OPN, BSP, and Bone Quality-Structural, Biochemical, and Biomechanical Assessment in OPN-/-, BSP-/-, and DKO Mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis.

Introduction: Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown. Methods: Here, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays. Results and discussion: We identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.

Detection of Genes Encoding Microbial Surface Component Recognizing Adhesive Matrix Molecules in Methicillin-Resistant Staphylococcus aureus Isolated from Pyoderma Patients.

Pyoderma is a common skin infection predominantly caused by Staphylococcus aureus. In addition to methicillin resistance, this pathogen is resistant to many other antibiotics, which ultimately limits the available treatment options. Therefore, the present study aimed to compare the antibiotic-resistance pattern, to detect the mecA gene and the genes encoding microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) in S. aureus isolates. A total of 116 strains were isolated from patients suffering with pyoderma. Disk diffusion assay was opted to perform antimicrobial susceptibility testing of the isolates. Out of the isolates tested, 23-42.2% strains appeared susceptible to benzylpenicillin, cefoxitin, ciprofloxacin and erythromycin. While linezolid was found to be the most effective anti-staphylococcal drug, followed by rifampin, chloramphenicol, clindamycin, gentamicin and ceftaroline. Out of 116 isolates, 73 (62.93%) were methicillin-resistant S. aureus (MRSA). Statistically significant (p ≤ 0.05) differences in antibiotic resistance patterns between MRSA and methicillin-susceptible S. aureus (MSSA) were found. A significant association of resistance to ceftaroline, rifampin, tetracycline, ciprofloxacin, clindamycin, trimethoprim-sulfamethoxazole and chloramphenicol was found in MRSA. However, no significant difference was observed between MRSA and MSSA for resistance against gentamicin, erythromycin or linezolid. All cefoxitin-resistant S. aureus, nonetheless, were positive for the mecA gene. femA was found in all the MRSA isolates. Among other virulence markers, bbp and fnbB were found in all the isolates, while can (98.3%), clfA and fnbA (99.1%) were present predominately in MRSA. Thus, this study offers an understanding of antibiotic resistance MSCRAMMs, mecA, and femA gene patterns in locally isolated strains of S. aureus.

Bone sialoprotein promotes lung cancer osteolytic bone metastasis via MMP14-dependent mechanisms.

Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.

Polymeric and Composite Carriers of Protein and Non-Protein Biomolecules for Application in Bone Tissue Engineering.

Conventional intake of drugs and active substances is most often based on oral intake of an appropriate dose to achieve the desired effect in the affected area or source of pain. In this case, controlling their distribution in the body is difficult, as the substance also reaches other tissues. This phenomenon results in the occurrence of side effects and the need to increase the concentration of the therapeutic substance to ensure it has the desired effect. The scientific field of tissue engineering proposes a solution to this problem, which creates the possibility of designing intelligent systems for delivering active substances precisely to the site of disease conversion. The following review discusses significant current research strategies as well as examples of polymeric and composite carriers for protein and non-protein biomolecules designed for bone tissue regeneration.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Angiogenesis In Vitro and In Ovo.

Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration.

Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP-/-), OPN-/- and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP-/- mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN-/- bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP-/- mice and decreased it in OPN-/-, without affecting structurally WT and DKO bone. Vibrated BSP-/- mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN-/- endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

The Effects of Polyphenol, Tannic Acid, or Tannic Acid in Combination with Pamidronate on Human Osteoblast Cell Line Metabolism.

BACKGROUND: This study investigates the effect of tannic acid (TA) combined with pamidronate (PAM) on a human osteoblast cell line. METHODS: EC50 for TA, PAM, and different combination ratios of TA and PAM (25:75, 50:50, 75:25) were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The combination index value was utilized to analyze the degree of drug interaction, while trypan blue assay was applied to analyze the cells proliferation effect. The mineralization and detection of bone BSP and Osx genes were determined via histochemical staining and PCR test, respectively. RESULTS: The EC50 of osteoblasts treated with TA and a 75:25 ratio of TA and PAM were more potent with lower EC50 at 0.56 µg/mL and 0.48 µg/mL, respectively. The combination of TA and PAM (75:25) was shown to have synergistic interaction. On Day 7, both TA and PAM groups showed significantly increased proliferation compared with control and combination groups. On Day 7, both the TA and combination-treated groups demonstrated a higher production of calcium deposits than the control and PAM-treated groups. Moreover, on Day 7, the combination-treated group showed a significantly higher expression of BSP and Osx genes than both the TA and PAM groups. CONCLUSION: Combination treatment of TA and PAM at 75:25 ameliorated the highest enhancement of osteoblast proliferation and mineralization as well as caused a high expression of BSP and Osx genes.

Extracellular Matrix Components Regulate Bone Sialoprotein Expression in MDA-MB-231 Breast Cancer Cells.

Bone sialoprotein (BSP) has become a target in breast cancer research as it is associated with tumor progression and metastasis. The mechanisms underlying the regulation of BSP expression have been largely elusive. Given that BSP is involved in the homing of cancer cells in bone metastatic niches, we addressed regulatory effects of proteolytic cleavage and extracellular matrix components on BSP expression and distribution in cell culture models. Therefore, MDA-MB-231 human breast cancer cells were kept in 2D and 3D spheroid cultures and exposed to basement membrane extract in the presence or absence of matrix metalloproteinase 9 or the non-polar protease, dispase. Confocal imaging of immunofluorescence samples stained with different antibodies against human BSP demonstrated a strong inducing effect of basement membrane extract on anti-BSP immunofluorescence. Similarly, protease incubation led to acute upregulation of anti-BSP immunofluorescence signals, which was blocked by cycloheximide, suggesting de novo formation of BSP. In summary, our data show that extracellular matrix components play an important function in regulating BSP expression and hint at mechanisms for the formation of bone-associated metastasis in breast cancer that might involve local control of BSP levels by extracellular matrix degradation and release of growth factors.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues ex vivo.

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

OBJECTIVE: To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. MATERIALS AND METHODS: Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. RESULTS: The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. CONCLUSIONS: Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

A repeated triple lysine motif anchors complexes containing bone sialoprotein and the type XI collagen A1 chain involved in bone mineralization.

While details remain unclear, initiation of woven bone mineralization is believed to be mediated by collagen and potentially nucleated by bone sialoprotein (BSP). Interestingly, our recent publication showed that BSP and type XI collagen form complexes in mineralizing osteoblastic cultures. To learn more, we examined the protein composition of extracellular sites of de novo hydroxyapatite deposition which were enriched in BSP and Col11a1 containing an alternatively spliced "6b" exonal sequence. An alternate splice variant "6a" sequence was not similarly co-localized. BSP and Col11a1 co-purify upon ion-exchange chromatography or immunoprecipitation. Binding of the Col11a1 "6b" exonal sequence to bone sialoprotein was demonstrated with overlapping peptides. Peptide 3, containing three unique lysine-triplet sequences, displayed the greatest binding to osteoblastic cultures; peptides containing fewer lysine triplet motifs or derived from the "6a" exon yielded dramatically lower binding. Similar results were obtained with 6-carboxyfluorescein (FAM)-conjugated peptides and western blots containing extracts from osteoblastic cultures. Mass spectroscopic mapping demonstrated that FAM-peptide 3 bound to 90 kDa BSP and its 18 to 60 kDa fragments, as well as to 110 kDa nucleolin. In osteoblastic cultures, FAM-peptide 3 localized to biomineralization foci (site of BSP) and to nucleoli (site of nucleolin). In bone sections, biotin-labeled peptide 3 bound to sites of new bone formation which were co-labeled with anti-BSP antibodies. These results establish the fluorescent peptide 3 conjugate as the first nonantibody-based method to identify BSP on western blots and in/on cells. Further examination of the "6b" splice variant interactions will likely reveal new insights into bone mineralization during development.

The Effect of Transforming Growth Factor Beta 1 on the Mineralization of Human Cementoblasts.

INTRODUCTION: Transforming growth factor beta 1 (TGF-ß1) plays an important role in bone mineralization and has been reported to promote osteoblast proliferation and differentiation. However, there is no report about the effects of TGF-ß1 on human cementoblasts. The purpose of this study was to clarify the effect of TGF-ß1 on the proliferation and differentiation of the human cementoblast cell line (HCEM) in vitro. METHODS: HCEM cells were stimulated with TGF-ß1 at concentrations of 0.05, 0.5, 5, and 10 ng/mL. A proliferation assay was performed from 24-72 hours. The effect of TGF-ß1 on mineralization was analyzed by quantifying the area stained with alizarin red on days 7 and 14. Real-time polymerase chain reaction was used to assess the effect of TGF-ß1 on the mineralization-related genes alkaline phosphatase, bone sialoprotein, and type I collagen on days 3, 7, and 14. RESULTS: TGF-ß1 did not affect cell proliferation. TGF-ß1 together with the mineralization medium (consisting of ascorbic acid, dexamethasone, and ß-glycerophosphate) increased the alizarin red-stained area on days 7 and 14. Real-time polymerase chain reaction revealed that alkaline phosphatase messenger RNA expression was increased in TGF-ß1-stimulated HCEM cells in mineralization medium on days 3 and 7, whereas bone sialoprotein and type I collagen messenger RNA expression was increased on day 7. CONCLUSIONS: Although TGF-ß1 does not affect cell proliferation, it does promote cell differentiation and mineralization of HCEM cells.

Serum levels of bone sialoprotein, osteopontin, and ß2-microglobulin in stage I of multiple myeloma.

CONTEXT: The fluctuations of proteins in multiple myeloma (MM) are well-known markers for checking the status of the patients. AIMS: The objective of this study was to examine three proteins that have an important role in disease progression. SUBJECTS AND METHODS: The study was performed with two groups: 30 MM stage I patients' (14 females/16 males; aged 60.83 ± 12.38 years) as case group and 40 healthy individuals (18 females/22 males; aged 57.65 ± 6.43 years) as control group. Both groups have been matched in gender and age. Bone sialoprotein (BSP), osteopontin (OPN), and ß2-microglobulin (ß2M) were measured with an enzyme-linked immunosorbent assay. RESULTS: Serum BSP levels of MM-I patients was significantly higher than that of healthy controls (29.24 ± 5.57 vs. 20.89 ± 3.67, P = 0.001). OPN levels of MM-I patients were significantly lower than that of healthy individuals (12.03 ± 3.45 vs. 19.35 ± 4.67, P = 0.001). ß2M levels of patients and controls were similar (1.49 ± 0.67 vs. 1.29 ± 0.55, P = 0.193). CONCLUSIONS: The results suggested that myeloma cells may affect the production of BSP and OPN, which possibly contributes to osteoclastic bone resorption in MM-I patients. Their levels may be a useful biomarker for assessing bone destruction in MM-I patients and distinguishing MM-I from healthy individuals.

The relationship between BSP mRNA expression and 25(OH)D/OPG in peripheral blood of newly diagnosed T2DM patients with different bone mass.

INTRODUCTION: The objective of the study was to detect the levels of osteoprotegerin (OPG) and 25-hydroxyvitamin D [25(OH)D], as well as the expression of bone sialoprotein (BSP) mRNA, in the peripheral blood of patients with newly diagnosed type 2 diabetes mellitus (T2DM) under different bone mass conditions, and to explore its role and significance in the development process of T2DM combined with osteoporosis (OP). MATERIAL AND METHODS: A total of 225 patients hospitalised in the Endocrinology Department and General Department from May 2017 to May 2018 were enrolled and categorised into five groups: the pure T2DM group (group A, 45 patients), the bone mass reduction group (group B, 45 patients), the T2DM + bone mass reduction group (group C, 45 patients), the OP group (group D, 45 patients), and the T2DM + OP group (group E, 45 patients); meanwhile, age-matched healthy subjects undergoing physical examination in our hospital were collected as the normal control group (group NC, 45 cases). Logistic regression analysis was used to analyse the influencing factors of bone mass in patients with T2DM. RESULTS: Compared with group B, the expression levels of glycated haemoglobin (HbA1c), 25(OH)D, N-terminal propeptide of type I procollagen (PINP), fasting plasma glucose (FPG), fasting plasma insulin (FINS), high-density lipoprotein cholesterol (HDL-C), and BSP mRNA were significantly increased while OPG and b-collagen degradation products (b-CTX) were significantly decreased in group A. CONCLUSION: The expression of BSP mRNA and the decrease of 25(OH)D and OPG in peripheral blood may participate in the development of diabetes and osteoporosis.

Effect of bone sialoprotein coating on progression of bone formation in a femoral defect model in rats.

PURPOSE: In orthopedic and trauma surgery, calcium phosphate cement (CPC) scaffolds are widely used as substitute for autologous bone grafts. The purpose of this study was to evaluate bone formation in a femoral condyle defect model in rats after scaffold-coating with bioactive bone sialoprotein (BSP). Our hypothesis was that BSP-coating results in additional bone formation. METHODS: In 20 Wistar rats, defects of 3.0 mm diameter were drilled into the lateral femoral condyles of both legs. BSP-coated scaffolds or uncoated control scaffolds were implanted into the defects. After 4 and 8 weeks, five rats of each group were euthanized, respectively. µCT scans and histological analyses were performed. The ratio of bone volume-total volume (BV/TV) was analyzed and histological sections were evaluated. RESULTS: At week four, bone fraction reached 5.2 ± 1.7% in BSP-coated scaffolds and 4.5 ± 3.2% in the control (p = 0.06). While bone fraction of the BSP-group did not change much between week four and eight [week eight: 5.4 ± 3.8% (p = 0.53)], there was a tendency towards an increase in the control [week eight: 7.0 ± 2.2% (p = 0.08)]. No significant difference in bone fraction were observable between BSP-coated and uncoated scaffolds at week eight (p = 0.08). CONCLUSIONS: A significant superiority of BSP-coated scaffolds over uncoated scaffolds could not be proven. However, BSP-coating showed a tendency towards improving bone ingrowth in the scaffolds 4 weeks after implantation. This effect was only short-lived: bone growth in the control scaffolds tended to outpace that of the BSP-group at week eight.

Expression of BSP-GFPtpz Transgene during Osteogenesis and Reparative Dentinogenesis.

Bone sialoprotein (BSP) is a member of the SIBLING family with essential roles in skeletogenesis. In the developing teeth, although the expression and function of BSP in the formation of acellular cementum and periodontal attachment are well documented, there are uncertainties regarding the expression and function of BSP by odontoblasts and dentin. Reporter mice are valuable animal models for biological research, providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. In the present study, we examined the expression of a BSP-GFPtpz reporter mouse line during odontoblast differentiation, reparative dentinogenesis, and bone. In the developing teeth, BSP-GFPtpz was expressed at high levels in cementoblasts but not in odontoblasts or dentin. In bones, the transgene was highly expressed in osteoblasts at an early stage of differentiation. Interestingly, despite its lack of expression in odontoblasts and dental pulp during tooth development, the BSP-GFPtpz transgene was detected during in vitro mineralization of primary pulp cultures and during reparative dentinogenesis following pulp exposures. Importantly, under these experimental contexts, the expression of BSP-GFPtpz was still exclusive to DSPP-Cerulean, an odontoblast-specific reporter gene. This suggests that the combinatorial use of BSP-GFPtpz and DSPP-Cerulean can be a valuable experimental tool to distinguish osteogenic from dentinogenic cells, thereby providing an avenue to investigate mechanisms that distinctly regulate the lineage progression of progenitors into odontoblasts versus osteoblasts.

Bone sialoprotein-αvß3 integrin axis promotes breast cancer metastasis to the bone.

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvß3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvß3 integrin. Interestingly, we found the αvß3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvß3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvß3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvß3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvß3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT-PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvß3 integrin is one of the downstream factors regulated by BSP in the breast cancer-bone metastatic cascade.

Effects of losartan and atorvastatin on the development of early posttraumatic joint stiffness in a rat model.

BACKGROUND: After a trauma, exuberant tissue healing with fibrosis of the joint capsule can lead to posttraumatic joint stiffness (PTJS). Losartan and atorvastatin have both shown their antifibrotic effects in different organ systems. OBJECTIVE: The purpose of this study was the evaluation of the influence of losartan and atorvastatin on the early development of joint contracture. In addition to joint angles, the change in myofibroblast numbers and the distribution of bone sialoprotein (BSP) were assessed. STUDY DESIGN AND METHODS: In this randomized and blinded experimental study with 24 rats, losartan and atorvastatin were compared to a placebo. After an initial joint injury, rat knees were immobilized with a Kirschner wire. Rats received either losartan, atorvastatin or a placebo orally daily. After 14 days, joint angle measurements and histological assessments were performed. RESULTS: Losartan increased the length of the inferior joint capsule. Joint angle and other capsule length measurements did not reveal significant differences between both drugs and the placebo. At cellular level both losartan and atorvastatin reduced the total number of myofibroblasts (losartan: 191±77, atorvastatin: 98±58, placebo: 319±113 per counting field, p<0.01) and the percentage area of myofibroblasts (losartan: 2.8±1.8% [p<0.05], atorvastatin: 2.5±1.7% [p<0.01], vs control [6.4±4%], respectively). BSP was detectable in equivalent amounts in the joint capsules of all groups with only a trend toward a reduction of the BSP-stained area by atorvastatin. CONCLUSION: Both atorvastatin and losartan reduced the number of myofibroblasts in the posterior knee joint capsule of rat knees 2 weeks after trauma and losartan increased the length of the inferior joint capsule. However, these changes at the cellular level did not translate an increase in range of motion of the rats´ knee joints during early contracture development.

Systemic administration of probiotics reduces the severity of apical periodontitis.

AIM: To evaluate the effect of systemic administration of probiotics on the severity of apical periodontitis (AP). METHODOLOGY: Twenty-four male Wistar rats were used. AP was induced in the maxillary left/right first molars. The animals were arranged into groups: Control, Lactobacillus rhamnosus, and Lactobacillus acidophilus. Probiotics were administered orally for gavage (109 colony-forming units diluted in 5 mL of water for 30 days) during the development of AP. After 30 days, cardiac puncture was performed to analyse the complete blood count. Moreover, microbiological analysis of the root canal contents and saliva was performed. Then, the animals were euthanized and the jaw removed for histopathological and IL-10, IL-1ß and IL-6 immunolabeling analyses. After the Shapiro-Wilk test of normality, the Kruskal-Wallis followed by Dunn's test was performed for nonparametric data, and analysis of variance followed by the Tukey test was performed for parametric data (P < 0.05). RESULTS: No significance difference was observed in the blood profiles and in the counts of microorganisms from the saliva samples among the groups (P > 0.05). Total microorganism counts in the root canal, the inflammatory infiltrate and the immunostaining for IL-1ß and IL-6 in AP were significantly lower in the probiotic groups when compared with the control group (P < 0.05). IL-10 was significantly more immunolabled in the probiotic groups than in the control group (P < 0.05). CONCLUSION: Supplementation with probiotics (Lactobacillus rhamnosus and Lactobacillus acidophilus) had a significant effect on the severity of apical periodontitis in rats, demonstrating the anti-inflammatory effect of probiotics on the development of apical periodontitis.