Co-culture of osteoblasts and chondrocytes modulates cellular differentiation in vitro

Biological integration of cartilage grafts with subchondral bone remains a significant clinical challenge. We hypothesize that interaction between osteoblasts and chondrocytes is important in regenerating the osteochondral interface on tissue-engineered osteochondral grafts. We describe here a sequential co-culturing model which permits cell-cell contact and paracrine interaction between osteoblast and chondrocytes in 3-D culture. This model was used to determine the effects of co-culture on the phenotypic maintenance of osteoblasts and chondrocytes. It was found that while chondrocytes synthesized a type II collagen and glycosaminoglycan (GAG) matrix, GAG deposition was significantly lower in co-culture. Alkaline phosphatase activity was maintained in osteoblasts, but cell-mediated mineralization in co-culture was markedly lower compared to osteoblast controls. These results collectively suggest that interactions between osteoblasts and chondrocytes modulate cell phenotypes, and the importance of these interactions on osteochondral interface regeneration will be explored in future studies.     

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.     

Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.     

Osteogenic differentiation of immature osteoblasts: Interplay of cell culture media and supplements

Differentiation of immature osteoblasts to mature osteoblasts in vitro initially was induced by supplementing the medium with β-gylcerophosphate and dexamethasone. Later, ascorbic acid, vitamin D3, vitamin K3 and TGFβ1 were used in varying concentrations as supplements to generate a mature osteoblast phenotype. We tested the effects of several combinations of cell culture media, seeding protocols and osteogenic supplements on osteogenic differentiation of human primary osteoblasts. Osteogenic differentiation was analyzed by staining alkaline phosphatase (ALP) with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) and by von Kossa staining of deposited calcium phosphate. The combinations of culture media and supplements significantly influenced osteogenic differentiation, but the seeding protocol did not. Staining of ALP and calcium phosphate could be achieved only if our own mix of osteogenic supplements was used in combination with Dulbecco's modified Eagle medium or if a commercial mix of osteogenic supplements was used in combination with osteoblast growth medium. Especially for von Kossa, we observed great variations in the staining intensity. Because osteogenic differentiation is a complex process, the origin of the osteoblasts, cell culture media and osteogenic supplements should be established by preliminary experiments to achieve optimal differentiation. Staining of ALP or deposited calcium phosphate should be supplemented with qRT-PCR studies to learn more about the influence of specific supplements on osteogenic markers.     

A new protein expressed in bone marrow cells and osteoblasts with implication in osteoblast recruitment

To study osteoblast recruitment from bone marrow cells, a rat femur cDNA library was screened by in situ hybridization for novel mRNA sequences that are frequently expressed in both marrow cells and osteoblasts. One isolated clone, called RP59, is described here. Northern blots indicated two bands of 2.6 and 2.8 kb in femur and spleen, tissues containing high amounts of immature mesenchymal cells, and no or little expression in other tissues. The cDNA sequence revealed a reading frame for a repetitive protein composed of arrays of 14-mers and phased phosphorylation sites. Antisera versus RP59 detected a single band of 90 kDa by Western blotting of femur extract. Immunohistochemistry indicated strong RP59 presence in the cytoplasm of bone marrow cells and weaker presence in nuclei of osteoblasts. Intermediate stages were found between strongly labeled, round, free bone marrow cells and weaker labeled, fibroblast-like young osteoblasts associated with bone matrix. These data indicated that marrow cells with high RP59 content were recruited into growing bone tissue. RP59 may help to study the transition of bone marrow cell to osteoblast in more detail.     

Hydrocortisone increases the rate of differentiation of cultured human osteoblasts

Reduced bone formation is the main finding in glucocorticoid-induced osteoporosis. The aim of this study was to determine whether differentiation of cultured human osteoblasts is inhibited by high concentrations of hydrocortisone. We measured the levels of mRNAs for three markers of cellular differentiation, type 1 collagen (COL1), alkaline phosphatase (ALP), and osteocalcin (OC), in four lines of human osteoblasts from female donors cultured with doses of hydrocortisone from 0 microM to 4 microM. The change in ALP/COL1 mRNA ratio over a given time was used to determine the average rate of differentiation of the cells in a culture. Although basal expression profiles and their changes with time were different for the different cell lines, all cell lines showed a dose-dependent rise in the rate of increase of ALP mRNA relative to COL1 mRNA. However, increase in OC mRNA with time, seen here only in young donor hOBs, was significantly inhibited by 4 microM hydrocortisone, indicating that hydrocortisone can inhibit OC expression while promoting cellular differentiation. The data suggest that increasing concentrations of glucocorticoid, including concentrations similar to plasma levels in patients receiving oral glucocorticoid therapy, increase the rate of cellular differentiation.     

Co-culture with neurotrophic factor secreting cells induced from adipose-derived stem cells: Promotes neurogenic differentiation

Adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) can be equally proper in the treatment of neurodegenerative diseases. However, ADSCs have practical benefits. In this study, we attempted to induce the secretion of neurotrophic factors (NTF) in human ADSCs. We then evaluated the effects of co-culture with NTF secreting cells in neural differentiation of human ADSCs. Isolated human ADSCs were induced to neurotrophic factors secreting cells. To evaluate the in vitro effects of NTF-secreting ADSCs on neurogenic differentiation of ADSCs, we used neurogenic induction medium (control group), the combination of neurogenic medium and conditioned medium, or co-cultured NTF-secreting ADSCs which were encapsulated in alginate beads (co-culture) for 7 days. ELISA showed increased (by about 5 times) release of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in NTF-secreting ADSCs compared to human ADSCs. Real time RT-PCR analysis revealed that NTF-secreting ADSCs highly expressed NGF and BDNF. In addition, co-culture with NTF-secreting ADSCs could also promote neuronal differentiation relative to gliogenesis. Overall, NTF-secreting ADSCs secrete a range of growth factors whose levels in culture could promote neuronal differentiation and could support the survival and regeneration in a variety of neurodegenerative diseases.     

Co-culture with endothelial progenitor cells promotes survival,migration, and differentiation of osteoclast precursors

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.     

Continuous activation of G alpha q in osteoblasts results in osteopenia through impaired osteoblast differentiation

We explored the role of G alpha(q)-mediated signaling on skeletal homeostasis by selectively expressing a constitutively active G alpha(q) (mutation of Q209L) in osteoblasts. Continuous signaling via G alpha(q) in mouse osteoblastic MC3T3-E1 cells impaired differentiation. Mice that expressed the constitutively active G alpha(q) transgene in cells of the osteoblast lineage exhibited severe osteopenia in cortical and trabecular bones. Osteoblast number, bone volume, and trabecular thickness were reduced in transgenic mice, but the osteoclasts were unaffected. Osteoblasts from transgenic mice showed impaired differentiation and matrix formation. In the presence of a protein kinase C inhibitor GF109203X, this impairment was not seen, indicating mediation by the protein kinase C pathway. We propose that continuous activation of the G alpha(q) signal in osteoblasts plays a crucial, previously unrecognized role in bone formation.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells via an estrogen-receptor-dependent pathway

TAK-778, a derivative of ipriflavone, has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating by which mechanism TAK-778 exerts its effect. Considering the evidences that its precursors act via classical estrogen-receptor (ER)-mediated signaling, in the present study, we tested the hypothesis that TAK-778 induces osteogenesis in human bone marrow cell culture via an ER-dependent pathway. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing: TAK-778 (10(-5) M), Tamoxifen (10(-5) M), TAK-778 (10(-5) M) + Tamoxifen (10(-5) M), and vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. At 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by two-way ANOVA and Duncan's multiple range test. TAK-778 did not affect cell viability. Cell number was reduced by TAK-778. Total protein content, ALP activity, and bone-like formation were increased by TAK-778. In general, Tamoxifen did not have any effect on cell behavior. However, when cells were cultured in medium containing both TAK-778 and Tamoxifen, the effect of TAK-778 on osteoblast differentiation was inhibited. The present results show that TAK-778 enhances osteoblast differentiation in human bone marrow cell culture, at least in part, via an ER-dependent pathway, since its effect was inhibited by Tamoxifen, a well-known estrogen receptor antagonist.     

Effect of calcitonin gene-related peptide on osteoblast differentiation in an osteoblast and endothelial cell co-culture system

We have investigated the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on the differentiation of OB (osteoblasts) in co-culture with HUVEC (human umbilical vein endothelial cells). Primary human MOB (mandibular OB) and OB-like cells (MG-63) were either cultured directly or indirectly co-cultured with HUVEC at a 1:1 ratio. Expression of OC (osteocalcin) was measured by ELISA, and expression of ALP (alkaline phosphatase) and collagen mRNA was measured by quantitative fluorescent PCR. For mineralization nodus, OB were stained with Alizarin Red-S. When co-cultured with HUVEC, expression of OC and ALP mRNA were increased in MG-63 (P<0.01), and the expression of OC, ALP and collagen mRNA were increased in MOB (P<0.01 or 0.05). When treated with CGRP, OC and ALP mRNA and mineralization nodus numbers were increased in the MG-63 co-culture system (P<0.01 or 0.05); OC, ALP and collagen mRNA, and mineralization nodus numbers were increased in the MOB co-culture system (P<0.01 or 0.05). The effect of CGRP regulation on the differentiation of OB is not only direct but also indirect, via its effect on HUVEC and stimulation of OB.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells

TAK-778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK-778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK-778 on human bone marrow cells. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing TAK-778 (10(-7), 10(-6), and 10(-5) M, each) or vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778 in all time period evaluated in a dose-dependent way. The effect of TAK-778 on total protein content, ALP activity and bone-like formation was a dose-dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in a time and dose-dependent way. It means that TAK-778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery.     

Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function

Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis.