Faculty Bibliography

INTRODUCTION: Histological studies of immature human permanent necrotic teeth with or without apical periodontitis after revascularization have not been reported. This case report describes the histological findings of tissue formed in the canal space of an immature permanent tooth #9 with irreversible pulpitis without apical periodontitis after revascularization. METHODS: An immature human permanent tooth #9 was fractured 3.5 weeks after revascularization and could not be retained. The tooth was extracted and prepared for routine histological and immunohistochemical evaluation in order to examine the nature of tissue formed in the root canal following the revascularization procedure. RESULTS: At 3.5 weeks after revascularization, more than one half of the canal was filled with loose connective tissue similar to the pulp tissue. A layer of flattened odontoblast-like cells lined along the predentin. Layers of epithelial-like cells, similar to the Hertwig's epithelial root sheath, surrounded the root apex. No hard tissue was formed in the canal. CONCLUSIONS: Based on the histological findings in the present case, regeneration of pulp-like tissue is possible after revascularization. In this case, both the apical papilla and the Hertwig's epithelial root sheath survived in an immature permanent tooth despite irreversible pulpitis but without apical periodontitis.

The current study involves fabrication and characterization of bio-composite scaffolds containing chitosan (CS), nano-hydroxyapatite (nHAp) and Cu-Zn alloy nanoparticles (nCu-Zn) by freeze drying technique. The fabricated composite scaffolds (CS/nHAp and CS/nHAp/nCu-Zn) were characterized by SEM, EDX, XRD and FT-IR studies. The addition of nCu-Zn in the CS/nHAp scaffolds significantly increased swelling, decreased degradation, increased protein adsorption, and increased antibacterial activity. The CS/nHAp/nCu-Zn scaffolds had no toxicity towards rat osteoprogenitor cells. So the developed CS/nHAp/nCu-Zn scaffolds have advantageous and potential applications over the CS-nHAp scaffolds for bone tissue engineering.

Bisphosphonates are therapeutic agents in the treatment of post-menopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, inappropriate femoral fractures, and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction. We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of adult mice injected with alendronate (10 microg/100 g/wk) for 8 weeks. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. After elimination of pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation, indicating the need for pre-osteoclasts for alendronate's effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts, whereas it enhanced ephrinB1 protein in pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression. Thus, alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1, which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF-like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real-time RT-PCR and promoter reporter assays revealed that EGF-like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR-dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR-induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co-repressors HDAC4 and 6 and over-expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down-regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage.

While the epidermal growth factor receptor (EGFR)-mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro-computed tomography (microCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfr(f/f)), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre Egfr(Wa5/f) mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit-fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr(Dsk5/+) mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

CONTEXT: Chronic high levels of PTH may be associated with up-regulation of proteases and cytokines. Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory cytokine, produced predominantly by macrophages and endothelial cells, and is expressed in adipose tissue. More recently it has been shown that PTH administration increases MCP-1 expression in osteoblasts. OBJECTIVES: Because both PTH and MCP-1 levels are higher in obesity, the goal was to determine whether the high MCP-1 occurs only in the presence of high serum PTH and is independent of adiposity and examine its relationship with bone mineral density (BMD) and turnover. DESIGN, SETTING, AND PARTICIPANTS: In this case-control clinical design, 111 eligible women were categorized into four groups: leaner women [body mass index (BMI) 23 +/- 2 kg/m(2)] with normal or higher PTH and obese (BMI 44 +/- 7 kg/m(2)) with normal or higher PTH. RESULTS: Serum MCP-1 levels were higher (P < 0.01) in the high (PTH = 74.9 +/- 27.0 pg/ml, MCP-1 = 421.5 +/- 157.0 pg/ml) compared with normal PTH (PTH = 32.5 +/- 10.4 pg/ml, MCP-1 = 322.5 +/- 97.8 pg/ml) group, independent of BMI. C-reactive protein and adiponectin were influenced only by BMI and not PTH. MCP-1 was positively associated with osteocalcin and propeptide of type 1 collagen in the leaner (r > 0.3, P < 0.05) but not the obese women and was not associated with BMD in either group. CONCLUSIONS: Together these data suggest that MCP-1 is higher only in the presence of increased PTH and that adiposity alone cannot explain the higher MCP-1 levels in obesity.

The epidermal growth factor receptor (EGFR) has vital roles in developmental and pathological processes. However, its function in the skeletal system has not been well studied. Our previous research demonstrated that its ligands are potent mitogens for osteoprogenitors but strongly suppress osteoblast differentiation in vitro. To address the in vivo role of EGFR in bone, we constructed transgenic and pharmacological mouse models and used microCT and histomorphometry to analyze their bone phenotype. To eliminate the pre/osteoblastic EGFR activity, we generated 3.6 kb collagen1a1-Cre Egfr^Wa5/flox (Col-Cre Egfr^Wa5/f) mice harboring an EGFR dominant negative allele, Wa5. At 3-, 5- and 7-months old, these mice exhibited a remarkable 20-40% decrease in tibial trabecular bone volume (BV/TV) with a reduction in trabecular number (17-32%) and trabecular thickness (10%) in both genders. Histological analyses of 7-month old female mice revealed that bone formation and mineralization activities were reduced and bone resorption was elevated. These mice exhibited decreases in osteoblast surface (35%), osteoid surface (34%), mineral apposition rate (MAR, 15%), bone formation rate (BFR, 24%), and an increase in osteoclast surface (78%). Similarly, administration of an EGFR inhibitor into wild type mice caused a 25% reduction in trabecular BV/TV. Histomorphometric analyses suggested similar changes in bone formation and resorption parameters as Col-Cre Egfr^Wa5/fmice. In contrast, 3-month old Egfr^Dsk5/+ mice with an EGFR constitutively active allele displayed increases in trabecular BV/TV (36-43%) accompanied by an increase in trabecular number (28-35%) and a decrease in trabecular separation (9-17%) in both genders. These structural changes were due to increases in osteoblast surface (37%), osteoid surface (35%), MAR (18%) and BFR (34%) and a decrease in osteoclast surface (27%). Interestingly, we found that EGFR inhibitor dramatically reduced the number of CFU-F (52%!

Parathyroid hormone (PTH) is the major hormone regulating calcium homeostasis, but it also has a significant role as an anabolic hormone in bone when administered by intermittent injection. The mechanisms for the bone anabolic effect of PTH are not understood. However, it has been established that the anabolic effects of PTH require osteoclast activity. PTH acts through a single receptor on the osteoblast to regulate many known genes. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when PTH(1-34) is injected intermittently in rats for 14 days. Through further in vivo studies, we found that rats treated with PTH(1-34) for 14 days showed a significant increase in serum MCP-1 levels 2 h after PTH injection (159 pg/ml) compared to basal levels obtained prior to the last injection (65 pg/ml). Furthermore, serum MCP-1 levels increase with the duration of treatment. Compared to vehicle treatment, PTH(1-34) treatment in rats for 1, 7 and 14 days showed a 24%, 53% and 79% increase in serum MCP-1 levels 2 h after injection, respectively. Using immunohistochemistry, increased MCP-1 expression is evident in trabecular osteoblasts by the second PTH(1-34) injection with peak staining occurring between 3 and 7 days of treatment. Male and female MCP-1 knockout mice injected daily with 80 ug/kg PTH(1-34) for 6 weeks showed significantly reduced anabolic effects of PTH compared to wild type mice as measured by pQCT and micro-CT. PTH-treated male MCP-1 null mice have significantly less trabecular bone mineral density (means, mg/cm³, vehicle: 454; PTH: 486) than the corresponding PTH-treated wild type mice (means, mg/cm³, vehicle: 452; PTH: 521). Trabecular bone volume (BV/TV) increased 27% in PTH-treated wild type mice with a corresponding increase in trabecular thickness (Tb.Th), however no increase in BV/TV or Tb.Th was observed in the PTH-treated MCP-1 null mice. Hi!

Parathyroid hormone (PTH) regulates the transcription of many genes involved in bone remodeling in osteoblasts. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. We have previously shown that Mmp-13 gene expression is highly induced by PTH treatment in osteoblastic UMR 106-01 cells, as well as primary osteoblasts. Here, we show that p300/CBP-associated factor (PCAF), in addition to p300 and Runx2, is required for PTH activation of Mmp-13 transcription. PCAF was increasingly recruited to the MMP-13 proximal promoter region after PTH treatment, and this was associated with an increase in RNA polymerase II recruitment and histone acetylation. In addition, PTH treatment increased the acetylation of PCAF, a process that required p300. Knockdown of PCAF, p300, or Runx2 by siRNA decreased Mmp-13 mRNA expression after PTH treatment in both UMR 106-01 cells and primary osteoblasts. We found that there is a mutual dependence between p300 and PCAF to be recruited to the Mmp-13 promoter after PTH treatment. In promoter-reporter assays, p300 and PCAF had an additive effect on PTH stimulation of MMP-13 promoter activity, and this required their histone acetyltransferase activity. Our findings demonstrate that PCAF acts downstream of PTH signaling as a transcriptional coactivator that is required for PTH stimulation of MMP-13 transcription. PCAF cooperates with p300 and Runx2 to mediate PTH activation of MMP-13 transcription.

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.