Evaluation of different culture techniques of osteoblasts on 3D scaffolds

Bones adjust their structure to withstand the mechanical demands they experience. It is suggested that flow-derived shear stress may be the most significant and primary mediator of mechanical stimulation. In this study, we designed and fabricated a fluid flow cell culture system that can load shear stress onto cells cultured on 3D scaffolds. We evaluated the effect of different culture techniques, namely, (1) continuous perfusion fluid flow, (2) intermittent perfusion fluid flow, and (3) static condition, on the proliferation of osteoblasts seeded on partially deproteinized bones. The flow rate was set at 1 ml/min for all the cells cultured using flow perfusion and the experiment was conducted for 12 days. Scanning electron microscopy analysis indicated an increase in cell proliferation for scaffolds subjected to fluid shear stress. In addition, the long axes of these cells lengthened along the flowing fluid direction. Continuous perfusion significantly enhanced cell proliferation compared to either intermittent perfusion or static condition. All the results demonstrated that fluid shear stress is able to enhance the proliferation of cells and change the form of cells.     

Protein kinase G and focal adhesion kinase converge on Src/Akt/β-catenin signaling module in osteoblast mechanotransduction

Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.     

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.     

Different effects of intermittent and continuous fluid shear stresses on osteogenic differentiation of human mesenchymal stem cells

A reasonable mechanical microenvironment similar to the bone microenvironment in vivo is critical to the formation of engineering bone tissues. As fluid shear stress (FSS) produced by perfusion culture system can lead to the osteogenic differentiation of human mesenchymal stem cells (hMSCs), it is widely used in studies of bone tissue engineering. However, effects of FSS on the differentiation of hMSCs largely depend on the FSS application manner. It is interesting how different FSS application manners influence the differentiation of hMSCs. In this study, we examined the effects of intermittent FSS and continuous FSS on the osteogenic differentiation of hMSCs. The phosphorylation level of ERK1/2 and FAK is measured to investigate the effects of different FSS application manners on the activation of signaling molecules. The results showed that intermittent FSS could promote the osteogenic differentiation of hMSCs. The expression level of osteogenic genes and the alkaline phosphatase (ALP) activity in cells under intermittent FSS application were significantly higher than those in cells under continuous FSS application. Moreover, intermittent FSS up-regulated the activity of ERK1/2 and FAK. Our study demonstrated that intermittent FSS is more effective to induce the osteogenic differentiation of hMSCs than continuous FSS.     

Mechanical loading by fluid shear stress enhances IGF-1 receptor signaling in osteoblasts in a PKCzeta-dependent manner

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-alpha-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCKzeta function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-zeta-dependent manner.     

Mechanical Loading by Fluid Shear Stress Enhances IGF-1 Receptor Signaling in Osteoblasts in A PKC ζ -Dependent Manner

Maintenance of optimal bone physiology requires the coordinated activity of osteoclasts that resorb old bone and osteoblasts that deposit new bone. Mechanical loading of bone and the resulting movement of interstitial fluid within the spaces surrounding bone cells is thought to play a key role is maintaining optimal bone mass. One way in which fluid movement may promote bone formation is by enhancing osteoblast survival. We have shown previously that application of fluid flow to osteoblasts in vitro confers a protective effect by inhibiting osteoblast apoptosis (Pavalko et al., 2003, J. Cell Physiol., 194: 194-205). To investigate the cellular mechanisms that regulate the response of osteoblasts to fluid shear stress, we have examined the possible interaction between fluid flow and growth factors in MC3T3-E1 osteoblast-like cells. We found that insulin-like growth factor-I (IGF-I) was significantly more effective at preventing TNF-$\alpha$-induced apoptosis when cells were first subjected to mechanical loading by exposure to either unidirectional or oscillatory fluid flow compared to cells that were maintained in static culture. Additionally, downstream signaling in response to treatment with IGF-I, including ERK and Akt activation, was enhanced in cells that were subjected to fluid flow, compared to cells maintained in static culture. Furthermore, we found that PKC$\zeta$ activity is essential for fluid shear stress sensitization of IGF-IR, since a specific inhibitor of PCK$\zeta$ function blocked the flow-enhanced IGF-I-activated Akt and ERK phosphorylation. Together, our results suggest that fluid shear stress may regulate IGF-I signaling in osteoblasts in a PKC-$\zeta$-dependent manner.     

MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation

Mechanical signals are important regulators of skeletal homeostasis, and strain-induced oscillatory fluid flow is a potent mechanical stimulus. Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm² triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway. mechanotransduction; bone; marrow     

Single Bout Short Duration Fluid Shear Stress Induces Osteogenic Differentiation of MC3T3-E1 Cells via Integrin β1 and BMP2 Signaling Cross-Talk

Fluid shear stress plays an important role in bone osteogenic differentiation. It is traditionally believed that pulsed and continuous stress load is more favorable for fracture recovery and bone homeostasis. However, according to our clinical practice, we notice that one single stress load is also sufficient to trigger osteogenic differentiation. In the present study, we subject osteoblast MC3T3-E1 cells to single bout short duration fluid shear stress by using a parallel plate flow system. The results show that 1 hour of fluid shear stress at 12 dyn/cm² promotes terminal osteogenic differentiation, including rearrangement of F-actin stress fiber, up-regulation of osteogenic genes expression, elevation of alkaline phosphatase activity, secretion of type I collagen and osteoid nodule formation. Moreover, collaboration of BMP2 and integrin β1 pathways plays a significant role in such differentiation processes. Our findings provide further experimental evidence to support the notion that single bout short duration fluid shear stress can promote osteogenic differentiation.     

Indirect osteoblast differentiation by liposomal clodronate

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.     

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.     

Cyclic fluid shear stress promotes osteoblastic cells proliferation through ERK5 signaling pathway

Fluid shear stress plays an important role in bone remodeling, however, the mechanism of mechanotransduction in bone tissue remains unclear. Recently, ERK5 has been found to be involved in multiple cellular processes. This study was designed to investigate the potential involvement of ERK5 in the proliferative response of osteoblastic cells to cyclic fluid shear stress. We reported here that cyclic fluid shear stress promoted ERK5 phosphorylation in MC3T3-E1 cells. Inhibition of ERK5 phosphorylation attenuated the increased expression of AP-1 and cyclin D1 and cell proliferation induced by cyclic fluid flow, but promoted p-16 expression. Further more, we found that cyclic fluid shear stress was a better stimuli for ERK5 activation and cyclin D1 expression compared with continuous fluid shear stress. Moreover, the pharmacological ERK5 inhibitor, BIX02189, which inhibited ERK5 phosphorylation in a time-dependent manner and the suppression lasted for at least 4 h. Taken together, we demonstrate that ERK5/AP-1/cyclin D1 pathway is involved in the mechanism of osteoblasts proliferation induced by cyclic fluid shear stress, which is superior in promoting cellular proliferation compared with continuous fluid shear stress.     

Effects of Zinc Transporter on Differentiation of Bone Marrow Mesenchymal Stem Cells to Osteoblasts

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step during bone formation. However, the exact mechanisms regulating the early stages of osteogenic differentiation remain unknown. In the present study, we found that ZnT7, a member of the zinc transporter family SLC30A(ZnTs), was downregulated during dexamethasone-induced differentiation of rat MSCs into osteoblasts. Dexamethasone treatment resulted in significantly lower levels of ZnT7 compared with cocultured cells without dexamethasone. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Overexpression of ZnT7 decreased the expression of the osteoblast alkaline phosphatase, type I collagen, as well as calcium deposition in mesenchymal cells. In contrast, knockdown of ZnT7 using siRNA promoted gene expression associated with osteoblast differentiation and matrix mineralization in vitro. Moreover, according to the ZnT7 inhibition or activation experiments, Wnt and ERK signaling pathways were found to be important signal transduction pathways in mediating the osteogenic effect of MSCs, and this effect is intensified by a decrease in the level of ZnT7 induced by dexamethasone. These findings suggest that ZnT7 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and marking the expression level of ZnT7 may be useful in the detection of early osteogenic differentiation.     

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways

Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss.     

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO₂) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl₄ or Zirconium (IV) oxynitrate (ZrO(NO₃)₂) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.     

Focal adhesion kinase signaling pathway is involved in mechanotransduction in MG-63 cells

Interstitial fluid flow, generated upon induced movement of extracellular fluid after mechanical loading, activates many signal transduction pathways in bone cells. The mechanisms of mechanobiology in bone tissue are still not clearly understood. Recently focal adhesion kinase (FAK) was shown to be involved in mechanotransduction in a number of cells. This study was designed to characterize the functional roles of FAK in mediating osteoblast response to mechanical steady-state fluid shear stress (FSS). We reported here that FSS (15 dynes/cm²) induced activation of FAK and formation of FAK·Grb2·Sos ternary complex in MG-63 cells, which was necessary for activation of the downstream mitogen-activated protein kinase pathway signaling molecules extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). Our results also showed that transfection of FAK (F397Y) plasmid, a negative mutant of FAK, blocked the increased expression of binding factor alpha 1, osterix, osteocalcin and alkaline phosphatase induced by FSS in MG-63 cells. These results demonstrate that FAK signaling is critical for FSS-induced activation of ERK and JNK, and for promotion of osteoblast differentiation and osteogenesis via its association with Grb2/Sos complex.     

Functional evaluation of cultured rabbit osteoblast-like cells

For the improvement of the adult osteoblast culture, the osteoblasts of young adult rabbit endosteal from long bones were isolated by collagenase digesting separation. 0.1% of type-I collagen precoated culture flasks were used as substrate for isolated bone cell growth. Morphological examination of cultured cells under a phase-contrast microscope, SEM and TEM observations showed a structure similar to osteoblast in vivo. Histochemical examination of alkaline phosphatase demonstrated 97% purity of cultured osteoblasts. The presence of calcium deposit activity in cultured cells was demonstrated by Van Kossa stain. High activity of alkaline phosphatase and inorganic pyrophosphatase in cultured osteoblasts as determined by biochemical analysis. High calcium uptake in cultured osteoblasts was demonstrated by radioisotope labelled 45CaCl12. According to these methods, it was indicated that the cells isolated from young rabbit long bone endosteal were osteoblast-like and still maintained their biological function. Our system for culturing osteoblast-like cells is a successful attempt in growing bone tissue in vitro starting from isolated bone cells. Therefore, this modified method for bone cell culture on collagen precoated culture flasks could be used as the experimental model in studies concerning the osteoblasts in vitro.     

Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial-derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization; and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited; and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.     

Mediators of the biphasic responses of bone to intermittent and continuously administered parathyroid hormone

Parathyroid hormone (PTH) has biphasic effects on bone: continuous treatment is catabolic whereas intermittent treatment is anabolic. The mechanism(s) responsible for these differing effects are still unclear, partly because of the previous non-availability of a model system in which effects on both formation and resorption indices could be studied concomitantly. In cultured marrow cells from 6-week old C57BL/6 mice, we demonstrated that 4 days of intermittent PTH treatment increased mRNA for osteoblast differentiation markers (Runx2, alkaline phosphatase (AP), and type I procollagen (COL1A1) whereas continuous treatment resulted in production of large numbers of TRAP-positive multinucleated osteoclasts. Although IGF-I mRNA did not increase after intermittent treatment, it was consistently higher than after continuous treatment, and the addition of an anti-IGF-I neutralizing antibody prevented the increase in bone formation indices observed with intermittent treatment. By contrast, after continuous treatment, gene expression of RANK ligand (RANKL) was increased and that of osteoprotegerin (OPG) was decreased, resulting in a 25-fold increase in the RANKL/OPG ratio. In this model system, the data suggest that intermittent PTH treatment enhances osteoblast differentiation through an IGF-I dependent mechanism and continuous PTH treatment enhances osteoclastogenesis through reciprocal increases in RANKL and decreases in OPG.