Characteristics of mineral particles in the human bone/cartilage interface

Bone and cartilage consist of different organic matrices, which can both be mineralized by the deposition of nano-sized calcium phosphate particles. We have studied these mineral particles in the mineralized cartilage layer between bone and different types of cartilage (bone/articular cartilage, bone/intervertebral disk, and bone/growth cartilage) of individuals aged 54 years, 12 years, and 6 months. Quantitative backscattered electron imaging and scanning small-angle X-ray scattering at a synchrotron radiation source were combined with light microscopy to determine calcium content, mineral particle size and alignment, and collagen orientation, respectively. Mineralized cartilage revealed a higher calcium content than the adjacent bone (p<0.05 for all samples), whereas the highest values were found in growth cartilage. Surprisingly, we found the mineral platelet width similar for bone and mineralized cartilage, with the exception of the growth cartilage sample. The most striking result, however, was the abrupt change of mineral particle orientation at the interface between the two tissues. While the particles were aligned perpendicular to the interface in cartilage, they were oriented parallel to it in bone, reflecting the morphology of the underlying organic matrices. The tight bonding of mineralized cartilage to bone suggests a mechanical role for the interface of the two elastically different tissues, bone and cartilage.     

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage matrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.     

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.     

Cyclic compression of articular cartilage explants is associated with progressive consolidation and altered expression pattern of extracellular matrix proteins.

In this study, we investigated the biosynthetic response of full thickness, adult bovine articular cartilage explants to 45 h of static and cyclic unconfined compression. The cyclic compression of articular cartilage resulted in a progressive consolidation of the cartilage matrix. The oscillatory loading increased protein synthesis ([35S]methionine incorporation) by as much as 50% above free swelling control values, but had an inhibitory influence on proteoglycan synthesis ([35SO4] incorporation). As expected, static compression was associated with a dose-dependent decrease in biosynthetic activity. ECM oligomeric proteins which were most affected by mechanical loading were fibronectin and cartilage oligomeric matrix protein (COMP). Static compression at all amplitudes caused a significant increase in fibronectin synthesis over free swelling control levels. Cyclic compression of articular cartilage at 0.1 Hz and higher was consistently associated with a dramatic increase in the synthesis of COMP as well as fibronectin. The biosynthetic activity of chondrocytes appears to be sensitive to both the frequency and amplitude of the applied load. The results of this study support the hypothesis that cartilage tissue can remodel its extracellular matrix in response to alterations in functional demand.     

The organ culture and grafting of lamprey cartilage and teeth

Summary Cartilage from larval (ammocoetes) and adult (prespawning upstream migrant) lamprey was successfully maintained both when cultured in vitro or grafted in vivo (on the chorioallantoic membrane (CAM) of host chick embryos). In addition teeth from adult lamprey were successfully cultured in vitro. Cartilages were cultured in supplemented Lebovitz's L15 medium at 15 and 20°C for periods of up to 56 d and in supplemented BGJ _b medium at 37° C for periods of up to 14 d. Cartilages were also grafted onto the CAM for up to 16 d. Both the cultured and grafted cartilages retained their structural and cellular integrity as verified histologically. The viability of the cartilage, even after extended culture periods, was demonstrated ultrastructurally by the presence of chondrocytes displaying abundant rough endoplasmic reticulum, mitochondria, and Golgi apparatii with associated vesicles. In addition the cartilages were shown to be metabolically active in vitro by the incorporation of radioactive sulfur into the matrix. Some cell outgrowth from other tissues, such as connective tissue, muscle, and gill when left adjacent to the cartilage, occurred over time in cultures. No cell outgrowth was observed in CAM-grafted tissue nor was there any invasion of the agnathan tissue by chorioallantoic blood vessels. Teeth cultured in L15-supplemented media for up to 14 d at either 15 or 20° C retained their structural and cellular integrity as observed histologically, with no apparent cell outgrowth. With the successful culture of these tissues, their development, biochemistry, and physiology, potentially of great importance in understanding early vertebrate evolution, can be better understood. Much of the field equipment was generously provided by Howard Dickson, Dept. Anatomy, Dalhousie University. Access to the fish ladder was graciously provided by the Department of Fisheries and Oceans Canada and their officers especially Elmer Jefferson who provided excellent assistance. This work was funded by NSERC grant #A5056 and a Dalhousie Research Development Fund grant to B. K. H, and Dalhousie and I. W. Killam fellowships to R. J. L.     

The calcified amorphous layer of the skin of Bufo marinus (Amphibia: Anura)

The integument of Bufo marinus is surveyed. It is similar to that of other anurans and contains the three chromatophores common to anurans. The skin of these toads contains more than 28% minerals deposited as small crystals in a mucopolysaccharide-positive amorphous layer between the stratum compactum and the stratum spongiosum. These crystals reveal a high content of calcium and phosphorus aggregated in and near membrane-bound vesicles which have an appearance very similar to matrix vesicles. Electron microscopy gives the appearance that these vesicles are associated with flbroblasts located between alternating bundles of collagen. Histochemical studies indicate that the amorphous layer possesses many of the characteristics commonly associated with mineralized cartilage or bone.     

The Effect of Bovine Parathyroid Hormone Withdrawal on MC3T3-E1 Cell Proliferation and Phosphorus Metabolism

Hypocalcemia and hypophosphatemia are common complications after parathyroidectomy (PTX). Sudden removal of high circulating levels of parathyroid hormone (PTH) causes decreased osteoclastic resorption resulting in a decreased bone remodeling space. These phenomena are likely due to an increased influx of calcium and phosphorus into bone. However, there are currently no data to support this hypothesis. In this study, we found that PTX significantly reduced levels of PTH, calcium and phosphate. Compared with preoperative levels, after 1 year, postoperative PTH, calcium and phosphate levels were 295.6 ± 173.7 pg/mL (P < 0.05), 86.62 ± 15.98 mg/dL (P < 0.05) and 5.56 ± 2.03 mg/dL (P < 0.05), respectively. We investigated continuous bovine PTH administration as well as withdrawal of bovine PTH stimulation in the mouse osteoblast precursor cell line MC3T3-E1. MC3T3-E1 cells were cultured with continuous bovine PTH treatment for 20 days or with transient bovine PTH treatment for 10 days. High doses of continuous bovine PTH exposure strongly reduced cell proliferation, alkaline phosphatase activity and the number of mineralized calcium nodules. However, withdrawal of bovine PTH (100 ng/mL) significantly increased the number of mineralized calcium nodules and caused a rapid decline in calcium and phosphorus content of culture medium. In conclusion, continuous exposure to bovine PTH inhibited osteoblast differentiation and reduced the formation of mineralized nodules. However, this inhibition was removed and mineralized nodule formation resumed with withdrawal of bovine PTH. According to the results of our clinical examinations and in vitro experiments, we hypothesize that the sudden removal of high levels of PTH may cause an increased influx of calcium and phosphorus into bone after PTX.     

Ontogenetic development of an exceptionally preserved Devonian cartilaginous skeleton

Cartilaginous vertebrate skeletons leave few records as fossils, unless mineralized. Here, we report outstanding preservation of early stages of cartilage differentiation, present in the Devonian vertebrate Palaeospondylus gunni. In large specimens of Palaeospondylus, enlarged, hypertrophic cell spaces (lacunae) are dominant in the cartilage matrix, each defined by thin mineralized matrix, where phosphorus and calcium co-occur. This is comparable to living endochondral cartilage, where cell hypertrophy and matrix mineralization mark the end of an ontogenetic process of cell growth and division before bone formation. New information from small individuals of Palaeospondylus demonstrates that the skeleton comprises mostly unmineralized organic matrix with fewer hypertrophic cell spaces, these occurring only in the central regions of each element. Only here has the surrounding matrix begun to mineralize, differing from the larger specimens in that phosphorus is dominant with little associated calcium at these earlier stages. This reflects cellular control of mineralization in living tissues through phosphate accumulation around hypertrophic cells, with later increase in calcium in the cartilaginous matrix. These features are always associated with endochondral bone development, but in the Palaeospondylus skeleton, this bone never develops. This skeletal state is thus far unique among vertebrates, with two alternative explanations: either later stages of endochondral bone development have been lost in Palaeospondylus, or, in a stepwise acquisition of the mineralized skeleton, these late stages have not yet evolved.     

Ryanodine receptor 1 mediated dexamethasone-induced chondrodysplasia in fetal rats

BackgroundOsteoarthritis is caused by cartilage dysplasia and has fetal origin. Prenatal dexamethasone exposure (PDE) induced chondrodysplasia in fetal rats by inhibiting transforming growth factor β (TGFβ) signaling. This study aimed to determine the effect of dexamethasone on fetal cartilage development and illustrate the underlying molecular mechanism.MethodsDexamethasone (0.2 mg/kg.d) was injected subcutaneously every morning in pregnant rats from gestational day (GD) 9 to GD21. Harvested fetal femurs and tibias at GD21 for immunofluorescence and gene expression analysis. Fetal chondrocytes were treated with dexamethasone (100, 250 and 500 nM), endoplasmic reticulum stress (ERS) inhibitor, and ryanodine receptor 1 (RYR1) antagonist for subsequent analyses.ResultsIn vivo, prenatal dexamethasone exposure (PDE) decreased the total length of the fetal cartilage, the proportion of the proliferation area and the cell density and matrix content in fetal articular cartilage. Moreover, PDE increased RYR1 expression and intracellular calcium levels and elevated the expression of ERS-related genes, while downregulated the TGFβ signaling pathway and extracellular matrix (ECM) synthesis in fetal chondrocytes. In vitro, we verified dexamethasone significantly decreased ECM synthesis through activating RYR 1 mediated-ERS.ConclusionsPDE inhibited TGFβ signaling pathway and matrix synthesis through RYR1 / intracellular calcium mediated ERS, which ultimately led to fetal dysplasia. This study confirmed the molecular mechanism of ERS involved in the developmental toxicity of dexamethasone and suggested that RYR1 may be an early intervention target for fetal-derived adult osteoarthritis.     

Collagen type XV and the ‘osteogenic status’

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

BIOMINERALIZATION OF THE STALKS OF ANTHOPHYSA VEGETANS (CHRYSOPHYCEAE)1

Mineralized stalks of the chryophyte Anthophysa ve-getans (O. F. Müller) Stein were examined by electron microscopy, and the composition of the stalks was determined by energy dispersive x-ray microanalysis. Colonies grown in water from a local lake (Dowdy Lake) had mineralized stalks that were colorless and composed mainly of spherules of calcium phosphate up to 0.7 μm in diameter. The addition of 0.1 mM MnCl₂ to the culture medium resulted in deeply orange-brown stalks mineralized primarily with minute granules of a manganese compound. The addition of 0.1 mM FeCl₃ to the culture media resulted in light yellow stalks mineralized mainly with small granules of an iron compound. The addition of 0.1 mM MgCl₂ or KCl resulted in stalks that were similar in appearance and composition to the colonies grown in culture medium alone. These results show that the composition of the mineralized stalks of A. vegetans is dependent on the elements present in the culture medium. This is also the first time that calcium phosphate has been reported as a mineralization product in the chrysophytes.     

Ultrastructural localization of alkaline phosphatase in the cyanobacteriaCoccochloris peniocytis andAnabaena cylindrica

Summary Histochemical techniques applied at the ultrastructural level have established the periplasmic space as the site of cell bound alkaline phosphatase activity inAnabaena cylindrica andCoccochloris peniocytis. For localization of activity unfixed cells were reacted with calcium nitrate, which acts as the initial capture reagent. After this deposition, the cells were suspended in 2% lead nitrate to convert the calcium phosphate to more electron dense lead phosphate. The majority of cell bound activity appeared to be associated with layer 3 of the cell wall. InA. cylindrica a secondary site of cell bound activity appeared to be in the sheath. Placement in a phosphate free medium caused a substantial increase in the enzyme activity ofA. cylindrica while the activity present in log phase cells ofC. peniocytis was similar to that found in phosphate starved cells.C. peniocytis also secretes the enzyme into the surrounding medium.     

Localization of transforming growth factor beta receptor II interacting protein-1 in bone and teeth: implications in matrix mineralization

Transforming growth factor beta receptor II (TGFβR-II) interacting protein 1 (TRIP-1) is a WD-40 protein that binds to the cytoplasmic domain of the TGF-β type II receptor in a kinase-dependent manner. To investigate the role of TRIP-1 in mineralized tissues, we examined its pattern of expression in cartilage, bone, and teeth and analyzed the relationship between TRIP-1 overexpression and mineralized matrix formation. Results demonstrate that TRIP-1 was predominantly expressed by osteoblasts, odontoblasts, and chondrocytes in these tissues. Interestingly, TRIP-1 was also localized in the extracellular matrix of bone and at the mineralization front in dentin, suggesting that TRIP-1 is secreted by nonclassical secretory mechanisms, as it is devoid of a signal peptide. In vitro nucleation studies demonstrate a role for TRIP-1 in nucleating calcium phosphate polymorphs. Overexpression of TRIP-1 favored osteoblast differentiation of undifferentiated mesenchymal cells with an increase in mineralized matrix formation. These data indicate an unexpected role for TRIP-1 during development of bone, teeth, and cartilage.     

The head cartilage of cephalopods. I. Architecture and ultrastructure of the extracellular matrix

The morphology of head cartilage of the cephalopods Sepia officinalis and Octopus vulgaris has been studied on samples fixed and embedded for light- and electron microscopy and on fresh frozen sections viewed by polarizing microscopy. The organization of extracellular matrix (ECM) varies in different regions of the head cartilage. Superficial zones are made up of densely packed collagenous laminae parallel to the cartilage surface, while radially arranged laminae form a deeper zone where territorial and interterritorial areas are present. A compact arrangement of banded collagen fibrils (10-25 nm in diameter) forms the laminae of the superficial zones and of the interterritorial areas; a loose three-dimensional network of fibrils (10-20 nm) with many proteoglycan aggregates forms the territorial areas. A pericellular matrix surrounds the bodies of extremely branched territorial chondrocytes. Peculiar anchoring devices (ADs) are dispersed with variable orientation within the ECM. A perichondrium is present, but often connectival and muscular bundles are fused with the superficial layers of cartilage. Some vessels were also observed within the superficial inner zone and clusters of hemocyanin molecules were demonstrated both in the ECM and in some cells. The cephalopod head cartilage seems to share some morphological characteristics with both hyaline cartilage and bone tissue of vertebrates.     

Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.     

Cartilage responses to a novel triaxial mechanostimulatory culture system

We have developed a novel mechanically active cartilage culture device capable of modulating the interplay between compression and shear, at physiologic stress levels (2-5 MPa). This triaxial compression culture system subjects cylindrical cartilage explants to pulsatile axial compression from platen contact, plus pulsatile radially transverse compression from external fluid compression. These compressive loads can be independently modulated to impose stress states that resemble normal physiologic loading, and to investigate perturbations of individual components of the multi-axial stress state, such as increased shear stress. Based on the observation that joint incongruity predisposes cartilage to premature degeneration, we hypothesized that cartilage extracellular matrix (ECM) synthesis would be inhibited under conditions of low transverse buttressing (high shear stress). To test this hypothesis, we compared ECM synthesis in human cartilage explants exposed to axial compression without transverse compression (high shear stress), versus explants exposed to axial compression plus an equal level of transverse compression (low shear stress). Both total (35)SO(4) incorporation and aggrecan-specific (35)SO(4) incorporation were significantly inhibited by axial compression, relative to axial plus transverse compression.     

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.     

Culture of cells from tissues of adult and larval sea lamprey

Methods were developed for the culture of cells derived from tissues of the sea lamprey (Petromyzon marinus). Cultures were initiated from gill, liver, muscle and gut from larvae and newly transformed individuals and brain, heart, kidney and ovary from sexually mature adults. The lamprey cells were viable for up to six months in culture and cells from ovary, muscle, gut, gill and liver were propagated for multiple passages. For all cultures except liver, optimal cell attachment and spreading was obtained on surfaces coated with fibronectin and collagen. Optimal liver cell attachment was achieved on basement membrane. Cells synthesizing DNA were detected by precursor incorporation in five week-old cultures derived from adult and larval tissues. Metabolic labeling experiments with [³⁵S]-methionine demonstrated that cultures initiated from liver and ovary continued to synthesize and release proteins into the medium for several weeks. Ultrastructural examination revealed the presence of ciliated cells in cultures from brain and the accumulation of lipid in epithelial cells derived from liver and gill.