A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

Uptake of bone-related matrix proteins into implanted deproteinized bovine bone mineral

Deproteinized bovine bone mineral (DBBM) (Bio-Oss®, Geistlich-Pharma, Wohlhusen, Switzerland) is widely used as a bone substitute for the preservation or augmentation of bone volume. After implantation near native bone, new bone may form around the DBBM particles. Since DBBM is very resistant to resorption, it will hardly ever be replaced by bone and, therefore, the mechanical stability largely depends on the extent of bridging between the newly formed bone and the DBBM particles. The molecular factors responsible for the deposition of new bone to the DBBM particles have not been determined. The aim of this study was, therefore, to test the hypothesis that DBBM implanted near bone take up bone-related matrix proteins that are involved in cell-matrix interactions. Cylindrical biopsies harvested from tooth extraction sites filled with DBBM particles were fixed in aldehydes, decalcified, and embedded in LR White resin. Thin sections were incubated with antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two bone proteins involved in cell attachment, signaling, and mineralization. High-resolution immunogold labeling was used to examine protein distribution. BSP and OPN were immunodetected in all DBBM particles and yielded an identical distribution pattern. Most gold particles were found over the peripheral DBBM matrix, although some peripheral regions lacked immunolabeling. The bulk of the interior DBBM portion was mainly free of labeling with the exception of the peripheral matrix of some osteocyte lacunae and canaliculi. It is concluded that DBBM selectively takes up at least BSP and OPN after its implantation at a bone site. BSP and OPN or other molecules accommodating in DBBM may modulate events associated with cell attachment and differentiation.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Systemic response of the GH/IGF-I axis in timely versus delayed fracture healing

The GH-IGF axis has profound effects on the local and systemic regulation of bone metabolism and may be important for quality of fracture healing. To test the hypothesis that deficiency of the GH/IGF axis may play a role in the pathogenesis of fracture non-union we investigated whether alterations of serum concentrations of the GH-IGF axis could be related to failed fracture healing compared to timely fracture healing in trauma patients. Serum probes were prospectively collected from 186 patients with surgical treatment of long bone fractures up to 6 months after surgery. Samples from 14 patients with atrophic type of non-union have been compared to 14 matched patients with normal bone healing. Postoperative time courses of serum concentrations have been analyzed using commercially available chemiluminescence sandwich assays (GH), fully automated assay systems (IGF-I, IGFBP-3) or sandwich immunometric assays (ALS). Comparison between both collectives revealed significantly lower serum concentrations of GH dependent ALS during early (1st week after surgery) and of both IGFBP-3 and ALS during late stages of fracture healing (6 and 8 weeks after surgery) in non-union patients, coinciding clinically with failed fracture healing. Tendentially lower serum levels of IGF-I in the non-union group over the entire investigation period were statistically not significant. We have been able to show time courses of serum concentrations of the GH/IGF-I axis during normal and failed fracture healing in humans. An impairment of the GH/IGF-I axis might be involved in the biochemical mechanisms determining delayed or failed fracture healing.

Targeted cell replacement with bone marrow cells for airway epithelial regeneration

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.

Bone-marrow derived progenitor cells are associated with psychosocial determinants of health after controlling for classical biological and behavioral cardiovascular risk factors

BACKGROUND: Circulating progenitor cells have been implicated with maintaining vascular integrity. Low counts are found in adults with high cardiovascular risk and are associated with impaired endothelial function. It remains unknown whether psychosocial risk factors are independently related to counts of circulating progenitor cells. METHODS: We investigated a random sample of 468 adult industrial employees (mean age 41.2 years, 89% men) of Caucasian origin. Cardiovascular risk factors (blood pressure, LDL, HDL and C-reactive protein), health behavior (smoking, alcohol and physical exercise), psychological variables (effort-reward imbalance social support, negative affectivity) and interaction terms served as predictors of circulating progenitor cells (CD34+ CD31dim) as enumerated by flow-cytometry. FINDINGS: Psychosocial variables were independently associated with progenitor cell counts. The association with risk factors increased with age (explained variance in 18-36 year olds R(2)=0.17, p=0.55; age 36.1-46 R(2)=0.32, p=0.001; age>46 R(2)=0.27, p<0.001). Data revealed a shift from a larger association between behavioral and psychosocial variables and cell counts to a stronger association between biological variables and cell counts in older individuals. A significant interaction was observed between smoking and effort-reward imbalance in middle-aged subjects, those with both risk factors present had lower cell counts. In older employees, the interaction between biological risk factors and smoking was related to lower cell counts. INTERPRETATION: In working middle-aged and older men, psychosocial risk factors were related to circulating counts of progenitor cells. Smoking interacted negatively with psychosocial risk factors (middle-aged men) or with biological risk factors (older employees).

IGF-I treatment in adults with type 1 diabetes: effects on glucose and protein metabolism in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp

Type 1 diabetes is associated with abnormalities of the growth hormone (GH)-IGF-I axis. Such abnormalities include decreased circulating levels of IGF-I. We studied the effects of IGF-I therapy (40 microg x kg(-1) x day(-1)) on protein and glucose metabolism in adults with type 1 diabetes in a randomized placebo-controlled trial. A total of 12 subjects participated, and each subject was studied at baseline and after 7 days of treatment, both in the fasting state and during a hyperinsulinemic-euglycemic amino acid clamp. Protein and glucose metabolism were assessed using infusions of [1-13C]leucine and [6-6-2H2]glucose. IGF-I administration resulted in a 51% rise in circulating IGF-I levels (P < 0.005) and a 56% decrease in the mean overnight GH concentration (P < 0.05). After IGF-I treatment, a decrease in the overnight insulin requirement (0.26+/-0.07 vs. 0.17+/-0.06 U/kg, P < 0.05) and an increase in the glucose infusion requirement were observed during the hyperinsulinemic clamp (approximately 67%, P < 0.05). Basal glucose kinetics were unchanged, but an increase in insulin-stimulated peripheral glucose disposal was observed after IGF-I therapy (37+/-6 vs. 52+/-10 micromol x kg(-1) x min(-1), P < 0.05). IGF-I administration increased the basal metabolic clearance rate for leucine (approximately 28%, P < 0.05) and resulted in a net increase in leucine balance, both in the basal state and during the hyperinsulinemic amino acid clamp (-0.17+/-0.03 vs. -0.10+/-0.02, P < 0.01, and 0.25+/-0.08 vs. 0.40+/-0.06, P < 0.05, respectively). No changes in these variables were recorded in the subjects after administration of placebo. These findings demonstrated that IGF-I replacement resulted in significant alterations in glucose and protein metabolism in the basal and insulin-stimulated states. These effects were associated with increased insulin sensitivity, and they underline the major role of IGF-I in protein and glucose metabolism in type 1 diabetes.

Diagnostic approach to hypercalcemia: relevance of parathyroid hormone and parathyroid hormone-related protein measurements

Background: Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTH-rP) are two potent hypercalcemic hormones that act on the same targets. Autonomous secretion of the former is involved in primary hyperparathyroidism (PHPT), whereas the latter is responsible for humoral hypercalcemia of malignancy (HHM). Methods: From 250 consecutive, hypercalcemic serum samples sent to our laboratory for assessment of intact PTH, we were able to obtain clinical information, as well as an additional plasma sample for PTH-rP measurement, in 134 patients. At the time of sampling, patients could be classified into seven groups: cancer without known bone metastases (CaNoMeta, n=36), cancer with bone metastases (CaMeta, n=9), no evidence of cancer (noEvCa, n=71), sarcoidosis (Sarc, n=3), end-stage renal disease (ESRD, n=12), vitamin D overdose (VIT-D, n=2), and hyperthyroidism (Thyr, n=1). Results: In the CaNoMeta group, 29/36 patients had elevated PTH-rP levels, 9/36 patients had inappropriately elevated PTH levels, and 5/36 had elevated levels of both hormones. In the CaMeta group, three of the nine patients had inappropriately elevated PTH levels, two of them with concomitantly elevated PTH-rP levels. In the NoEvCa group, 63/71 patients had an inappropriate elevation of PTH levels and were diagnosed as having PHPT. Four of the 71 patients had elevated levels of both PTH and PTH-rP; three of them were in poor health and died within a short period of time. All of the ESRD patients had very high PTH and normal PTH-rP levels, except for one woman with high PTH-rP and undetectable PTH levels; she died from what later turned out to be a recurrent bladder carcinoma. In the Sarc, Vit-D, and Thyr groups, both PTH and PTH-rP levels were normal. Conclusions: (1) Elevated PTH-rP levels are a common finding in cancer patients without bone metastases. Intact PTH, however, should always be measured in hypercalcemic patients with malignancy because concurrent primary hyperparathyroidism is not rare. (2) Primary hyperparathyroidism accounts for hypercalcemia in 90% of patients without evidence of cancer whose PTH-rP levels may also be found to be elevated in a few cases, even some with surgically demonstrated parathyroid adenoma.

PTH-related protein is released into the mother's bloodstream during location: evidence for beneficial effects on maternal calcium-phosphate metabolism

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradiometric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/l), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 +/- 10 vs. 13 +/- 9 micrograms/l; p = 0.018) and so was PTHrP (2.8 +/- 0.35 vs. 0.52 +/- 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 +/- 0.02 mmol/l) to B (1.22 +/- 0.02, mmol/l), whereas total Ca corrected for protein (2.18 +/- 0.02 mmol/l) or uncorrected (2.18 +/- 0.02 mmol/l) significantly rose during lactation (2.31 +/- 0.02 mmol/l, p = 0.003 and 2.37 +/- 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 +/- 0.38 vs. 2.11 +/- 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 +/- 2.3 vs. 24 +/- 1.9 ng/ml and 29.5 +/- 6.0 vs. 21.9 +/- 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 +/- 0.03 vs. 0.86 +/- 0.05 mmol/l GF, p = 0.003 and 1.25 +/- 0.03 vs. 0.96 +/- 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 +/- 7.1 vs. 52.6 +/- 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 +/- 0.003 vs. 0.017 +/- 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.

In vitro evaluation of demineralized freeze-dried bone allograft in combination with enamel matrix derivative

BACKGROUND Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. METHODS DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. RESULTS Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days. CONCLUSION The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein.

Dominant-negative mutations in the homopentameric extracellular matrix glycoprotein cartilage oligomeric matrix protein (COMP) result in inappropriate intracellular retention of misfolded COMP in the rough endoplasmic reticulum of chondrocytes, causing chondrocyte cell death, which leads to two skeletal dysplasias: pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). COMP null mice show no adverse effects on normal bone development and growth, suggesting a possible therapy involving removal of COMP mRNA. The goal of this study was to assess the ability of a hammerhead ribozyme (Ribo56, designed against the D469del mutation) to reduce COMP mRNA expression. In COS7 cells transfected with plasmids that overexpress wild-type or mutant COMP mRNA and Ribo56, the ribozyme reduced overexpressed normal COMP mRNA by 46% and mutant COMP mRNA by 56% in a dose-dependent manner. Surprisingly, the use of recombinant adenoviruses to deliver wild-type or mutant COMP mRNA and Ribo56 simultaneously into COS7 cells proved problematic for the activity of the ribozyme to reduce COMP expression. However, in normal human costochondral cells (hCCCs) infected only with adenoviruses expressing Ribo56, expression of endogenous wild-type COMP mRNA was reduced in a dose-dependent manner by 50%. In chondrocytes that contain heterozygous COMP mutations (D469del, G427E and D511Y) that cause PSACH, Ribo56 was more effective at reducing COMP mRNA (up to 70%). These results indicate that Ribo56 is effective at reducing mutant and wild-type COMP levels in cells and suggests a possible mode of therapy to reduce the mutant protein load.

Expression and regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis

Factors involved in regulating tissue specific gene expression play a major role in cell differentiation. In order to further understand the differentiation events occurring during hematopoiesis, a myeloid specific gene was characterized, the expression pattern during hematopoiesis was analyzed, and the mechanisms governing its regulation were assessed. Previously, our laboratory isolated an anonymous cDNA clone, pD-D1, which displayed preferential expression in myeloid cells. From nucleotide sequencing of overlapping cDNA clones I determined that the D-D1 message encodes a hematopoietic proteoglycan core protein (HpPG). The expression pattern of the gene was assessed by in situ hybridization of bone marrow and peripheral blood samples. The gene was shown to be expressed, at variable levels, in all leukocytes analyzed, including cells from every stage of neutrophil development. In an attempt to ascertain the differentiation time point in which the HpPG gene is initially expressed, more immature populations of leukemic myeloblasts were assessed by northern blot analysis. Though the initial point of expression was not obtained, an up-regulatory event was discovered corresponding to a time point in which granule genesis occurs. This finding is consistent with prior observations of extensive packaging of proteoglycans into the secretory granules of granule producing hematopoietic cells. The HpPG gene was also found to be expressed at low levels in all stages of lymphocyte development analyzed, suggesting that the HpPG gene is initially expressed before the decision for myeloid-lymphoid differentiation. To assess the mechanism for the up-regulatory event, a K562 in vitro megakaryocytic differentiation system was used. Nuclear run-off analyses in this system demonstrated the up-regulation to be under transcriptional control. In addition, the HpPG gene was found to be down regulated during macrophage differentiation of HL60 cells and was also shown to be transcriptionally controlled. These results indicate that there are multiple points of transcriptional regulation of the HpPG gene during differentiation. Furthermore, the factors regulating the gene at these time points are likely to play an important role in the differentiation of granule producing cells and macrophages. ^

Proteomic analysis of porcine bone-conditioned medium.

PURPOSE Autografts are considered to support bone regeneration. Paracrine factors released from cortical bone might contribute to the overall process of graft consolidation. The aim of this study was to characterize the paracrine factors by means of proteomic analysis. MATERIALS AND METHODS Bone-conditioned medium (BCM) was prepared from fresh bone chips of porcine mandibles and subjected to proteomic analysis. Proteins were categorized and clustered using the bioinformatic tools UNIPROT and PANTHER, respectively. RESULTS Proteomic analysis showed that BCM contains more than 150 proteins, of which 43 were categorized into "secreted" and "extracellular matrix." Growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration, eg, pleiotrophin, galectin-1, transforming growth factor beta (TGF-β)-induced gene (TGFBI), lactotransferrin, insulin-like growth factor (IGF)-binding protein 5, latency-associated peptide forming a complex with TGF-β1, and TGF-β2, were discovered. CONCLUSION The present results demonstrate that cortical bone chips release a large spectrum of proteins with the possibility of modulating cellular aspects of bone regeneration. The data provide the basis for future studies to understand how these paracrine factors may contribute to the complex process of graft consolidation.

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.