αSMA Osteoprogenitor Cells Contribute to the Increase in Osteoblast Numbers in Response to Mechanical Loading.

Bone is a dynamic tissue that site-specifically adapts to the load that it experiences. In response to increasing load, the cortical bone area is increased, mainly through enhanced periosteal bone formation. This increase in area is associated with an increase in the number of bone-forming osteoblasts; however, the origin of the cells involved remains unclear. Alpha-smooth muscle actin (αSMA) is a marker of early osteoprogenitor cells in the periosteum, and we hypothesized that the new osteoblasts that are activated by loading could originate from αSMA-expressing cells. Therefore, we used an in vivo fate-mapping approach in an established axial loading model to investigate the role of αSMA-expressing cells in the load-induced increase in osteoblasts. Histomorphometric analysis was applied to measure the number of cells of different origin on the periosteal surface in the most load-responsive region of the mouse tibia. A single loading session failed to increase the number of periosteal αSMA-expressing cells and osteoblasts. However, in response to multiple episodes of loading, the caudal, but not the cranial, periosteal surface was lined with an increased number of osteoblasts originating from αSMA-expressing cells 5 days after the initial loading session. The proportion of osteoblasts derived from αSMA-labeled progenitors increased by 70% (p < 0.05), and the proportion of αSMA-labeled cells that had differentiated into osteoblasts was doubled. We conclude that αSMA-expressing osteoprogenitors can differentiate and contribute to the increase in periosteal osteoblasts induced by mechanical loading in a site-specific manner.

Mineral trioxide aggregate improves healing response of periodontal tissue to injury in mice.

BACKGROUND AND OBJECTIVE: Mineral trioxide aggregate (MTA) is a biomaterial used in endodontic procedures as it exerts beneficial effects on regenerative processes. In this study, we evaluate the effect of MTA on healing of periodontal ligament (PDL) and surrounding tissue, following injury, in a transgenic mouse model and on the differentiation of murine mesenchymal progenitor cells in vitro. MATERIAL AND METHODS: We used an inducible Cre-loxP in vivo fate mapping approach to examine the effects of MTA on the contributions of descendants of cells expressing the αSMA-CreERT2 transgene (SMA9+ ) to the PDL and alveolar bone after experimental injury to the root furcation on the maxillary first molars. Col2.3GFP was used as a marker to identify mature osteoblasts, cementoblasts and PDL fibroblasts. The effects of MTA were examined 2, 17 and 30 days after injury and compared histologically with sealing using an adhesive system. The effects of two dilutions of medium conditioned with MTA on proliferation and differentiation of mesenchymal progenitor cells derived from bone marrow (BMSC) and periodontal ligament (PDLC) in vitro were examined using the PrestoBlue viability assay, alkaline phosphatase and Von Kossa staining. The expression of markers of differentiation was assessed using real-time PCR. RESULTS: Histological analyses showed better repair in teeth restored with MTA, as shown by greater expansion of SMA9+ progenitor cells and Col2.3GFP+ osteoblasts compared with control teeth. We also observed a positive effect on differentiation of SMA9+ progenitors into osteoblasts and cementoblasts in the apical region distant from the site of injury. The in vitro data showed that MTA-conditioned medium reduced cell viability and osteogenic differentiation in both PDLC and BMSC, indicated by reduced von Kossa staining and lower expression of osteocalcin and bone sialoprotein. In addition, cultures grown in the presence of MTA had marked decreases in SMA9+ and Col2.3GFP+ areas as compared with osteogenic medium, confirming reduced osteogenesis. CONCLUSION: MTA promotes regeneration of injured PDL and alveolar bone, reflected as contribution of progenitors (SMA9+ cells) into osteoblasts (Col2.3GFP+ cells). In vitro, MTA-conditioned medium fails to promote osteogenic differentiation of both PDLC and BMSC.

Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis.

Our study aimed to determine the functional activity of different osteoclast progenitor (OCP) subpopulations and signals important for their migration to bone lesions, causing local and systemic bone resorption during the course of collagen-induced arthritis in C57BL/6 mice. Arthritis was induced with chicken type II collagen (CII), and assessed by clinical scoring and detection of anti-CII antibodies. We observed decreased trabecular bone volume of axial and appendicular skeleton by histomorphometry and micro-computed tomography as well as decreased bone formation and increased bone resorption rate in arthritic mice in vivo. In the affected joints, bone loss was accompanied with severe osteitis and bone marrow hypercellularity, coinciding with the areas of active osteoclasts and bone erosions. Flow cytometry analysis showed increased frequency of putative OCP cells (CD3- B220- NK1.1- CD11b-/lo CD117+ CD115+ for bone marrow and CD3- B220- NK1.1- CD11b+ CD115+ Gr-1+ for peripheral haematopoietic tissues), which exhibited enhanced differentiation potential in vitro. Moreover, the total CD11b+ population was expanded in arthritic mice as well as CD11b+ F4/80+ macrophage, CD11b+ NK1.1+ natural killer cell and CD11b+ CD11c+ myeloid dendritic cell populations in both bone marrow and peripheral blood. In addition, arthritic mice had increased expression of tumour necrosis factor-α, interleukin-6, CC chemokine ligand-2 (Ccl2) and Ccl5, with increased migration and differentiation of circulatory OCPs in response to CCL2 and, particularly, CCL5 signals. Our study characterized the frequency and functional properties of OCPs under inflammatory conditions associated with arthritis, which may help to clarify crucial molecular signals provided by immune cells to mediate systemically enhanced osteoresorption.

Gene-expression analysis of cementoblasts and osteoblasts.

BACKGROUND AND OBJECTIVE: Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. MATERIAL AND METHODS: OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. RESULTS: Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. CONCLUSION: The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Bone-specific overexpression of NPY modulates osteogenesis.

OBJECTIVES: Neuropeptide Y (NPY) is a peptide involved in the regulation of appetite and energy homeostasis. Genetic data indicates that NPY decreases bone formation via central and peripheral activities. NPY is produced by various cell types including osteocytes and osteoblasts and there is evidence suggesting that peripheral NPY is important for regulation of bone formation. We sought to investigate the role of bone-derived NPY in bone metabolism. METHODS: We generated a mouse where NPY was over-expressed specifically in mature osteoblasts and osteocytes (Col2.3NPY) and characterized the bone phenotype of these mice in vivo and in vitro. RESULTS: Trabecular and cortical bone volume was reduced in 3-month-old animals, however bone formation rate and osteoclast activity were not significantly changed. Calvarial osteoblast cultures from Col2.3NPY mice also showed reduced mineralization and expression of osteogenic marker genes. CONCLUSIONS: Our data suggest that osteoblast/osteocyte-derived NPY is capable of altering osteogenesis in vivo and in vitro and may represent an important source of NPY for regulation of bone formation. However, it is possible that other peripheral sources of NPY such as the sympathetic nervous system and vasculature also contribute to peripheral regulation of bone turnover.

Defining a visual marker of osteoprogenitor cells within the periodontium.

BACKGROUND AND OBJECTIVE: Cells with osteoprogenitor potential are present within periodontal tissues during development and in postnatal life. To identify an osteoprogenitor population, this study utilized a transgenic model in which an alpha-smooth muscle actin (alphaSMA) promoter directed green fluorescent protein (GFP) expression. MATERIAL AND METHODS: Observation of GFP expression was complemented with analysis of osteogenic differentiation by determining the expression of RNA of bone markers, by histochemical staining for alkaline phosphatase and by the detection of mineralized nodules using xylenol orange. Flow cytometry was utilized to determine the proliferative potential and cell-surface phenotype of cultured alphaSMA-positive cells. RESULTS: alphaSMA-GFP expression was detected within the dental follicle and in the apical region of the root (i.e. areas rich in vascularization) but not in mature bone. alphaSMA-GFP expression was observed during the early stages of primary cultures derived from the dental follicle and periodontal ligament and was diminished in areas undergoing mineralization. Intense alkaline phosphatase activity and the presence of mineralized nodules was observed 2 wk after osteogenic induction. Consequently, the expression of bone sialoprotein, osteocalcin and dentin matrix protein-1 was increased. Flow cytometry revealed that in vitro expansion enriched for an alphaSMA-GFP-positive population in which 55-65% of cells expressed the cell-surface markers Thy1(+) and Sca1(+). The alphaSMA-GFP-positive population exhibited high proliferative and osteogenic potentials when compared with an alphaSMA-GFP-negative population. CONCLUSION: Our data indicate that the alphaSMA promoter can be used to identify a population of osteoprogenitor cells residing within the dental follicle and periodontal ligament that can differentiate into mature osteoblasts.

FGF2 Enhances Odontoblast Differentiation by αSMA+ Progenitors In Vivo.

The goal of this study was to examine the effects of early and limited exposure of perivascular cells expressing α (αSMA) to fibroblast growth factor 2 (FGF2) in vivo. We performed in vivo fate mapping by inducible Cre-loxP and experimental pulp injury in molars to induce reparative dentinogenesis. Our results demonstrate that early delivery of exogenous FGF2 to exposed pulp led to proliferative expansion of αSMA-tdTomato+ cells and their accelerated differentiation into odontoblasts. In vivo lineage-tracing experiments showed that the calcified bridge/reparative dentin in FGF2-treated pulps were lined with an increased number of Dspp+ odontoblasts and devoid of BSP+ osteoblasts. The increased number of odontoblasts derived from αSMA-tdTomato+ cells and the formation of reparative dentin devoid of osteoblasts provide in vivo evidence for the stimulatory effects of FGF signaling on odontoblast differentiation from early progenitors in dental pulp.

FGF Signaling Prevents the Terminal Differentiation of Odontoblasts.

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis, with conflicting results regarding their effects on odontoblast differentiation. Our recent studies showed that the effects of FGF2 on cells in odontoblast lineage were stage-specific and depended on the stage of cell maturity. Continuous exposure of pulp cells to FGF2 inhibited odontoblast differentiation, whereas early and limited exposure of pulp cells to FGF2 resulted in marked increases in odontoblast differentiation. The purpose of this study was to evaluate the cellular and molecular mechanisms regulating the inhibitory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of the addition of FGF2 during the differentiation/mineralization phase of the in vitro growth of pulp cultures derived from a series of green fluorescent protein reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation. Our results showed that this treatment first stimulated the differentiation of remaining progenitors in pulp cultures into functional odontoblasts but prevented their differentiation into mature odontoblasts. In addition, this treatment inhibited expression of markers of osteogenesis. Furthermore, we demonstrated that the inhibitory effects of FGF2 on odontoblast differentiation were mediated through activation of FGFR/MEK/Erk1/2 signaling and downregulation of bone morphogenetic protein signaling, with negative and positive roles in the expression of Dmp1 and Dspp, respectively, during the advanced stage of odontoblast differentiation.

αSMA-Expressing Perivascular Cells Represent Dental Pulp Progenitors In Vivo.

The goal of this study was to examine the contribution of perivascular cells to odontoblasts during the development, growth, and repair of dentin using mouse molars as a model. We used an inducible, Cre-loxP in vivo fate-mapping approach to examine the contributions of the descendants of cells expressing the αSMA-CreERT2 transgene to the odontoblast lineage. In vivo lineage-tracing experiments in molars showed the contribution of αSMA-tdTomato+ cells to a small number of newly formed odontoblasts during primary dentinogenesis. Using an experimental pulp exposure model in molars to induce reparative dentinogenesis, we demonstrate the contribution of αSMA-tdTomato+ cells to cells secreting reparative dentin. Our results demonstrate that αSMA-tdTomato+ cells differentiated into Col2.3-GFP+ cells composed of both Dspp+ odontoblasts and Bsp+ osteoblasts. Our findings identify a population of mesenchymal progenitor cells capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis. This population also makes a small contribution to odontoblasts during primary dentinogenesis.

Enhanced Dentinogenesis of Pulp Progenitors by Early Exposure to FGF2.

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis. Although there appears to be a general agreement on the effects of FGF signaling on the proliferation of pulp cells, there are conflicting results regarding its effects on odontoblast differentiation. We recently examined the effects of continuous exposure of dental pulp cells to FGF2 and showed that the effects of FGF2 on differentiation of progenitor cells into odontoblasts were stage specific and dependent on the stage of cell maturity. The purpose of this study was to gain further insight into cellular and molecular mechanisms regulating the stimulatory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of early and limited exposure of pulp cells from a series of green fluorescent protein (GFP) reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation to FGF2. Our results showed that early and limited exposure of pulp cells to FGF2 did not have significant effects on the extent of mineralization but induced significant increases in the expression of Dmp1 and Dspp and the number of DMP1-GFP(+) and DSPP-Cerulean(+) odontoblasts. Our results also showed that the stimulatory effects of FGF2 on odontoblast differentiation were mediated through FGFR/MEK/Erk1/2 signaling, increases in Bmp2, and activation of the BMP/BMPR signaling pathway. These observations show that early and limited exposure of pulp cells to FGF2 alone promotes odontoblast differentiation and provides critical insight for applications of FGF2 in dentin regeneration.

Col1a1-driven transgenic markers of osteoblast lineage progression.

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated nodules. The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2.3-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.

Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage.

Green fluorescent protein (GFP)-expressing transgenic mice were produced containing a 3.6-kilobase (kb; pOBCol3.6GFPtpz) and a 2.3-kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6-kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3-kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz-positive cells first appeared in spindle-shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz-positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice.

Two transgenic mouse lines were generated with a DNA construct bearing a 2.3-kilobase (kb) fragment of the rat alpha1 type I collagen promoter driving a truncated form of the herpes thymidine kinase gene (Col2.3Atk). Expression of the transgene was found in osteoblasts coincident with other genetic markers of early osteoblast differentiation. Mice treated with ganciclovir (GCV) for 16 days displayed extensive destruction of the bone lining cells and decreased osteoclast number. In addition, a dramatic decrease in bone marrow elements was observed, which was more severe in the primary spongiosum and marrow adjacent to the diaphyseal endosteal bone. Immunostaining for transgene expression within the bone marrow was negative and marrow stromal cell cultures developed normally in the presence of GCV until the point of early osteoblast differentiation. Our findings suggest that the early differentiating osteoblasts are necessary for the maintenance of osteoclasts and hematopoiesis. Termination of GCV treatment produced an exaggerated response of new bone formation in cortical and trabecular bone. The Col2.3deltatk mouse should be a useful model to define the interrelation between bone and marrow elements as well as a model to analyze the molecular and cellular events associated with a defined wave of osteogenesis on termination of GCV treatment.

Osteoblastic response to the defective matrix in the osteogenesis imperfecta murine (oim) mouse.

This work examines the cellular pathophysiology associated with the weakened bone matrix found in a murine model of osteogenesis imperfecta murine (oim). Histomorphometric analysis of oim/oim bone showed significantly diminished bone mass, and the osteoblast and osteoclast histomorphometric parameters were increased in the oim/oim mice, compared with wild-type (+/+) mice. To assess osteoblast activity, a rat Col1a1 promoter linked to the chloramphenicol acetyltransferase reporter transgene was bred into the oim model. At 8 d and 1 month of age, no difference in transgene activity between oim and control mice was observed. However, at 3 months of age, chloramphenicol acetyl transferase activity was elevated in oim/oim;Tg/Tg, compared with +/+;Tg/Tg and oim/+;Tg/Tg. High levels of urinary pyridinoline crosslinks in the oim/oim;Tg/Tg mice were present at all ages, reflecting continuing high bone resorption. Our data portray a state of ineffective osteogenesis in which the mutant mouse never accumulates a normal quantity of bone matrix. However, it is only after the completion of the rapid growth phase that the high activity of the oim/oim osteoblast can compensate for the high rate of bone resorption. This relationship between bone formation and resorption may explain why the severity of osteogenesis imperfecta decreases after puberty is completed. The ability to quantify high bone turnover and advantages of using a transgene that reflects osteoblast lineage activity make this a useful model for studying interventions designed to improve the bone strength in osteogenesis imperfecta.

Human bone marrow stromal cells are efficiently transduced by vesicular stomatitis virus-pseudotyped retrovectors without affecting subsequent osteoblastic differentiation.

This study tested the transduction efficiency of human bone marrow stromal cells (hBMSCs) with vesicular stomatitis virus (VSV)-pseudotyped retrovectors and their subsequent osteogenic differentiation in vitro. Two different retrovectors encoding beta-galactosidase (beta-gal) or enhanced green fluorescent protein (eGFP) as marker genes were examined for transduction of hBMSCs. hBMSCs were obtained from bone marrow filtrates of normal donors (aged 5-35 years), cultured in alpha-minimal essential medium (alpha-MEM) containing 10% fetal calf serum and infected with retrovectors soon after the adherent cells started to form individual colonies. Transduced hBMSCs were observed to express eGFP protein 4-7 days after infection in primary cultures, and the majority of hBMSCs were eGFP-positive. hBMSCs were also stained for beta-gal in the secondary cultures and virtually all hBMSCs expressed beta-gal activity. Transduced hBMSCs were examined for their osteogenic potential. These cells were found to express markers of osteogenic differentiation, including alkaline phosphatase, type I collagen, bone sialoprotein, decorin, and osteocalcin, as strongly as uninfected control cells. Mineralization was also induced by dexamethasone in transduced cells as well as control cells. These results demonstrate that hBMSCs are highly susceptible to infection with VSV-pseudotyped retrovectors with the majority of cultured cells expressing the viral transgenes without antibiotic selection. Transduced cells retain their osteogenic potential in vitro. hBMSCs are a promising cellular vehicle for systemic human gene therapy and VSV-pseudotyped retrovectors should be effective for their in vitro transduction prior to cellular engraftment.

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.

Our previous studies have demonstrated that promoter-green fluorescent protein (GFP) transgenes can be used to identify and isolate populations of cells at the preosteoblastic stage (pOBCol3.6GFP) and at the mature osteoblastic stage (pOBCol2.3GFP) in living primary bone cell cultures. This strategy forms the basis for appreciating the cellular heterogeneity of lineage and relating gene function to cell differentiation. A weakness of this approach was the lack of a selective marker for late osteoblasts and mature osteocytes in the mineralized matrix. In this study, we have examined the expression of DMP-1 mRNA in murine marrow stromal and calvarial osteoblast cultures, and in bone, and calvaria in vivo. Furthermore, we have generated transgenic mice utilizing a mouse DMP1 cis-regulatory system to drive GFP as a marker for living osteocytes. Transgene expression was directed to mineralized tissues and showed a high correlation with the expression of the endogenous gene. Osteocyte-restricted expression of GFP was observed in histological sections of femur and calvaria and in primary cell cultures. Generation of this transgenic model will facilitate studies of gene expression and biological functions in these terminally differentiated bone cells.

[Dynamic aggregation of thrombocytes in acute myocardial infarct]. / Dinamika agregacije trombocita u akutnom infarktu miokarda.

The dynamics of circulating platelet aggregates (CPA) in acute myocardial infarction (MI) was correlated to its extent, localization and clinical outcome. Creatine kinase (CK) and CPA were measured in 30 patients with acute MI 24 hours after its onset, and on the third, fifth and eighth day following the accident. Twelve patients had anteroseptal MI, another 12 had inferior, and the remaining 6 had non-Q wave MI. 24 hours after the accident CPA values differed significantly between the three groups (p < 0.05). The values of CPA increased with increasing CK. In all the patients CPA and CK returned to normal or almost normal values on days 8 and 5 following MI, respectively. Eleven patients (37%) had heart failure: 8 of them (73%) had anteroseptal MI and 3 had inferior MI (27%). In 10 patients with heart failure (91%) and only in 2 out of 19 patients without heart failure (10.5%), CPA peaked on days 3 or 5 after MI (p < 0.05). In all other patients CPA declined steadily from the initial value. These results suggest that platelet aggregability is significantly associated with the severity of MI and with heart failure.

In vivo identification of periodontal progenitor cells.

The periodontal ligament contains progenitor cells; however, their identity and differentiation potential in vivo remain poorly characterized. Previous results have suggested that periodontal tissue progenitors reside in perivascular areas. Therefore, we utilized a lineage-tracing approach to identify and track periodontal progenitor cells from the perivascular region in vivo. We used an alpha-smooth muscle actin (αSMA) promoter-driven and tamoxifen-inducible Cre system (αSMACreERT2) that, in combination with a reporter mouse line (Ai9), permanently labels a cell population, termed 'SMA9'. To trace the differentiation of SMA9-labeled cells into osteoblasts/cementoblasts, we utilized a Col2.3GFP transgene, while expression of Scleraxis-GFP was used to follow differentiation into periodontal ligament fibroblasts during normal tissue formation and remodeling following injury. In uninjured three-week-old SMA9 mice, tamoxifen labeled a small population of cells in the periodontal ligament that expanded over time, particularly in the apical region of the root. By 17 days and 7 weeks after labeling, some SMA9-labeled cells expressed markers indicating differentiation into mature lineages, including cementocytes. Following injury, SMA9 cells expanded, and differentiated into cementoblasts, osteoblasts, and periodontal ligament fibroblasts. SMA9-labeled cells represent a source of progenitors that can give rise to mature osteoblasts, cementoblasts, and fibroblasts within the periodontium.