Physiology and Pharmacology Publications

Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development.

Matthew R McCann et al. — Fri, 03 May 2013 09:20:49 PDT

Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration.

Acute Vibration Induces Transient Expression of Anabolic Genes in the Murine Intervertebral Disc

Matthew R. McCann et al. — Thu, 02 May 2013 13:15:51 PDT

One- and three-dimensional growth of hydroxyapatite nanowires during sol-gel-hydrothermal synthesis.

Daniel O Costa et al. — Tue, 30 Oct 2012 05:26:13 PDT

Nanoscale hydroxyapatite (HA) is an optimal candidate biomaterial for bone tissue engineering because of its bioactive and osteoconductive properties. In this study, micro- and nanoscale HA particles with rod- and wirelike morphology were synthesized by a novel sol-gel-hydrothermal process. Sol-gel chemistry was used to produce a dry gel containing amorphous calcium phosphate (ACP), which was used as a precursor material in a hydrothermal process. The sol-gel-hydrothermal products were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) to determine particle morphology, crystal structure, and the presence of chemical functional groups. A pure HA crystal was synthesized, which underwent both one- and three-dimensional growth, resulting in tunable microrod and nanorod, and wire morphologies. The effects of solution pH and reaction time on particle diameter and length were assessed. Particle diameter ranged from 25 to 800 nm and decreased with an increase in solution pH, whereas both particle length and diameter increased as the hydrothermal process was prolonged. Nanowire HA powders (10-50 wt %) were mixed with poly(ε-caprolactone) (PCL) to produce PCL/HA composites. Fracture surfaces of PCL/HA composites showed a well-dispersed and homogeneous distribution of HA nanowires within the PCL matrix. Mechanical testing revealed a significant (p < 0.05) increase in the Young's and compressive moduli of PCL/HA composites compared to PCL alone, with 50 wt % HA producing a 3-fold increase in Young's modulus from 193 to 665 MPa and 2-fold increase in compressive modulus from 230 to 487 MPa. These HA nanowires can be used to reinforce polymer composites and are excellent biomaterials for tissue engineering of bone.

Control of surface topography in biomimetic calcium phosphate coatings.

Daniel O Costa et al. — Tue, 30 Oct 2012 05:26:11 PDT

The behavior of cells responsible for bone formation, osseointegration, and bone bonding in vivo are governed by both the surface chemistry and topography of scaffold matrices. Bone-like apatite coatings represent a promising method to improve the osteoconductivity and bonding of synthetic scaffold materials to mineralized tissues for regenerative procedures in orthopedics and dentistry. Polycaprolactone (PCL) films were coated with calcium phosphates (CaP) by incubation in simulated body fluid (SBF). We investigated the effect of SBF ion concentration and soaking time on the surface properties of the resulting apatite coatings. CaP coatings were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), and energy dispersive X-ray spectrometry (EDX). Young's modulus (E(s)) was determined by nanoindentation, and surface roughness was assessed by atomic force microscopy (AFM) and mechanical stylus profilometry. CaP such as carbonate-substituted apatite were deposited onto PCL films. SEM and AFM images of the apatite coatings revealed an increase in topographical complexity and surface roughness with increasing ion concentration of SBF solutions. Young's moduli (E(s)) of various CaP coatings were not significantly different, regardless of the CaP phase or surface roughness. Thus, SBF with high ion concentrations may be used to coat synthetic polymers with CaP layers of different surface topography and roughness to improve the osteoconductivity and bone-bonding ability of the scaffold.

P2X₇-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells.

Matthew W Grol et al. — Tue, 30 Oct 2012 05:26:06 PDT

The P2X₇ receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X₇ function exhibit altered bone formation. Moreover, activation of P2X₇ in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X₇ agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X₇ receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X₇, failed to elicit a sustained increase in proton efflux. Specific P2X₇ receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²⁺ was required during P2X₇ receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X₇ receptors on osteoblast-like cells triggers a dramatic, Ca²⁺-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.

Visualization of the solubilization process of the plasma membrane of a living cell by waveguide evanescent field fluorescence microscopy.

Abdollah Hassanzadeh et al. — Tue, 30 Oct 2012 05:06:03 PDT

Waveguide evanescent field fluorescence microscopy (WEFF) is a novel microscopy technology that allows imaging of a cell's plasma membrane in the vicinity of a glass substrate with high axial resolution, low background and little photobleaching. Time-lapse imaging can be performed to investigate changes in cell morphology in the presence or absence of chemical agents. WEFF microscopy provides a method to investigate plasma membranes of living cells and allows a comparison to simplified model membranes immobilized on planar substrates. The interaction of the nonionic detergent Triton X-100 with plasma membranes of osteoblasts in an aqueous environment was investigated. Solubilization of the membranes very close to the waveguide surface was visualized and related to the three-stage solubilisation model proposed for liposomes and supported lipid bilayers. Findings for the plasma membranes of cells are in excellent agreement with results reported for these artificial model systems.

One- and three-dimensional growth of hydroxyapatite nanowires during sol-gel-hydrothermal synthesis.

Daniel O Costa et al. — Mon, 29 Oct 2012 05:10:51 PDT

Control of surface topography in biomimetic calcium phosphate coatings.

Daniel O Costa et al. — Mon, 29 Oct 2012 05:05:46 PDT

P2X₇-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells.

Matthew W Grol et al. — Mon, 29 Oct 2012 04:55:42 PDT

Bioactive and Biodegradable Nanocomposites and Hybrid Biomaterials for Bone Regeneration

Bedilu A. Allo et al. — Mon, 29 Oct 2012 04:50:47 PDT

Strategies for bone tissue engineering and regeneration rely on bioactive scaffolds to mimic the natural extracellular matrix and act as templates onto which cells attach, multiply, migrate and function. Of particular interest are nanocomposites and organic-inorganic (O/I) hybrid biomaterials based on selective combinations of biodegradable polymers and bioactive inorganic materials. In this paper, we review the current state of bioactive and biodegradable nanocomposite and O/I hybrid biomaterials and their applications in bone regeneration. We focus specifically on nanocomposites based on nano-sized hydroxyapatite (HA) and bioactive glass (BG) fillers in combination with biodegradable polyesters and their hybrid counterparts. Topics include 3D scaffold design, materials that are widely used in bone regeneration, and recent trends in next generation biomaterials. We conclude with a perspective on the future application of nanocomposites and O/I hybrid biomaterials for regeneration of bone.

Finite-Element Modeling of Viscoelastic Cells During High-Frequency Cyclic Strain

Jaques S. Milner et al. — Mon, 29 Oct 2012 04:46:04 PDT

Mechanotransduction refers to the mechanisms by which cells sense and respond to local loads and forces. The process of mechanotransduction plays an important role both in maintaining tissue viability and in remodeling to repair damage; moreover, it may be involved in the initiation and progression of diseases such as osteoarthritis and osteoporosis. An understanding of the mechanisms by which cells respond to surrounding tissue matrices or artificial biomaterials is crucial in regenerative medicine and in influencing cellular differentiation. Recent studies have shown that some cells may be most sensitive to low-amplitude, high-frequency (i.e., 1–100 Hz) mechanical stimulation. Advances in finite-element modeling have made it possible to simulate high-frequency mechanical loading of cells. We have developed a viscoelastic finite-element model of an osteoblastic cell (including cytoskeletal actin stress fibers), attached to an elastomeric membrane undergoing cyclic isotropic radial strain with a peak value of 1,000 µstrain. The results indicate that cells experience significant stress and strain amplification when undergoing high-frequency strain, with peak values of cytoplasmic strain five times higher at 45 Hz than at 1 Hz, and peak Von Mises stress in the nucleus increased by a factor of two. Focal stress and strain amplification in cells undergoing high-frequency mechanical stimulation may play an important role in mechanotransduction.

Lesion of cholinergic neurons in nucleus basalis enhances response to general anesthetics

Leung L. Stan — Tue, 09 Oct 2012 07:30:57 PDT

Acetylcholine in the brain has been associated with consciousness and general anesthesia effects. We tested the hypothesis that the integrity of the nucleus basalis magnocellularis (NBM) affects the response to general anesthetics. Cholinergic neurons in NBM were selectively lesioned by bilateral infusion of 192IgG-saporin in adult, male Long–Evans rats, and control rats were infused with saline. Depletion of choline-acetyltransferase (ChAT)-immunoreactive cells in the NBM and decrease in optical density of acetylcholinesterase (AChE) staining in the frontal and visual cortices confirmed a significant decrease in NBM cholinergic neurons in lesioned as compared to control rats. AChE staining in the hippocampus and ChAT-positive neurons in the medial septum–vertical limb of the diagonal band were not different between lesioned and control rats. When a general anesthetic was administered, lesioned compared to control rats showed significantly longer duration of loss of righting reflex (LORR) after propofol (5 or 10 mg/kg i.v.), pentobarbital (20 or 40 mg/kg i.p.) but not halothane (2%). However, the behavioral excitation, as indicated by horizontal movements, induced by halothane was reduced in lesioned as compared to control rats. Reversible inactivation of NBM with GABAA receptor agonist muscimol increased slow waves in the neocortex during awake immobility, and prolonged the duration of LORR and loss of tail-pinch response after propofol, pentobarbital and halothane. In summary, lesion of NBM cholinergic neurons or inactivation of the NBM prolonged the LORR response to general anesthetic drugs.

Durand et al 2012 Supplemental figures

M. Durand et al. — Tue, 09 Oct 2012 07:10:49 PDT

Objective. Our objective was to compare the osteoclastogenic capacity of peripheral blood mononuclear cells (PBMCs) from patients with osteoarthritis (OA) to that of PBMCs from self-reported normal individuals.

Methods. PBMCs from 140 patients with OA and 45 healthy donors were assayed for CD14⁺ expression and induced to differentiate into osteoclasts (OCs) over 3 weeks in vitro. We assessed the number of the OCs, their resorptive activity, OC apoptosis, and expression of the following cytokine receptors: receptor activator of nuclear factor κB (RANK), interleukin-1 receptor type I (IL-1R1) and IL-1R2. A ridge logistic regression classifier was developed to discriminate OA patients from controls.

Results. PBMCs from OA patients gave rise to more OCs that resorbed more bone surface than did PBMCs from controls. The number of CD14⁺ precursors was comparable in both groups, but there was less apoptosis in OCs obtained from OA patients. Although no correlation was found between osteoclastogenic capacity and clinical or radiologic scores, levels of IL-1R1 were significantly lower in cultures from patients with OA compared to controls. OC apoptosis and expression levels of IL-1R1 and IL-1R2 were used to build a multivariate predictive model for OA.

Conclusion. During 3 weeks of culture under identical conditions, monocytes from patients with OA display enhanced capacity to generate OCs compared to cells from controls. Enhanced osteoclastogenesis is accompanied by increased resorptive activity, reduced OC apoptosis and diminished IL-1R1 expression. These findings support the possibility that generalized changes in bone metabolism affecting OCs participate in the pathophysiology of OA.

Vestibular stimulation enhances hippocampal long-term potentiation via activation of cholinergic septohippocampal cells

Leung L. Stan — Tue, 09 Oct 2012 07:05:57 PDT

Vestibular stimulation induced acetylcholine release in the hippocampus, and acetylcholine is known to facilitate long-term potentiation (LTP) in the hippocampus. Thus, we hypothesize that vestibular stimulation enhances LTP in CA1 in freely behaving rats, and this enhancement depends on the activation of septohippocampal cholinergic neurons. Field excitatory postsynaptic potentials were recorded in CA1 area of behaving rats following stimulation of the basal dendritic afferents. LTP was induced by a single stimulation train (100 pulses at 200 Hz) during passive whole-body rotation or during awakeimmobility. LTP induced during rotation was significantly larger than that induced during immobility. Pretreatment with cholinergic antagonist atropine sulfate (50 mg/kg i.p.) abolished the facilitation of LTP during rotation as compared to immobility. Selective lesion of cholinergic cells in the medial septum (MS) with 192 IgG-saporin (0.49 microg in 1.4 microl) also abolished the difference in LTP induced during rotation and immobility, which was found in sham-lesion rats. 192 IgG-saporin lesioned rats, as compared to sham-lesion rats, revealed a depletion of MS cells immunopositive to choline acetyltransferase and paling of acetylcholinesterase staining in the hippocampus, without significant change in the number of parvalbumin-immunopositive cells. We conclude that enhancement of LTP during vestibular stimulation is mediated by the activation of cholinergic septohippocampal cells. This is the first direct evidence that vestibular stimulation facilitates hippocampal synaptic plasticity via a cholinergic input.

Long Telomeres Bypass the Requirement for Telomere Maintenance in Human Tumorigenesis

Michael A. S. Taboski et al. — Fri, 03 Feb 2012 17:46:49 PST

Despite the importance of telomere maintenance in cancer cell survival via the elongation of telomeres by telomerase reverse transcriptase (TERT) or alternative lengthening of telomeres (ALT), it had not been tested directly whether telomere maintenance is dispensable for human tumorigenesis. We engineered human tumor cells containing loxP-flanked hTERT to enable extensive telomere elongation prior to complete hTERT excision. Despite unabated telomere erosion, hTERT-excised cells formed tumors in mice and proliferated in vitro for up to 1 year. Telomerase reactivation or ALT was not observed, and the eventual loss of telomeric signal coincided with loss of tumorigenic potential and cell viability. Crisis was averted via the reintroduction of active but not inactive hTERT. Thus, telomere maintenance is dispensable for human tumorigenesis when telomere reserves are long. Yet, despite telomere instability and the presence of oncogenic RAS, human tumors remain susceptible to crisis induced by critically short telomeres.

Osteopontin Signals through Calcium and Nuclear Factor of Activated T Cells (NFAT) in Osteoclasts: A Novel RGD-dependent Pathway Promoting Cell Survival

Natsuko Tanabe et al. — Mon, 02 Jan 2012 15:38:45 PST

Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca(2+)-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca(2+) (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) suppressed Ca(2+) oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca(2+)-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.

Male Reproductive System Defects and Subfertility in a Mutant Mouse Model of Oculodentodigital Dysplasia

M. Gregory et al. — Wed, 16 Nov 2011 16:29:57 PST

Oculodentodigital dysplasia (ODDD) is a dysmorphogenesis syndrome resulting from mutations in the GJA1 gene encoding the gap junction protein, connexin43 (CX43). In the testis this connexin localizes between Leydig cells, Sertoli cells and between Sertoli cells and germ cells. It is essential for Sertoli cell differentiation and spermatogenesis. This study explored male fertility in Gja1(Jrt) /+ mice which carry a dominant mutation that causes an amino acid substitution (G60S) in CX43. Gja1(Jrt) /+ mice mimic the phenotype of ODDD. Immunodetection methods revealed a reduction of both total CX43 and CX43 in membrane plaques in mutant testes. Correspondingly, intercellular coupling along the tubules was diminished as revealed by fluorescent dye transfer. Light and electron microscopy revealed loss of germ cells and sloughing of germ cells into the tubular lumen. There were also irregularities in size and shape of Sertoli cell nuclei. Analyses of cauda epididymal sperm indicated significant decreases in sperm count and sperm velocity parameters associated with sperm vigour, and significantly lower sperm head movement parameters associated with progressiveness. A significant decrease was also observed in total per cent motility. These results further confirm a critical role for CX43 in spermatogenesis and sperm maturation and support the possibility of subfertility in ODDD human males.

WNT2 Regulates DNA Synthesis in Mouse Granulosa Cells through Beta-catenin

Hong-Xing Wang et al. — Sun, 13 Nov 2011 16:19:44 PST

WNTs are secreted extracellular signaling molecules that transduce their signals by binding to G protein-coupled receptors of the frizzled (FZD) family. They control diverse developmental processes, such as cell fate specification, cell proliferation, cell differentiation, and apoptosis. Although WNT signaling has been shown to be essential for development of the ovary, its mechanistic role in folliculogenesis within the adult ovary has not been studied extensively. Therefore, the objective of this study was to investigate the regulation and function of WNT2 signaling in mouse granulosa cells. Immunostaining identified WNT2 as being expressed in granulosa cells throughout folliculogenesis, but with varying signal strength: in sequential sections, WNT2 immunoreactivity was strongest in healthy antral follicles but weak in atretic follicles. Knockdown of WNT2 expression using transfected short interfering RNA decreased DNA synthesis in granulosa cells, whereas WNT2 overexpression using a recombinant viral vector enhanced it. WNT2 knockdown led to accumulation of glycogen synthase kinase-3beta (GSK3B) in the cytoplasm but reduced the expression of beta-catenin. Conversely, WNT2 overexpression reduced the expression of GSK3B in the cytoplasm and induced beta-catenin translocation from the membrane into the nucleus. Beta-catenin knockdown also inhibited DNA synthesis in granulosa cells and neutralized the effect of WNT2 overexpression. WNT2/beta-catenin signaling had a slight effect on the apoptosis of granulosa cells. Taken together, the data indicate that WNT2 regulates beta-catenin localization in granulosa cells, and WNT2/beta-catenin signaling contributes to regulating their proliferation.

EBP1 Is a Novel E2F Target Gene Regulated by Transforming Growth Factor-β

David Judah et al. — Mon, 05 Sep 2011 19:39:10 PDT

Regulation of gene expression requires transcription factor binding to specific DNA elements, and a large body of work has focused on the identification of such sequences. However, it is becoming increasingly clear that eukaryotic transcription factors can exhibit widespread, nonfunctional binding to genomic DNA sites. Conversely, some of these proteins, such as E2F, can also modulate gene expression by binding to non-consensus elements. E2F comprises a family of transcription factors that play key roles in a wide variety of cellular functions, including survival, differentiation, activation during tissue regeneration, metabolism, and proliferation. E2F factors bind to the Erb3-binding protein 1 (EBP1) promoter in live cells. We now show that E2F binding to the EBP1 promoter occurs through two tandem DNA elements that do not conform to typical consensus E2F motifs. Exogenously expressed E2F1 activates EBP1 reporters lacking one, but not both sites, suggesting a degree of redundancy under certain conditions. E2F1 increases the levels of endogenous EBP1 mRNA in breast carcinoma and other transformed cell lines. In contrast, in non-transformed primary epidermal keratinocytes, E2F, together with the retinoblastoma family of proteins, appears to be involved in decreasing EBP1 mRNA abundance in response to growth inhibition by transforming growth factor-β1. Thus, E2F is likely a central coordinator of multiple responses that culminate in regulation of EBP1 gene expression, and which may vary depending on cell type and context.

Regulation of Gene Expression by PI3K in Mouse Growth Plate Chondrocytes

Veronica Ulici et al. — Mon, 05 Sep 2011 19:01:36 PDT

BACKGROUND: Endochondral ossification, the process through which long bones are formed, involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. In a previous publication we showed that pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes.

METHODOLOGY/PRINCIPAL FINDINGS: Through the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1 and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures.

CONCLUSIONS/SIGNIFICANCE: Discovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering.