Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells

Although substance P (SP) is associated with osteoclast differentiation and bone resorption, little is known about the osteogenic differentiation-inducing effects of SP in periodontal ligament (PDL) cells. This study investigated whether PDL cells could differentiate into osteoblastic-like cells by SP. The expression of osteoblastic differentiation markers such as osteopontin (OPN), osteonectin (ON), osteocalcin (OCN) and bone sialoprotein (BSP) were evaulated by Western blotting. Additionally, SP-mediated heme oxygenase-1 (HO-1) pathways were further clarified.SP increased HO-1 and osteogenic differentiation in concentration- and time-dependent manners, as determined by OPN, ON, OCN and BSP expression. Furthermore, treatment with inhibitors of p38, ERK MAPK, and NF-κB abolished SP-induced osteogenic differentiation and HO-1 expression. SP-induced translocation of Nrf-2 was also observed. The combined results suggest that SP activates the stress-response enzymes HO-1 and Nrf-2, subsequently leading to upregulation of osteogenic differentiation in human PDL cells.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Confocal imaging and timing of secretion of matrix proteins by osteoblasts derived from avian long bone

Primary osteoblasts derived from avian long bone have been evaluated in terms of spatial and temporal expression of known osteoblastic marker proteins during the early phases of cell culture. Confocal imaging of matrix proteins revealed that osteocalcin, bone sialoprotein, osteopontin, and osteonectin were restricted to the cell interior at day 4 of culture; secretion and deposition into the extra-cellular matrix of bone sialoprotein and osteopontin was evident at 8 and 12 days of culture. Osteocalcin and osteonectin were not deposited in the matrix within the timeframe of the study. Total collagen levels produced and alkaline phosphatase activity were substantial by day 4 of culture, and increased from that point 4.0- and 5.5-fold, respectively, by culture day 12. The expression of type I collagen, PTHrP receptor, osteopontin, bone sialoprotein and osteocalcin was followed by Northern blot analysis. Type I collagen and osteopontin mRNA were expressed at constant levels throughout the culture period. Over the 12 days of culture both PTH/PTHrP receptor and bone sialoprotein mRNA expression were found to increase by 2.3- and 2.5-fold, respectively. In contrast, the expression of osteocalcin message decreased by 2.5-fold by day 8 of culture.     

Time course of osteopontin, osteocalcin, and osteonectin accumulation and calcification after acute vessel wall injury.

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Characterisation of the temporal sequence of osteoblast gene expression during estrogen-induced osteogenesis in female mice

Osteoblast differentiation under in vitro conditions is associated with increased expression of non-collagenous bone proteins including osteocalcin, osteopontin, and osteonectin, the exact function of which remain poorly understood. To determine whether these proteins play an important role in the formation of mineralised bone matrix by osteoblasts in vivo, we analysed the time-course of their expression during estrogen-induced osteogenesis in female mice, and compared this with the formation of new cancellous bone. Female mice were sacrificed prior to or following treatment with 17beta-estradiol for up to 32 days (500 microg/animal/week). Total RNA was extracted from femurs, and changes in expression of genes for a range of osteoblast-derived proteins assessed by Northern blot analysis. In parallel experiments, the time course of cancellous bone formation was determined by measuring bone mineral density (BMD) of the distal femur. Estrogen led to a rapid increase in BMD, which reached significance by Day 16. This was preceded by three-fold increases in expression of alkaline phosphatase (ALP) and type I collagen (COL I) at Days 8 and 12 respectively. In contrast, osteocalcin, osteopontin, and osteonectin expression showed no change during this initial period, although modest increases were observed at later times (i.e., Days 20 and 24). Our results suggest that osteocalcin, osteopontin, and osteonectin are not involved in the initial phase of the osteogenic response to estrogen, suggesting that these non-collagenous bone proteins do not play a direct role in the formation of mineralised bone matrix by osteoblasts in vivo.     

Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I,osteopontin, and osteocalcin gene expression in chicken osteoblasts

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)₂ D₃ showed a 2–10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α₁(I) and α₂(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)₂ D₃ at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)₂ D₃ treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)₂ D₃ in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.     

Induction of a program gene expression during osteoblast differentiation with strontium ranelate

Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.     

Functional roles of the tissue inhibitor of metalloproteinase 3 (TIMP-3) during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells

The tissue inhibitor of the metalloproteinase-3 (TIMP-3) gene was isolated as a gene involved in the process of ascorbate-induced differentiation of mouse MC3T3-E1 cells by the differential display method. The functional roles of TIMP-3 were characterized by establishing stable cell lines, which constitutively expressed the TIMP-3 gene. The TIMP-3 transfectants produced type I collagen at the same level as that of normal cells in response to ascorbic acid 2-phosphate (AscP). However, the expression of the other osteoblastic marker proteins such as alkaline phosphatase (ALPase), osteopontin (OP), osteocalcin (OC), osteonectin (ON) and matrix metalloproteinases (MMPs) remained at a low level even in the presence of AscP. Furthermore, no mineralization of the extracellular matrix (ECM) occurred with the transfectants. Remodeling ECM through TIMPs and MMPs is concluded to be required for osteoblastic differentiation.     

Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

 Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading. Accepted: 9 July 1996     

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.     

Homocysteine attenuates the expression of osteocalcin but enhances osteopontin in MC3T3-E1 preosteoblastic cells

It has been pointed out that very high plasma levels of homocysteine are characteristic of homocystinuria, a rare autosomal recessive disease accompanied by the early onset of generalized osteoporosis. However, it is unclear by which mechanism hyperhomocysteine induces osteoporosis, although it is known to interfere with the formation of cross-links in collagen, an essential process in bone formation. Therefore, we investigated the effect of homcysteine on expression of osteocalcin and osteopontin in MC3T3-E1 preosteoblastic cells. Confluent cells were grown in RPMI 1640 containing 10% fetal calf serum with or without homocysteine in an atmosphere of 95% humidified air, 5% CO2 at 37 degrees C. The secretion of osteocalcin from the cells increased time-dependently until the end of culture (day 34), but 500 microM homocysteine led to an approximately 61% decrease for osteocalcin after 19 days of culture as compared with the control. On the other hand, osteopontin was not inhibited by 500 microM homocysteine but rather activated, and ranged from 134%-209% of the control level in the period from 10 days until the end of culture. From analysis of RT-PCR for mRNA of osteocalcin and osteopontin at the end of the culture, homocysteine levels of 100 and 500 microM significantly increased the expression of osteopontin mRNA with the control (p < 0.05). In contrast, the expression of osteopontin mRNA was suppressed in a dose-dependent manner, showing a mirror image of the effect on osteopontin mRNA. These findings suggest that hyperhomocystenemia appears to be an independent risk factor for osteoporosis by disturbing osteoblast function.     

Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.     

Generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide LL-37 treated monocytes

Background

Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair.

Methods and Findings

Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK).

Conclusion

Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.     

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow-derived mesenchymal stem cells, by upregulating the associated genes

Objectives: Chitosan is widely used as a scaffold for bone tissue engineering. However, up‐to‐date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan‐coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow. Materials and methods: Tissue culture plates coated with chitosan at different coating densities were used to evaluate the effects on hMSC adhesion and osteoblast differentiation. hMSCs were induced to differentiate into osteoblasts on the chitosan‐coated plates and were evaluated using established techniques: alkaline phosphatase assay, demonstration of presence of calcium and real time PCR. Results: The cells adhered to plates of lower coating density of chitosan, but formed viable cell aggregates at higher coating density (100 μg/sq.cm). Coating density of 25 μg/sq.cm, supporting cell adhesion was chosen for osteoblast differentiation experiments. Differentiating hMSCs showed higher mineral deposition and calcium content on chitosan‐coated plates. Chitosan upregulated genes associated with calcium binding and mineralization such as collagen type 1 alpha 1, integrin‐binding sialoprotein, osteopontin, osteonectin and osteocalcin, significantly. Conclusions: We demonstrate for the first time that chitosan enhanced mineralization by upregulating the associated genes. Thus, the study may help clinical situations promoting use of chitosan in bone mineralization, necessary for healing non‐union fractures and more.