high-density culture would respond in a similar fashion while maintaining phenotypic stability. Isolated bovine articular chondrocytes were seeded in high-density culture on Millicell filters and subjected to mechanical vibrations 48 h after seeding. Mechanical vibrations enhanced chondrocyte proliferation at frequencies above 350 Hz, with the peak response occurring at a 1g amplitude for a duration of 30 min. Under these conditions, the gene expression of cartilage-specific and dedifferentiation markers (collagen II, collagen I, and aggrecan) were unchanged by the imposed stimulus. To determine the effect of accumulated extracellular matrix (ECM) on this proliferative response, selected cultures were stimulated under the same conditions after varying lengths of preculture. The amount of accumulated ECM (collagen and proteoglycans) decreased this proliferative response, with the cultures becoming insensitive to the stimulus after 1 week of preculture. Thus, mechanical vibration can serve as an effective means preferentially to stimulate the proliferation of chondrocytes during culture, but its effects appear to be limited to the early stages where ECM accumulation is at a minimum.     

Alteration of cartilage matrix morphology with histological processing

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neontal rats was seen by light microscopy to: (1) consist of elements, 0·5 μm thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.     

Histochemical evidences on the chronological alterations of the hypertrophic zone of mandibular condylar cartilage

The hypertrophic chondrocytes lack the ability to proliferate, thus permitting matrix mineralization as well as vascular invasion from the bone in both the mandibular condyle and the epiphyseal cartilage. This study attempted to verify whether the histological appearance of the hypertrophic chondrocytes is in a steady state during postnatal development of the mouse mandibular condyle. Type X collagen immunohistochemistry apparently distinguished the fibrous layer described previously as the "articular zone," "articular layer," and "resting zone" from the hypertrophic zone. Interestingly, the ratio of the type X collagen-positive hypertrophic zone in the entire condyle seemed higher in the early stages but decreased in the later stages. Some apparently compacted cells in the hypertrophic zone showed proliferating cell nuclear antigen (PCNA) immunoreaction, indicating the potential for cell proliferation at the early stages. As the mice matured, in contrast, they further enlarged and assumed typical features of hypertrophic chondrocytes. Apoptotic cells were also discernible in the hypertrophic zone at the early but not later stages. Consistent with morphological configurations of hypertrophic chondrocytes, immunoreactions for alkaline phosphatase, osteopontin, and type I collagen were prominent at the later stage, but not the early stage. Cartilaginous matrices demonstrated scattered patches of mineralization at the early stage, but increased in their volume and connectivity at the later stage. Thus, the spatial and temporal occurrence of these immunoreactions as well as apoptosis likely reflect the prematurity of hypertrophying cells at the early stage, and imply a physiological relevance during the early development of the mandibular condyles.     

Chondrocytes interconnecting tracks and cytoplasmic projections observed within the superficial zone of normal human articular cartilage--a transmission electron microscopy, atomic force microscopy, and two-photon excitation microscopy studies

The morphology of the normal human and rat articular cartilage was assessed using transmission electron microscopy (TEM), atomic force microscopy (AFM), and two-photon excitation (2PE) microscopy. Spurr-embedded sections from fixed human cartilage were simultaneously evaluated using TEM and AFM. The presences of tracks among the chondrocytes from the superficial zone of the cartilage were observed. In order to ratify the presence of interconnecting tracks among superficial zone chondrocytes, whole fixed human and rat cartilage, as well as fresh whole rat cartilage, were examined under the 2PE. In all cases, these tracks were observed. In addition, porous matrix, well-defined lacunae, and cytoplasmic projections anchored to the extracellular matrix (ECM) were also detected. We conclude that normal human and rat flattened superficial chondrocytes might be interconnected by tracks running through the ECM. In addition, cytoplasmic projections were observed anchored to the ECM. All these structures may possibly be related to cell/cell and ECM/chondrocytes signaling. Our findings provide new information that possibly will be of relevant importance for a more profound study of normal cartilage physiology and eventually, the pathogenesis of osteoarthritis.     

The effects of ionizing radiation on the growth plate in rat tibiae

The deleterious effects of ionizing radiation on the growth plate continue to be cause for concern. This study evaluated the ionizing radiation effects on bone development and growth plate in the tibia of rats. All animals were submitted to ionizing radiation on the left leg. The animals were divided into two groups and euthanized 30 and 60 days after radiation. The tibiae were removed and separated into groups: control 30 days, irradiated 30 days, control 60 days and irradiated 60 days. Animals in each group (n = 7) were used for macroscopic and histological analysis. The irradiated tibiae showed arrested growth, angular deformity and limb length discrepancy when compared with nonirradiated tibiae. There was statistical difference between control and radiation groups in all the parameters analyzed, except in the lateral‐medial thickness of the distal epiphysis. Histological analysis showed evident changes in the growth plate, which was thicker in the Groups irradiated for 30 days, and irradiated for 60 days, compared with their respective controls. The growth plate showed wide areas with disorganized zones of chondrocytes and severely reduced calcification zone. It was concluded that ionizing radiation damaged the growth plate, compromised the endochondral ossification process, and resulted in complete arrest of bone development.     

Tumor necrosis factor-alpha induced DNA cleavage in human articular chondrocytes may involve multiple endonucleolytic activities during apoptosis

Apoptosis has been documented in chondrocytes both in the growth plates of young, healthy cartilages and in osteoarthritic cartilages; little, however, is known about apoptosis in chondrocytes of normal adult articular cartilage. For the current study, apoptosis in adult chondrocytes was evaluated by labeling DNA fragments using the ISEL in situ end labeling of 3'-recessed strand breaks) or TUNEL (5'-recessed or blunt-ended strand breaks with terminal deoxynucleotidyl transferase-mediated nick end labeling) techniques in primary cultures of chondrocytes in monolayer. Apoptosis was induced in the chondrocytes by either Tumor Necrosis Factor alpha (TNF alpha), Interleukin 1-beta (IL-1 beta), or anti-Fas antibody but only after 48 hours in culture. At 4 and 24 hours, there was no detectable DNA fragmentation. With TNF alpha, IL1 beta, and anti-Fas antibody, chondrocytes show evidence of at least two types of DNA strand breaks within the same cell (as assessed by simultaneous labeling with ISEL and TUNEL). Therefore, some pathways leading to apoptosis in chondrocytes appear to involve more than one type of endonuclease activity. When the chondrocytes were cultured as explants with the articular matrix intact (ex vivo), neither IL-1 beta, TNF alpha, the anti-Fas antibody, nor fibronectin fragments were able to induce apoptosis in the chondrocytes. In normal human adult cartilage that was untreated and uncultured (in situ), DNA fragmentation was undetectable; however, a significant number of chondrocytes in osteoarthritic cartilage did contain strand breaks. These data suggest that apoptosis occurs in chondrocytes in which the matrix has been disrupted experimentally or destroyed by the osteoarthritic disease process. The results of these studies suggest that the ECM may be an essential survival factor for chondrocytes.     

Stereology for anisotropic cells: Application to growth cartilage*

A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth–Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume-weighted mean cell volume is estimated from vertical sections via point-sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.     

Electron spectroscopic diffraction and imaging of the early and mature stages of calcium phosphate formation in the epiphyseal growth plate

A review of different models of biomineralization in collagen-rich hard tissues shows that further investigations of crystal formation are necessary. The electron spectroscopic diffraction (ESD) mode of operation of an energy-filtering electron microscope offers the possibility of being able to avoid the background from inelastic scattering in selected-area electron diffraction patterns. First experiments on the different stages of mineralization in the epiphyseal growth plate have only indicated the presence of apatite. The ESD mode can be complemented by the electron spectroscopic imaging mode and by elemental mapping of calcium.     

Effects of caloric restriction on development of the proximal growth plate and metaphysis of the caput femoris in spontaneously hypertensive rats: microscopic and computer-assisted image analyses

We previously demonstrated that caloric restriction (CR) reduced the prevalence of osteonecrosis in caput femoris of spontaneously hypertensive rats (SHR), a model of human Perthes' disease. The effects of CR on the development and pathology in the proximal femoral growth plate (GP) and adjacent structures in SHR were investigated by morphometric and computer-assisted image analyses. From 6 weeks of age, the food intake of SHR was restricted to 65% of the mean intake of ad libitum fed control SHR (SHR-AL). Wistar Kyoto rats (WKY), from which the SHR strain was isolated, fed ad libitum were also included as controls. CR reduced prevalence of chondromucinous degeneration and dysarray of cartilage cell columns in the GP, becoming prevalent between 10 and 20 weeks in the SHR-AL group, attaining the same levels of the WKY group. Thicknesses of non-calcifying cell columns in the GP were greater in the SHR-AL than WKY group; CR slightly reduced the thickness, but incompletely. Thicknesses of calcifying cell columns did not significantly differ among the three groups. CR decreased volume density and mean thicknesses of the trabecular bone in areas adjacent to GPs, and was greater in the SHR-AL than the WKY group. The present morphologic analysis suggested that CR ameliorates dysplastic changes of trabecular bones in areas adjacent to the GP, rather than modulating the ossification process in the GP. The CR paradigm might give insight into the pathogenesis of, and a therapeutic strategy for, human Perthes' disease.     

Confocal laser scanning microscopy of chondrocytes in vitro: Cytoskeletal changes after quinolone treatment

The use of quinolone antibiotics would be significant for chronically diseased children (e.g., cysticfibrosis) as a prophylactic long-term treatment. However, quinolones were shown to cause cartilage damage in experimental animals when administered during certain developmental stages. In the present study, the effect of quinolones on chondrocytes was studied in a cell culture model in order to avoid animal experiments, to investigate the influence of single factors, and to open up the possibility to test human tissue. Chondrocytes were obtained from hipjoint cartilage of 3 to 4-weeks-old rats and cultured in control medium or quinolone-supplemented medium. It was shown that quinolones heavily disturbed adhesion of chondrocytes to the culture dish, accompanied by changes in cell shape and cytoskeletal morphology. Reduction of filamentous actin (stress fibers) and disintegration of vimentin fibers was demonstrated by immunofluorescence and evaluated by con-focal laser scanning microscopy. In contrast, distribution and amount of the adhesion molecule integrin α did not change. Results of the present study indicate that quinolones disturb the adherence mechanism of chondrocytes and lead to cytoskeleton changes.     

超薄膜生长过程中分形凝聚体的计算机模拟

利用计算机模拟了超薄膜中分形凝聚体的生长 ,考虑了薄膜生长过程中的团簇扩散和旋转。开始的团簇生长采用不同允许扩散步长下准圆形团状生长的分子动力学模型。研究了基底上分形凝聚体中团簇的扩散和旋转对表面形貌的影响 ,引进了团簇的扩散和旋转因子。结果表明 :初始的小团簇经过无规则的扩散和旋转逐渐凝聚成具有分支状的凝聚体。团簇的旋转对分形结构的维数值没有太大的影响。