Synovial bone sialoprotein indicates aseptic failure in total joint arthroplasty.

BACKGROUND: Until today, a reliable diagnostic discrimination between periprosthetic joint infections (PJI) and aseptic failure (AF) after total joint arthroplasty (TJA) remains challenging. Nearly all recent research focused on synovial markers to be elevated in PJI rather than in AF patients. In this study, synovial bone sialoprotein (sBSP) was investigated in PJI and AF arthroplasty patients before revision surgery. METHODS: sBSP and C-reactive protein (CRP) were determined in synovial fluid samples of PJI (n = 13) patients fulfilling the MSIS criteria and AF (n = 25) patients. Beside descriptive analysis and comparison, computed statistics determined the area under the receiver operating characteristics curve (AUC) to evaluate the discrimination ability of the tested synovial markers. RESULTS: In patients with PJI according to the MSIS criteria, mean sBSP was significantly lower: 14.8 ng/ml (95% CI 5.5-24.1) vs. 38.2 ng/ml in the AF group (95% CI 31.1-45.3), p ≤ 0.001. Conversely, mean sCRP was significantly higher in PJI patients: 8.4 µg/ml (95% CI 0-17.2) vs. 1.8 µg/ml in the AF group (95% CI 0.9-2.8), p = 0.032. The AUC of sCRP in PJI patients was 0.71. The AUC of sBSP in AF revision arthroplasty patients was 0.83. The detection of osteolyses was not associated with higher sBSP concentrations. CONCLUSIONS: Considering the MSIS criteria, significantly higher sBSP concentrations were found in synovial fluid samples of AF compared to PJI patients. sCRP showed only fair, sBSP good discrimination potential. If it is not clear whether PJI is present or not, sBSP may be considered as an add-on synovial marker.

Impact of history of periodontitis on gene expression of bone-related factors in young patients.

Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-ß and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Impact of history of periodontitis on gene expression of bone-related factors in young patients

Abstract Although dental implants and bone regenerative procedures are important approaches for the reestablishment of esthetics and function in young patients with a history of generalized aggressive periodontitis (GAP), no predictable outcomes have been reported, and the host osteo-immunoinflammatory response may play a relevant role in this context. In view of the lack of molecular investigations into the bone tissue condition of young patients with periodontitis, the aim of this study was to evaluate the gene expression of bone-related factors in this population. Bone biopsies were obtained from the posterior mandible in 16 individuals previously diagnosed with GAP and on periodontal support therapy and from 17 periodontally healthy (PH) patients. The gene expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, receptor activator of the NF-κB ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OC), bone sialoprotein (BSP), and type I collagen (COL-I), important biomarkers of bone turnover, was evaluated by qRT-PCR. Lower TGF-β and OPG mRNA levels were observed in GAP patients compared to PH individuals (p ≤ 0.05). There were no between-group differences in levels of TNF-α, BSP, RANKL, OC, or COL-I mRNA (p>0.05). In young adults, a history of periodontal disease can negatively modulate the gene expression of important bone-related factors in alveolar bone tissue. These molecular outcomes may contribute to the future development of therapeutic approaches to benefit bone healing in young patients with history of periodontitis via modulation of osteo-immuno-inflammatory biomarkers.

Influence of photobiomodulation therapy on root development of rat molars with open apex and pulp necrosis.

This study aimed to evaluate the role of photobiomodulation (PBM) in apexification and apexogenesis of necrotic rat molars with an open apex. Rat molars were exposed to the oral environment for 3 weeks. Canals were rinsed with 2.5% NaOCl and 17% EDTA, filled with antibiotic paste and sealed. After 7 days, canals were rinsed and divided into six groups (n=6): mineral trioxide aggregate (MTA); blood clot (BC); human dental pulp stem cells (hDPSC); MTA+PBM; BC+PBM; and hDPSC+PBM. In hDPSC groups, a 1% agarose gel scaffold was used. Two groups were not exposed: healthy tooth+PBM (n = 6), healthy tooth (n = 3); and one was exposed throughout the experiment: necrotic tooth (n = 3). In PBM groups, irradiation was performed with aluminum gallium indium phosphide (InGaAlP) diode laser for 30 days within 24-h intervals. After that, the specimens were processed for histological and immunohistochemical analyses. Necrotic tooth showed greater neutrophil infiltrate (p < 0.05). Necrotic tooth, healthy tooth, and healthy tooth+PBM groups showed absence of a thin layer of fibrous condensation in the periapical area. All the other groups stimulated the formation of a thicker layer of fibers (p < 0.05). All groups formed more mineralized tissue than necrotic tooth (p < 0.05). PBM associated with MTA, BC, or hDPSC formed more mineralized tissue (p < 0.05). MTA+PBM induced apexification (p < 0.05). Rabbit polyclonal anti-bone sialoprotein (BSP) antibody confirmed the histological findings of mineralized tissue formation, and hDPSC groups exhibited higher percentage of BSP-positive cells. It can be concluded that PBM improved apexification and favored apexogenesis in necrotic rat molars with an open apex.

Elevated serum levels of bone sialoprotein (BSP) predict long-term mortality in patients with pancreatic adenocarcinoma.

Patients with pancreatic adenocarcinoma (PDAC) still face a very limited prognosis. At early stage, surgical tumor resection might offer long-term survival but disease recurrence is common and the existing stratification algorithms are often unsuitable to identify patients who particularly benefit from surgery. Here, we investigated the potential role of bone sialoprotein (BSP) as a circulating marker in patients undergoing resection of PDAC. We used ELISA to determine serum concentrations of BSP in a cohort of 132 PDAC patients as well as 39 healthy controls. Circulating BSP levels were significantly higher in PDAC patients compared to healthy controls. Notably, elevated preoperative BSP levels above the ideal cut-off value of 4743 pg/ml turned out as a significant predictor for an impaired postoperative survival. The potential of preoperative BSP levels as a prognostic marker was further underlined by uni- and multivariate Cox-regression analyses including various tumour- and patient-specific. Finally, high tumoral BSP expression was also associated with a significantly impaired long-term survival. In conclusion, we identified a novel role of circulating BSP as a biomarker in PDAC patients undergoing tumor resection. Such data might help to establish new preoperative stratification strategies to better identify patients who particularly benefit from tumor resection.

Effectiveness of rhBMP-2 association to autogenous, allogeneic, and heterologous bone grafts.

Proteins with osteoinductive potential, especially recombinant human bone morphogenetic protein (rhBMP)-2, have large effects on cell growth and their differentiation. The aim of this study was to assess repair of bone defects in rat calvaria with different types of grafts associated with rhBMP-2, through immunohistochemistry and micro computed tomography (CT) analyses. A total of 35 male Wistar rats were selected, each weighing ~250 g, with a waiting period of 6 weeks from the creation of the defect to the sacrifice, and divided into five groups (n = 7): autograft plus 5 µg rhBMP-2 (AuG/BMP-2); allograft plus 5 µg rhBMP-2 (AlG/BMP-2); xenograft (heterologous) plus 5 µg rhBMP-2 (XeG/BMP-2); 5 µg rhBMP-2 (BMP-2) and the control group (n = 7). The micro CT reveal that all groups associating different bone grafts with BMP-2 showed increased bone formation compared to the control. The immunostaining show that osteocalcin and bone sialoprotein were higher in groups with BMP-2 than control group; BMP was high expressed in AuG/BMP-2, AlG/BMP-2, and BMP-2; vascular endothelial growth factor (VEGF) was more expressed in groups with BMP-2; VEGF-R2 was low to moderate in AuG/BMP-2, XeG/BMP-2, and BMP-2, predominantly moderate in AlG/BMP-2 and low in the control; CD-31 was predominantly moderate in AuG/BMP-2, AlG/BMP-2, and XeG/BMP-2, low to moderate in BMP-2 and low in the control. The results revealed that rhBMP-2 improved bone repair when administered alone, or when associated with different bone grafts.

Influence of photobiomodulation therapy on root development of rat molars with open apex and pulp necrosis

Abstract This study aimed to evaluate the role of photobiomodulation (PBM) in apexification and apexogenesis of necrotic rat molars with an open apex. Rat molars were exposed to the oral environment for 3 weeks. Canals were rinsed with 2.5% NaOCl and 17% EDTA, filled with antibiotic paste and sealed. After 7 days, canals were rinsed and divided into six groups (n=6): mineral trioxide aggregate (MTA); blood clot (BC); human dental pulp stem cells (hDPSC); MTA+PBM; BC+PBM; and hDPSC+PBM. In hDPSC groups, a 1% agarose gel scaffold was used. Two groups were not exposed: healthy tooth+PBM (n = 6), healthy tooth (n = 3); and one was exposed throughout the experiment: necrotic tooth (n = 3). In PBM groups, irradiation was performed with aluminum gallium indium phosphide (InGaAlP) diode laser for 30 days within 24-h intervals. After that, the specimens were processed for histological and immunohistochemical analyses. Necrotic tooth showed greater neutrophil infiltrate (p < 0.05). Necrotic tooth, healthy tooth, and healthy tooth+PBM groups showed absence of a thin layer of fibrous condensation in the periapical area. All the other groups stimulated the formation of a thicker layer of fibers (p < 0.05). All groups formed more mineralized tissue than necrotic tooth (p < 0.05). PBM associated with MTA, BC, or hDPSC formed more mineralized tissue (p < 0.05). MTA+PBM induced apexification (p < 0.05). Rabbit polyclonal anti-bone sialoprotein (BSP) antibody confirmed the histological findings of mineralized tissue formation, and hDPSC groups exhibited higher percentage of BSP-positive cells. It can be concluded that PBM improved apexification and favored apexogenesis in necrotic rat molars with an open apex.

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies.

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.

[Experimental study of transforming growth factor-ß3 combined with dental pulp stem cells in promoting the implant's osseointegration].

Objective: To investigate the effect of transforming growth factor-ß3 (TGF-ß3) and dental pulp stem cells (DPSC) in promoting the implant's osteointegration. Methods: Thirty-three New Zealand white rabbits were randomly divided into phosphate buffer saline (PBS) group, DPSC group and TGF-ß3 + DPSC group (12 rabbits/group). Two teeth from the rabbits's mandibular incisors or molars were pulled out randomly, then implant were placed in the tooth extraction site immediately. In PBS group, the implant area was filled with Bio-Oss powder 0.30 g mixed by PBS 20 µl only; while the implant area was filled with Bio-oss powder 0.30 g and 1×10(8)/L DPSC 20 µl in DPSC group; in the the TGF-ß3+DPSC group the implant area was filled with Bio-Oss powder 0.30 g mixed with 1×10(8)/L DPSC 20 µl and 80 µg/L TGF-ß3 20 µl. Eighteen New Zealand rabbits were executed in the 4 weeks and 8 weeks respectively. The treated alveolar bone tissue and implant were collected for plastic section. Alizarin red staining (ARS), immunohistochemical detection (IHC) of bone sialoprotein (BSP), osteocalcin (OC) and type Ⅰ collagen (COL-Ⅰ) were performed after 4 weeks and 8 weeks. Combined bone lamelta width (CBLW) and implant bone contact rate (IBCR), trabecular width (TW) and trabecular area percentage (TA) were observed by histomorphometric measurement. Results: ARS staining: 4 weeks after the operation, the TGF-ß3+ DPSC group showed more red calcified nodules than the other two groups; 8 weeks after operation, the red calcified nodule was further increased. 4 weeks after the operation, the expression of BSP, OC and COL-Ⅰ was (0.35± 0.04), (0.36 ± 0.03) and (0.39 ± 0.01) respectively in TGF-ß3+ DPSC group, (0.27 ± 0.02), (0.24 ± 0.01) and (0.28±0.03) respectively in DPSC group, and (0.13±0.03), (0.15±0.02) and (0.16±0.02) respectively in PBS group. Eight weeks after operation, the expression of BSP, OC and COL-Ⅰ was (0.51±0.02), (0.49±0.03) and (0.53±0.02) respectively in TGF-ß3+DPSC group, (0.35±0.02), (0.37±0.01) and (0.38±0.01) respectively in DPSC group, and (0.21±0.03), (0.19±0.01) and (0.22±0.02) respectively in PBS group. After 4 weeks and 8 weeks, the expression of BSP, OC and COL-Ⅰ in TGF-ß3+DPSC group were significantly higher than the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05). Eight weeks after operation, the CBLW, IBCR, TW and TA around implant in TGF-ß3+ DPSC group were significantly higher than that in the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05). Conclusions: The DPSC has the potential osteogenic differentiation ability; TGF-ß3 can accelerate the osteogenic differentiation of DPSC to some extent; TGF-ß3 combined with DPSC can effectively promote the implant's osseointegration.

Cancer Secretome May Influence BSP and DSP Expression in Human Salivary Gland Cells.

One of the biggest challenges in managing head and neck cancers, especially salivary gland cancers, is the identification of secreted biomarkers of the disease that can be evaluated noninvasively. A relevant source of enriched tumor markers could potentially be found in the tumor secretome. Although numerous studies have evaluated secretomes from various cancers, the influence of the cancer secretome derived from salivary gland cancers on the behavior of normal cells has not yet been elucidated. Our data indicate that secretome derived from salivary gland cancer cells can influence the expression of two potential biomarkers of oral cancer-namely, bone sialoprotein (BSP) and dentin sialoprotein (DSP)-in normal salivary gland cells. Using routine immunohistochemistry, immunofluorescence, and immunoblotting techniques, we demonstrate an enrichment of BSP and DSP in human salivary gland (HSG) cancer tissue, unique localizations of BSP and DSP in HSG cancer cells, and enriched expression of BSP and DSP in normal salivary gland cells exposed to a cancer secretome. The secretome domain of the cancer microenvironment could alter signaling cascades responsible for normal cell proliferation, migration, and invasion, thus enhancing cancer cell survival and the potential for cancer progression. The cancer secretome may be critical in maintaining and stimulating "cancer-ness," thus potentially promoting specific hallmarks of metastasis.

DSPP Is Essential for Normal Development of the Dental-Craniofacial Complex.

The craniofacial skeleton is derived from both neural crest cells and mesodermal cells; however, the majority of the bone, cartilage, and connective tissue is derived from the neural crest. Dentin sialophosphoprotein (DSPP) is a precursor protein that is expressed by the connective tissues of the craniofacial skeleton, namely, bone and dentin with high expression levels in the dentin matrix. Gene ablation studies have shown severe dental defects in DSPP-null mutant mice. Therefore, to elucidate the role of DSPP on the developing dental-craniofacial complex, we evaluated phenotypic changes in the structure of intramembranous bone and dentin mineralization using 3 different age groups of DSPP-null and wild-type mice. Results from micro-computed tomographic, radiographic, and optical microscopic analyses showed defective dentin, alveolar and calvarial bones, and sutures during development. The impaired mineralization of the cranial bone correlated well with low expression levels of Runx2, Col1, and OPN identified using calvarial cells from DSPP-null and wild-type mice in an in vitro culture system. However, the upregulation of MMP9, MMP2, FN, and BSP was observed. Interestingly, the null mice also displayed low serum phosphate levels, while calcium levels remained unchanged. Alizarin red and von Kossa staining confirmed the dysfunction in the terminal differentiation of osteoblasts obtained from the developing calvaria of DSPP-null mice. Immunohistochemical analysis of the developing molars showed changes in Runx2, Gli1, Numb, and Notch expression in the dental pulp cells and odontoblasts of DSPP-null mice when compared with wild-type mice. Overall, these observations provide insight into the role of DSPP in the normal development of the calvaria, alveolar bone, and dentin-pulp complex.

Optimizing Collagen Scaffolds for Bone Engineering: Effects of Cross-linking and Mineral Content on Structural Contraction and Osteogenesis.

INTRODUCTION: Osseous defects of the craniofacial skeleton occur frequently in congenital, posttraumatic, and postoncologic deformities. The field of scaffold-based bone engineering emerged to address the limitations of using autologous bone for reconstruction of such circumstances. In this work, the authors evaluate 2 modifications of three-dimensional collagen-glycosaminoglycan scaffolds in an effort to optimize structural integrity and osteogenic induction. METHODS: Human mesenchymal stem cells (hMSCs) were cultured in osteogenic media on nonmineralized collagen-glycosaminoglycan (C-GAG) and nanoparticulate mineralized collagen-glycosaminoglycan (MC-GAG) type I scaffolds, in the absence and presence of cross-linking. At 1, 7, and 14 days, mRNA expression was analyzed using quantitative real-time -reverse-transcriptase polymerase chain reaction for osteocalcin (OCN) and bone sialoprotein (BSP). Structural contraction was measured by the ability of the scaffolds to maintain their original dimensions. Mineralization was detected by microcomputed tomographic (micro-CT) imaging at 8 weeks. Statistical analyses were performed with Student t-test. RESULTS: Nanoparticulate mineralization of collagen-glycosaminoglycan scaffolds increased expression of both OCN and BSP. Cross-linking of both C-GAG and MC-GAG resulted in decreased osteogenic gene expression; however, structural contraction was significantly decreased after cross-linking. Human mesenchymal stem cells-directed mineralization, detected by micro-CT, was increased in nanoparticulate mineralized scaffolds, although the density of mineralization was decreased in the presence of cross-linking. CONCLUSIONS: Optimization of scaffold material is an essential component of moving toward clinically translatable engineered bone. Our current study demonstrates that the combination of nanoparticulate mineralization and chemical cross-linking of C-GAG scaffolds generates a highly osteogenic and structurally stable scaffold.

Matrix metalloproteinase 20-dentin sialophosphoprotein interaction in oral cancer.

Matrix metalloproteinase 20 (MMP-20), widely regarded as tooth specific, participates with MMP-2 in processing dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein. In biochemical system, MMP-2, MMP-3, and MMP-9 bind with high affinity to, and are activated by, specific small integrin-binding ligand N-linked glycoproteins (SIBLINGs): bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively. Subsequent reports documented possible biological relevance of SIBLING-MMP interaction in vivo by showing that SIBLINGs are always coexpressed with their MMP partners. However, the cognate MMPs for 2 other SIBLINGs-DSPP and matrix extracellular phosphogylcoprotein-are yet to be identified. Our goal was to investigate MMP-20 expression and to explore preliminary evidence of its interaction with DSPP in oral squamous cell carcinomas (OSCCs). Immunohistochemistry analysis of sections from 21 cases of archived human OSCC tissues showed immunoreactivity for MMP-20 in 18 (86%) and coexpression with DSPP in all 15 cases (71%) positive for DSPP. Similarly, 28 (93%) of 30 cases of oral epithelial dysplasia were positive for MMP-20. Western blot and quantitative real-time polymerase chain reaction analysis on OSCC cell lines showed upregulation of MMP-20 protein and mRNA, respectively, while immunofluorescence showed coexpression of MMP-20 and DSPP. Colocalization and potential interaction of MMP-20 with dentin sialoprotein was confirmed by coimmunoprecipitation and mass spectrometry analysis of immunoprecipitation product from OSCC cell lysate, and in situ proximity ligation assays. Significantly, results of chromatin immunoprecipation revealed a 9-fold enrichment of DSPP at MMP-20 promoter-proximal elements. Our data provide evidence that MMP-20 has a wider tissue distribution than previously acknowledged. MMP-20-DSPP specific interaction, excluding other MMP-20-SIBLING pairings, identifies MMP-20 as DSPP cognate MMP. Furthermore, the strong DSPP enrichment at the MMP-20 promoter suggests a regulatory role in MMP-20 transcription. These novel findings provide the foundation to explore the mechanisms and significance of DSPP-MMP-20 interaction in oral carcinogenesis.

Effect of Porphyromonas gingivalis lipopolysaccharide on bone marrow mesenchymal stem cell osteogenesis on a titanium nanosurface.

BACKGROUND: Titanium (Ti) dental implants have been widely used for prosthetic reconstruction of dentition. Unfortunately, peri-implantitis can result in failure of dental implant osseointegration. Lipopolysaccharide (LPS) acts as a chronic inflammatory stimulus and maintains peri-implant inflammation, worsening the prognosis for implant osseointegration. The purpose of this study is to determine the effects of 10 M NaOH-modified Ti surface with nanonetwork structure on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in the context of Porphyromonas gingivalis LPS exposure. METHODS: Titanium disks treated with 10 M NaOH solution and control were incubated with BMMSCs and exposed to P. gingivalis LPS (0, 0.1, or 1 µg/mL). The effects of the modified nanonetwork structure on osteogenic differentiation of rat BMMSCs were evaluated in the context of different concentrations of P. gingivalis LPS exposure. RESULTS: Rat BMMSCs on the 10 M NaOH-modified Ti surface with nanonetwork structure had higher levels of osteogenesis-related gene expression and significantly greater cell proliferation, alkaline phosphatase activity, and extracellular matrix deposition and mineralization than cells on the untreated Ti surfaces, in all the groups with different doses of P. gingivalis LPS exposure. CONCLUSION: The 10 M NaOH-modified Ti surface with nanonetwork structure has better endotoxin tolerance under P. gingivalis LPS exposure than the non-modified surface.

Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17). MATERIAL AND METHODS: Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining. RESULTS: Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 . CONCLUSION: Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.

Comparison of mesenchymal stem cells isolated from pulp and periodontal ligament.

BACKGROUND: Cell-based therapy using mesenchymal stem cells (MSCs) seems promising to obtain regeneration of dental tissues. A comparison of tissue sources, including periodontal ligament (PDL) versus pulp (P), could provide critical information to select an appropriate MSC population for designing predictable regenerative therapies. The purpose of this study is to compare the proliferation and stemness and the MSC-specific and mineralized tissue-specific gene expression of P-MSCs and PDL-MSCs. METHODS: MSCs were obtained from PDL and P tissue of premolars (n = 3) extracted for orthodontic reasons. MSC proliferation was evaluated using a real-time cell analyzer for 160 hours. Telomerase activity was evaluated by a telomeric repeat amplification protocol assay based on enzyme-linked immunosorbent assay. Total RNA was isolated from the MSCs on day 3. A polymerase chain reaction (PCR) array was used to compare the expression of MSC-specific genes. The expression of mineralized tissue-associated genes, including Type I collagen (COL I), runt-related transcription factor 2 (RunX2), bone sialoprotein (BSP), and osteocalcin (OCN) messenger RNA (mRNA), was evaluated using quantitative real-time PCR. RESULTS: Higher proliferation potential and telomerase activity were observed in the P-MSCs compared to PDL-MSCs of premolar teeth. Fourteen of 84 genes related to MSCs were expressed differently in the PDL-MSCs versus the P-MSCs. The expressions of bone morphogenetic protein 2 (BMP2) and BMP6; sex-determining region Y-box 9 (SOX9); integrin, alpha 6 (ITGA6); melanoma cell adhesion molecule (MCAM); phosphatidylinositol glycan anchor biosynthesis, class S (PIGS); prominin 1 (PROM1); ribosomal protein L13A (RPL13A); and microphthalmia-associated transcription factor (MITF) were higher in the P-MSCs compared to the PDL-MSCs, and higher expression of matrix metalloproteinase 2 (MMP2), interleukin (IL)-6, insulin (INS), alanyl (membrane) aminopeptidase (ANPEP), and IL-10 were observed in the PDL-MSCs. However, there was no statistically significant difference in the expression of mineralized tissue-associated genes, including BSP and RunX2, between the P-MSCs and the PDL-MSCs. Higher expression of COL I and lower expression of OCN mRNA transcripts were noted in the PDL-MSCs compared to the P-MSCs. CONCLUSIONS: The results of this study suggest that MSCs isolated from P and PDL tissues show different cellular behavior. To increase the predictability of MSC-based regenerative treatment, differences in dental tissue-derived MSCs and favorable aspects of cell sources should be further clarified.

Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic rats

Objective The aim of this paper was to evaluate the repair of onlay autogenous bone grafts covered or not covered by an expanded polytetrafluoroethylene (e-PTFE) membrane using immunohistochemistry in rats with induced estrogen deficiency. Material and Methods Eighty female rats were randomly divided into two groups: ovariectomized (OVX) and with a simulation of the surgical procedure (SHAM). Each of these groups was again divided into groups with either placement of an autogenous bone graft alone (BG) or an autogenous bone graft associated with an e-PTFE membrane (BGM). Animals were euthanized on days 0, 7, 21, 45, and 60. The specimens were subjected to immunohistochemistry for bone sialoprotein (BSP), osteonectin (ONC), and osteocalcin (OCC). Results All groups (OVX+BG, OVX+BMG, SHAM+BG, and SHAM+BMG) showed greater bone formation, observed between 7 and 21 days, when BSP and ONC staining were more intense. At the 45-day, the bone graft showed direct bonding to the recipient bed in all specimens. The ONC and OCC showed more expressed in granulation tissue, in the membrane groups, independently of estrogen deficiency. Conclusions The expression of bone forming markers was not negatively influenced by estrogen deficiency. However, the markers could be influenced by the presence of the e-PTFE membrane. .

Effects of mineral trioxide aggregate mixed with hydration accelerators on osteoblastic differentiation.

INTRODUCTION: Despite good physical and biological properties, mineral trioxide aggregate (MTA) has a long setting time. A hydration accelerator could decrease the setting time of MTA. This study assessed the biocompatibility of MTA mixed with hydration accelerators (calcium chloride and low-dose citric acid) and investigated the effect of these materials on osteoblast differentiation. METHODS: Cell viability was evaluated by the EZ-Cytox assay kit (Daeil Lab Service, Seoul, Korea). The gene expressions of osteocalcin and bone sialoprotein were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. The mineralization behavior was evaluated with alizarin red staining. RESULTS: There was no statistically significant difference in cell viability between experimental groups. The messenger RNA level of osteogenic genes significantly increased in MTA mixed with hydration accelerators compared with the control and MTA mixed with water. MTA mixed with the hydration accelerators resulted in similar mineralization compared with MTA mixed with water. CONCLUSIONS: Hydration accelerators increase the osteogenic effect and show a similar effect on the mineralization of MTA, which may have clinical applications.

Immunohistochemical and histochemical analysis of newly formed tissues in root canal space transplanted with dental pulp stem cells plus platelet-rich plasma.

INTRODUCTION: Tissue regeneration in root canals after pulpectomy can be achieved by transplantation of autologous dental pulp stem cells and/or platelet-rich plasma. However, the identity of the newly formed tissue in the pulp space has been only examined by histologic analysis. This study aimed to apply immunohistochemistry and histochemistry to detect specific markers in the newly generated tissues after root canal regenerative treatment. METHODS: In our previous study, 32 root canals in 4 mature dogs were treated with a pulp regeneration procedure after pulpectomy using either blood clot, transplantation of dental pulp stem cells, platelet-rich plasma, or a combination of cells and plasma. In the present study, the tissues were examined for the expression of periostin to detect periodontal ligament tissue, nestin and dentin sialoprotein for odontoblasts, and bone sialoprotein and osteocalcin for bone tissues. Samples were also stained for tartrate-resistant acid phosphatase (TRAP) as a marker for osteoclastic lineages. RESULTS: Continuous periostin-positive tissue was observed extending from the periodontal ligament into the inner canal surface in which the mineral islands were surrounded by weak periostin staining. There was also positive staining for TRAP, bone sialoprotein, and osteocalcin in the canal space, suggesting the presence of bone tissue. A layer of mineralized tissue along the inner surface of the root canal was negative for TRAP, suggesting the tissue likely to be cementum. In all samples, no nestin-positive reaction was observed, whereas dentin sialoprotein was detected in PDL, dentinal tubules, and intracanal fibrous tissues. There was no difference between any of the 4 groups. CONCLUSIONS: The tissues formed in the dog mature root canals after regenerative endodontic procedures are not pulp tissues but mainly periodontal tissues.

Platelet-rich plasma derived from bone marrow aspirate promotes new cementum formation.

BACKGROUND: This study evaluates the influence of platelet-rich plasma derived from bone marrow aspirate (PRP-BMA) on the healing of periodontal fenestration defects in rats. METHODS: Periodontal fenestration defects were surgically created in the mandibles of 40 rats. The animals were randomly divided into two groups, control and PRP-BMA, in which defects were filled with blood clot or PRP-bma, respectively. Animals were euthanized at either 10 or 30 days post-surgery. Histologic, histometric, and immunohistochemical analyses were performed. Percentage of new bone area (NBA), area of bone trabeculae (ABT), new cementum (NC), and extension of remaining defect were histometrically evaluated. Proliferating cell nuclear antigen (PCNA), bone sialoprotein (BSP), osteocalcin (OCN), and tartrate-resistant acid phosphatase (TRAP) immunohistochemical staining were performed. Immunolabeled cells were quantified. Data were statistically analyzed (analysis of variance; Tukey, P <0.05). RESULTS: At 10 days, control and PRP-BMA groups presented similar amounts of NBA and ABT; NC formation was not observed. At 30 days, control and PRP-BMA groups presented similar amounts of NBA and ABT; the PRP-BMA group showed NC formation with collagen fibers inserted obliquely or perpendicularly to the root surface. NC formation was not observed in any control group specimen. PRP- BMA presented higher numbers of PCNA-positive and BSP-positive cells than control at 10 and 30 days post-surgery. No significant differences in the number of either OCN-positive or TRAP-positive cells were observed between groups at 10 or 30 days. CONCLUSION: PRP-BMA promoted NC formation with a functional periodontal ligament when applied at experimental periodontal fenestration defects.