Calcified Zone Research Articles

X-ray diffraction, electron microscopy, and Fourier transform infrared spectroscopy of apatite crystals isolated from chicken and bovine calcified cartilage.

Apatite crystals of the calcified zone of the subarticular cartilaginous growth plates of the long bones of young growing chickens and calves were isolated by low temperature reaction with hydrazine and plasma ashing and examined by electron microscopy, electron diffraction and microprobe analysis, and computer-generated deconvolution of the spectra obtained by Fourier transform infrared spectroscopy. The crystal habit was that of wide, very thin, relatively long rectangular plates, which tended to form small clusters of crystals, possibly because reaction with hydrazine alone did not remove all of the organic matrix constituents. Further reaction with low power plasma ashing released more of the isolated crystals although to a lesser extent than was possible with bone. Stereograms of the small clusters showed that many of the crystals in the small isolated aggregates of crystals were bent and/or curved. Together with the resultant overlap of individual adjacent crystals, they also produced images of sharp, very dense lines, reminiscent of the electron-dense needle or rod-like appearances frequently observed by transmission electron microscopy of thin sections of calcified cartilage and thought to represent the habit of the apatite crystals. No true rod or needle-like crystals were observed in the isolated crystals. Although the overall general apatitic structure of the apatite crystals was similar to that of the apatitic crystals of bone, the individual crystals were significantly larger than those of bone from the same specimen, and there were small but significant differences in the concentrations of acid phosphate and carbonate groups and in their short range order.

Rabbit Articular Cartilage Defects Treated With Autologous Cultured Chondrocytes

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.

Characteristics and effects of calcified degenerative zones on the formation of hard tissue barriers in amputated canine dental pulp

The purpose of this research was to study under undecalcified conditions the presence, ultrastructural features, and contributions of the degenerative zone beneath the necrotic zone and whether it had effects on the formation of reparative dentin in canine incisors and premolars. The research was conducted over a period of 14 days after experimental pulpotomy using calcium hydroxide as a pulp-capping agent. On the first day following pulp exposure and capping with calcium hydroxide, electron-dense spherical bodies were observed under the necrotic zone. Energy dispersive X-ray point analysis confirmed that these electron-dense deposits contained calcium and phosphorus. By the third day, varying amounts of minute von Kossa-positive granules could be observed light-microscopically between the two zones of necrosis and underlying vital pulp tissue. Migration and proliferation of pulpal cells, most probably mesenchymal cells, were observed adjacent to the von Kossa-positive zone. The ultrastructure of the von Kossa-positive zone consisted of degenerated cells, electron-dense spherical bodies, and electron-dense shortened dilating fibrils. By the seventh day, short cylindrical-shaped cells collected at the coronal end of the vital pulp tissue. By the fourteenth day, the specimens having a uniform von Kossa-positive zone exhibited rapid differentiation of odontoblasts and tubular dentin formation. In contrast, only some specimens having an irregular von Kossa-positive zone exhibited osteodentin formation and the beginning of odontoblast differentiation beneath the osteodentin. These findings suggest that this calcified degenerative zone has an important effect on the reparative process of pulp tissue after pulpotomy.

The effect of removal of the medical collateral ligament on subchondral bone and cartilage in the growing rat knee joint.

The growing knee joint in immature rats was chosen as a model to study quantitatively bone apposition and thickness of articular cartilage after surgical removal of the medial collateral ligament. In 32 rats, subchondral bone apposition was assessed by measuring double tetracycline labelling, and the thickness of calcified and uncalcified articular cartilage was measured by image analysis. The results indicate that there was a significant increase in subchondral bone growth after removal of the medial collateral ligament, but no change in thickness of the calcified and uncalcified zones of the articular cartilage in the medial knee compartment. Further, despite an obvious increase in valgus laxity on manual examination, there were no signs of articular cartilage degeneration in any animal. The study shows that traumatic injury to ligaments and joint instability during the growing period may influence the development of subchondral bone in the knee joint.

Sodium phosphate crystals in chondromalacic patellar cartilage

We report the finding of sodium- and phosphorus-based crystallisation in abnormal human articular cartilage. We prepared five chondromalacic, five osteoarthritic and four macroscopically normal specimens of patellar cartilage by a cryofracturing technique and examined them in a scanning electron microscope. An energy-dispersive X-ray microanalysis system was used to identify the crystals, which were found in only three of the five chondromalacic specimens. Star-shaped crystals were seen either individually or in clusters in the matrix of the cartilage. They consisted of sodium and phosphorus, and we have found no previous reports of such findings. The calcified zone, the bone, and the articular surface were free from crystals.

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia.

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

Two Monoclonal Antibodies Distinguish Growth Cartilage from Other Types of Cartilage and Other Tissues.

Two monoclonal antibodies (mAbs) were raised as probes to study the specificity of a growth cartilage (GC). Immunized antigen was a homogenate of cultured GC cells from young rabbit ribs. Raised mAbs were investigated as to their reactivity with undecalcified or decalcified paraffin-embedded rabbit tissues. By immunohistochemical examination of the young rabbit tissues, one mAb, mAb-A, reacted with non-osteogenic cartilage, muscle, bone marrow cells, dermis, epidermis, gut, tendon, lung, liver, heart, spleen, and thymus, but not with the growth cartilage. The other mAb, mAb-B, however, reacted specifically with late hypertrophic and calcified growth cartilage zones, but not with other non-osteogenic cartilage or other tissues.These two mAbs are useful probes to distinguish osteogenic cartilage from nonosteogenic cartilage and other tissues by negative reactivity as in mAb-A, and by positive reactivity as in mAb-B. Further-more, these results imply the existence of certain molecules to distinguish osteogenic cartilage from non-osteogenic cartilage.

Horizontal well case study under pattern waterflood. Investigation of horizontal well performance in a heterogenous carbonate oil reservoir.

The performance of two horizontal wells drilled in a major limestone oil reservoir in the Middle East was investigated. The wells were drilled to test the application of horizontal wells in the reservoir by replacing two watered out vertical wells.The reservoir is relatively thin (-45ft.) and highly heterogenous. The reservoir is characterised by significant vertical variation in horizontal permeability with the majority of the flow capacity situated in the botton 10-15% of the reservoir section. Furthermore, a thin, and possibly continuous, low vertical permeability dense calcified zone, caused by stylolitisation, has been suspected immediately above the high flow capacity formation.The Well A case is unique in that water breakthrough was observed in the vertical well, but to date, not in its replacement horizontal Well A (drilled in the upper half of the reservoir section). This is in spite of more than 24 months of production from Well A. This compares to the performance of the Well B replacement horizontal well in a nearby pattern where water production was recorded immediately after the well was brought on line.The performance of horizontal Wells A and B can be explained by examining the degree of vertical heterogeneity and layer segregation within the reservoir. The predicted shape and rate of advance of the water flood front in the Well A and B pattern element models can be successfully controlled using a combination of vertical layer heterogeneity and layer kv reduction. Specifically, it is layer permeability thickness product contrast and layer segregation that controls the shape of the advancing flood front. The greatest influence on well performance is observed when the vertical permeability of the dense calcified zone is drastically reduced.

The morphology of articular cartilage assessed by magnetic resonance imaging (MRI). Reproducibility and anatomical correlation.

Quantitative assessment of cartilage volume and thickness in a formalin-alcohol fixed specimen of a human patella was conducted with magnetic resonance imaging (MRI), as it is still unclear whether the morphology of normal and damaged cartilage can be accurately demonstrated with this technique. MR imaging was carried out at 1.0 T (section thickness 2 mm, in-plane-resolution 0.39-0.58 mm) with the following pulse sequences: 1) T1-weighted spin-echo, 2) 3D-MPRAGE, 3) 3D-FISP, 4) 3D-MTC-FISP, 5) 3D-DESS, 6) 3D-FLASH. Following imaging, the patella was sectioned perpendicular to the articular surface at intervals of 2 mm with a diamond band-saw. The volume of its cartilage was determined from the anatomical sections and the MR images, using a Vidas IPS 10 image analysing system (Kontron). Measurements were carried out with and without the low-signal layer in the transitional zone between the articular cartilage and the subchondral bone. If the low-signal layer was included, the volume was overestimated with MRI by 16 to 19%. Without the low-signal layer the volumes were less than those determined from the anatomical sections: T1-SE-18.2%, MPRAGE -22.6%, FISP -17.1%, MTC-FISP -9.5%, DESS -9.3% and FLASH -6.1%. The coefficient of variation for a 6-fold determination of the volume amounted to between 6.2% (T1-SE) and 2.6% (FLASH). The FLASH sequence allowed the most valid and reproducible assessment of the cartilage morphology. The remaining difference from the real volume of the cartilage may be due to the fact that the calcified zone of the cartilage is not delineated by MRI.

Applying a calcium phosphate layer on PEO/PBT copolymers affects bone formation in vivo

Recently a copolymer (PolyactiveR) has been introduced that combines elastomeric and bone-bonding properties. Since calcification of the copolymer is a prerequisite for bone bonding. Polyactive was precalcified in vitro in order to increase the bone-bonding rate. Precalcification was performed by subsequent incubation in Ca and P solutions and resulted in formation of a hydroxyapatite layer on the surface of the implant. Within one week after implantation this layer had disappeared from the surface and a new calcification zone was formed under the surface of the copolymer. Longer implantation periods showed that in precalcified implants bone was apposited along the walls of the pores, while in control implants new bone was first formed in the centre of the pores. Consequently, the percentage of bone contact was increased in precalcified implants, however, the amount of bone ingrowth was equal in both control and precalcified implants. Transmission electron microscopy showed the presence of an electron-dense layer at the bone implant interface, which was indicative for bone-bonding. It is concluded from these experiments that precalcification of PEO/PBT copolymers affected the direction of bone apposition and increased the bone-bonding rate.

Methotrexate osteopathy in infants with tumors of the central nervous system

Methotrexate osteopathy, previously reported as a complication of maintenance-therapy for acute lymphoblastic leukemia, is characterized by osteopenia, particularly involving the lower extremities, thick, dense provisional zones of calcification, growth arrest lines, and corner fractures resembling scurvy. In attempts to postpone radiotherapy in infants under three years of age, the multicentric German therapy protocol for childhood central nervous system tumors (HIT-89 protocol) has employed high cumulative methotrexate doses. Here we describe osteopathy in three patients as a toxic side effect after administration of cumulative methotrexate doses of 20 g/m2, 80 g/m2 and 135 g/m2. The high prevalence of this adverse effect in infants with tumors of the central nervous system may be attributed to the long-term therapy with high cumulative methotrexate-doses. Both factors may favor intracellular accumulation of methotrexate and formation of methotrexate-polyglutamates and may be responsible for bone toxicity. Apparently the susceptibility of the rapidly growing skeletal structures of infants under three years of age to this toxic side effect of methotrexate is remarkably high.

Effects of parathyroid hormone‐related peptide on chick growth plate chondrocytes

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH) have similar biological effects in vitro that are mediated through the PTH receptor. PTH receptors have been demonstrated in the zone of provisional calcification and the hypertrophic zone of the cartilaginous growth plate. The current study examined the biological effects of PTHrP on chick growth plate chondrocytes. Chondrocytes were exposed to varying doses of PTHrP for 24 h, and the incorporation of radioactive thymidine into DNA was used as an index of proliferation. A dose-dependent stimulation of proliferation was seen, with a maximal 27-fold increase at 50 nM PTHrP. A dose-dependent stimulation of cAMP was seen, with a maximal effect at a dose of 50 nM. Proteoglycan synthesis, measured by incorporation of radioactive sulfate, was stimulated, with a maximal effect of 65% at 1 nM. Collagen synthesis and alkaline phosphatase activity from both cellular and matrix vesicle sources decreased in a dose-dependent fashion, with a maximal inhibition of approximately 50% of the control value. The physiologic significance of the PTH and PTHrP-responsiveness of growth plate chondrocytes is uncertain at the present time. It is possible that PTH or PTHrP, or both, act as a systemic, developmental modulator of cellular proliferation and differentiation in the growth plate.

A monoclonal antibody distinguishes growth cartilage from other types of cartilage: a new probe for osteogenic cartilage.

Monoclonal antibodies (mAbs) were raised by injection of a homogenate of cultured growth cartilage (GC) cells from young rabbit ribs. These mAbs were examined by immunohistochemical staining for their reactivity to paraffin sections of rabbit tissues. The results showed that an mAb reacted preferentially with late hypertrophic and calcified costal GC zones. The mAb also reacted with hypertrophic GC adjacent to bone that existed in sternum and femur, but not to other cartilages, including resting cartilage, articular cartilage, auricular cartilage, nasal cartilage, tracheal cartilage and meniscus cartilage, or with other tissues, including tendon, skin, muscles, lung, liver, heart, thymus, spleen, eye and gut. It reacted with a wider area of the GC zone when the sections were decalcified, although its reactivity with the extended area was much less intensive than that with late hypertrophic and calcified GC zones. On treatment of the sections with bacterial collagenase, neither the reactive area nor its intensity were changed, while when treated with trypsin the reactivity was lost. These results suggest the existence of a certain molecule which distinguishes GC (osteogenic cartilage) from other (non-osteogenic) cartilage. This mAb is a useful probe for distinguishing osteogenic cartilage from non-osteogenic cartilage, and for studying differentiation steps of cartilage cells in endochondral bone formation. The mAb can also be used as a probe for clinical and stored specimens because it reacts with decalcified and paraffin-embedded human specimens.

Calcified cartilage zone and its dimensional relationship to the articular cartilage in the human temporomandibular joint of elderly individuals.

The aim was to describe the appearance of the calcified cartilage zone (CCZ) and to determine its dimensional relationship to the articular cartilage thickness in the normal human temporomandibular joint. An autopsy material comprising 21 joints from 12 elderly individuals was examined microscopically. The appearance of the CCZ was examined, and the thickness of the CCZ and of the total articular cartilage was measured in 18 different positions in each joint. The CCZ was outlined by a flat or gently undulating tidemark and an irregular osteochondral junction. The cellularity of the CCZ varied extensively. The cells were numerous in the CCZ when the overlying articular cartilage displayed high cellularity. Statistical analysis of the measurements demonstrated a relationship (p < 0.001) between the thickness of the CCZ and of the articular cartilage. Our findings, both qualitative and quantitative, indicate a close relationship between the physiology of the CCZ and of the overlying articular cartilage.

Biomineralization and Eggshells: Cell-Mediated Acellular Compartments of Mineralized Extracellular Matrix

Publisher Summary This chapter summarizes cell biology, morphological organization, crystallography, chemical composition, process of mineralization, and biological function of avian eggshells. It also discusses emerging concepts of biomineralization and speculates about the mechanism of eggshell assembly and its implications for fabrication of polymer-ceramic composites. The eggshell is a microenvironmental compartment for housing developing embryos of a number of species. This unique microenvironment provides physical protection to the embryo and regulates gas, water, and ionic exchange. The avian eggshell can be characterized as a multilayered, polymer–ceramic composite. The three main layers include an outer mucous layer, an intermediate calcified zone, and an inner fibrous membrane layer. The shell membranes are the most internal layer of the eggshell and are formed by two nonmineralized fibrillar sublayers, the outer membrana testae externa, and the inner membrana testae interna or putaminis. The most external layer of the eggshell is referred to as the cuticle. This proteinaceous layer covers the entire calcified portion of the shell to a depth of about 10 μ m. The complementary use of biological, chemical, and crystallographic approaches demonstrates that the avian eggshell is a very promising model for the study of biomineralization. Avian eggshell is one of the most rapidly mineralizing biological systems known.

Spaceflight and age affect tibial epiphyseal growth plate histomorphometry.

Growth plate histomorphometry of rats flown aboard the Soviet biosatellite COSMOS 2044, a 14-day spaceflight, was compared with that of control groups. In growth plates of flight animals, there was a significant increase in cell number per column and height of the proliferative zone and a reduction in height and cell number in the hypertrophy/calcification zone. No significant differences were found in matrix organization at the ultrastructural level of flight animals, indicating that although spaceflight continues to affect bone growth of 15-wk-old rats, extracellular matrix is not altered in the same manner as seen previously in younger animals. All groups showed growth plate characteristics attributed to aging: lack of calcification zone, reduced hypertrophy zone, and unraveling of collagen fibrils. Tail-suspended controls did not differ from other controls in any of the parameters measured. Our results suggest that growth plates of older rats are less responsive to unloading by spaceflight or suspension than those of younger rats and provide new evidence about the modifying effect of spaceflight on the growth plate.

Sequential Serum Aluminum and Urine Aluminum: Creatinine Ratio and Tissue Aluminum Loading in Infants With Fractures/Rickets

Aluminum toxicity is associated with the development of bone disorders, including fractures, osteopenia, and osteomalacia. Fifty-one infants with a mean (+/- SEM) birth weight of 1007 +/- 34 g, gestational age of 28.5 +/- 0.3 weeks, and serial radiographic documentation at 3, 6, 9, and 12 months for the presence (n = 16) or absence (n = 35) of fractures and/or rickets were studied at the same intervals to determine the serial changes in serum aluminum concentrations and urine aluminum-creatinine ratios. Autopsy bone samples were used to determine the presence of tissue aluminum. Serum aluminum concentrations from 46 infants were stable and similar between groups, with mean values between 15 and 22 micrograms/L. Urine aluminum-creatinine (micrograms per milligram) ratios from 14 infants were higher in infants with fractures and/or rickets (0.26 +/- 0.06 vs 0.12 +/- 0.04) at onset, and rate of decrease in aluminum-creatinine ratio was faster in infants without fractures and/or rickets. All but three infants were tolerating complete enteral feeding at all sampling points. One infant who received aluminum-containing antacid had marked increase in serum aluminum to 83 micrograms/L while urine aluminum-creatinine ratio increased from 0.09 to a peak of 8.53. Vertebrae from three infants at autopsy (full enteral feeding was tolerated for 37 and 41 days in two infants, respectively) showed aluminum deposition in the zone of provisional calcification and along the newly formed trabecula.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological characteristics of the life cycle of resting cartilage cells in mouse rib investigated in intrasplenic isografts.

Resting cartilages taken from 2-day-old mouse ribs were transplanted into spleens in order to carry out morphological investigations of the life cycles of their chondrocytes. The explants were isografted for periods of up to 60 days and examined at light and electron microscopic levels, using von Kossa's reaction or osmium-potassium ferrocyanide (OPF) fixation. By day 3 after transplantation, resting cartilage containing immature chondrocytes was well adapted to splenic tissue and by 7 days after transplantation these chondrocytes had changed into early hypertrophic chondrocytes containing large vacuoles, glycogen aggregates and abundant secretory organelles. It was also demonstrated by von Kossa's reaction that the initial calcification occurred in the territorial matrix during this period. In spite of the hypertrophic chondrocytes in the central zone being surrounded by an extensively calcified matrix during days 14-21 after transplantation, these cells had well-preserved organized organelles, except that Golgi-associated elements and endoplasmic reticulum revealed a tendency toward degenerative changes. With increased duration of the grafting period, from 30-60 days, the calcification zone progressed gradually, and the number of hypertrophic chondrocytes embedded in the calcified matrix decreased considerably. By day 60, degenerating hypertrophic chondrocytes of two types were distinguished:flattened cells containing large vacuoles, poorly developed Golgi apparatuses, and rough endoplasmic reticulum; and shrunken dark cells displaying terminal hypertrophy. During the present study, we observed no vascular invasion into the calcified matrix, or appearance of bone-related cells, and the morphological changes from the resting chondrocytes to cellular hypertrophy accompanied by the formation of a calcified matrix were observed at day 60. These findings indicate that resting cartilage cells of the mouse have the capacity for terminal differentiation when transplanted into the spleen.

In Vitro Studies on the Regulation of Endochondral Ossification by Vitamin D

The research described in this article has focused on the complex autocrine, paracrine, and endocrine regulation of endochondral ossification using vitamin D metabolites and TGF-beta as models. By comparing results from a number of laboratories utilizing a diverse array of in vivo and in vitro systems, a coherent picture is beginning to emerge. Vitamin D metabolites influence cell differentiation and maturation and have direct effects on cell function. Differentiation of the mesenchymal cells into chondroblasts is regulated by both 1,25-(OH)2D3 and 24,25-(OH)2D3, as well as by TGF-beta. The resting zone chondrocytes respond primarily to 24,25-(OH)2D3 in terms of matrix synthesis and matrix vesicle biochemistry. They synthesize both metabolites and other factors that stabilize matrix vesicle enzymes like AHSG. In addition to the paracrine role these factors may play in regulating the matrix, it is possible that they may influence the cells in the growth plate itself. Growth zone chondrocytes also synthesize both metabolites, but respond primarily to 1,25-(OH)2D3 for the parameters measured in the studies described. These cells also synthesize TGF-beta which further increases alkaline phosphatase activity, perhaps via an autocrine stimulation of the cell. While cells from the calcified zone have not yet been studied directly in culture, it is likely that they respond to paracrine signals from the avascular cartilage as well as to serum-derived factors. How the signals are transferred among the cells is unknown. Certainly one can postulate information flow in both upward and downward directions. The signal transduction mechanisms for the factors at the cellular level are complex. While it is known that 1,25-(OH)2D3 stimulates gene transcription and stabilization of mRNA for proteins like alkaline phosphatase, its nongenomic effects are only beginning to emerge. Membrane effects of this metabolite have been shown in intestine and kidney in conjunction with studies on Ca flux. It is becoming increasingly evident that other steroid hormones may operate in similar ways. Studies with the rat costochondral chondrocytes are the first to show that there are specific membrane effects for at least two vitamin D metabolites and that membrane enzymes, including those involved in phospholipid metabolism, can be differentially regulated by them. Furthermore, these experiments have provided for the first time a clear hypothesis for how cells can regulate events in the extracellular matrix after the matrix vesicles are produced and incorporated into the matrix.