In vitro and in vivo tests of hydrothermally synthesised hydroxyapatite coating

High pure and crystalline Hydroxyapatite (HA) coatings on titanium alloy were prepared by hydrothermal synthesis (HS) of plasma-sprayed (PS) precursors from brushite powders (HS-HA). In vitro and in vivo tests were done to evaluate its biological property. The HS-HA coating was compared with the current PS-HA coating. Cultures of the primary osteoblasts on these two HA coatings showed similar cell attachment, proliferation and alkaline phosphatase (ALP) expression. The cell morphology on the coatings was demonstrated by scanning electron microscopy (SEM). The cell spread well at 1 day after seeding culture and the extracellular matrix was secreted after 14 days culture. Histomorphometric analysis was conducted on samples implanted in femoral bone of four dogs for 1 and 3 months, and bone-implant contact percentage was evaluated by light microscopy. The calcium and phosphate distribution on the interface of bone-implant was analysed by SEM and electron dispersive X-ray (EDX) analysis. The results show the osteoconduction of HS-HA coated implants.     

Retention of estrogen receptors in vitro requires limited estradiol exposure in vivo

Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated.     

In vitro effects of dentin matrix protein-1 on hydroxyapatite formation provide insights into in vivo functions

Dentin matrix protein-1 (DMP1) is a mineralized tissue matrix protein synthesized by osteoblasts, hypertrophic chondrocytes, and ameloblasts as well as odontoblasts. DMP1 is believed to have multiple in vivo functions, acting both as a signaling molecule and a regulator of biomineralization. Using a cell-free system in vitro, we evaluated the action of DMP1 in the regulation of hydroxylapatite (HA) formation and crystal growth. The non-phosphorylated recombinant protein acted as an HA nucleator, increasing the amount of mineral formed in a gelatin gel HA growth system relative to protein-free controls. The recombinant protein phosphorylated in vitro had no detectable effect on HA formation and growth. In contrast, phosphorylated bovine DMP1 expressed in marrow stromal cells with an adenovirus vector containing 29.7 phosphates/mol was an effective inhibitor of HA formation and growth. The native full-length protein appeared to be absent or present in only small amounts in the extracellular matrix of bones and teeth. However, two highly phosphorylated fragments representing the N- and C-terminal portions of DMP1 have been identified, apparently arising from proteolytic cleavage of four X-Asp bonds. The highly phosphorylated C-terminal 57-kDa fragment (containing 42 phosphates/mol), like the non-phosphorylated DMP1, was an HA nucleator. These data suggest that, in its native form, DMP1 inhibits mineralization, but when cleaved or dephosphorylated, it initiates mineralization. These in vitro data are consistent with the findings in the DMP1 knockout mouse.     

Uncaria tomentosa reduces osteoclastic bone loss in vivo

BackgroundThe genus Uncaria (Rubiaceae) has several biological properties significant to human health. However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain.PurposeThe present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats and on osteoclastogenesis in vitro.MaterialsUTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization (ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro.ResultsThe analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tetracyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat attenuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-specific activity markers, such as cathepsin K and TRAP.ConclusionUTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential natural product for the treatment of certain osteolytic diseases, such as periodontitis.     

Degradation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in vivo and in vitro

1. Phosphoenolpyruvate carboxykinase (GTP) in the cytosol fraction of liver was labelled in young rats by the injection of [(3)H]leucine and then isolated with specific antibody. Antibody-antigen precipitates from ;pulse'-labelled animals and from animals in which the content of radioactive enzyme had been decreased by a period of degradation were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. No radioactive breakdown products were found. 2. (3)H-labelled phosphoenolpyruvate carboxykinase (GTP) was purified from rat liver and used to measure degradation in vitro. There was first a loss of catalytic activity, then a disappearance of immunological activity and finally a loss of solubility before any evidence of proteolytic cleavage. Proteolytic-cleavage fragments, when found, were also insoluble. 3. An analysis of the subcellular location of enzyme inactivation showed that phosphoenolpyruvate carboxykinase (GTP) was stable when incubated with liver cytosol fraction and was inactivated most rapidly by the microsomal fraction. 4. We propose that denaturation of the enzyme is the rate-limiting step in degradation in vivo, and precedes proteolytic cleavage when the enzyme is incubated with liver preparations in vitro.     

Periplaneta Americana extract inhibits osteoclastic differentiation in vitro

ObjectivesPeriplaneta americana extract (PAE) is proven to be promising in treating fever, wound healing, liver fibrosis, and cardiovascular disease. However, the role of PAE in skeletal disorders remains unclear. This study investigated whether PAE regulates osteoclastic differentiation in vitro via the culture using RAW264.7 cells and bone marrow derived macrophages (BMDMs).Materials and MethodsRAW264.7 cells and BMDMs were cultured and induced for osteoclastic differentiation supplementing with different concentrations of PAE (0, 0.1, 1, and 10 mg/mL). Cell counting kit‐8 (CCK‐8) assay was used to detect the cytotoxicity and cell proliferation. TRAP staining, actin ring staining, real‐time quantitative PCR (RT‐qPCR), and bone resorption activity test were performed to detect osteoclastic differentiation. RT‐qPCR and enzyme‐linked immunosorbent assay (ELISA) were conducted to assay the expression and secretion of inflammatory factors. RNA sequencing (RNA‐seq) and western blot analysis were carried out to uncover the underlying mechanism.ResultsCCK‐8 results showed that 10 mg/mL and a lower concentration of PAE did not affect cell proliferation. RT‐qPCR analysis verified that PAE down‐regulated the osteoclastic genes Nfatc1, Ctsk, and Acp5 in macrophages. Moreover, PAE restrained the differentiation, formation, and function of osteoclasts. Besides, RT‐qPCR and ELISA assays showed that PAE decreased inflammatory genes expression and reduced the secretion of inflammatory factors, including IL1β, IL6, and TNFα. Subsequent RNA‐seq analysis identified possible genes and signaling pathways of PAE‐mediated osteoclastogenesis suppression.ConclusionsOur study indicates that PAE has inhibitive effects on osteoclastogenesis and may be a potential therapeutic alternative for bone diseases.

Periplaneta americana extract (PAE), the animal medicine material extracted from the insects Periplaneta americana, is proven to possess a variety of pharmacological functions. However, the role of PAE in skeletal disorders remains unclear. In this study, we found that PAE decreased osteoclast genes expression Nfatc1, Ctsk, and Acp5 in macrophages. Besides, PAE restrained the differentiation, formation, and function of osteoclasts. Moreover, PAE suppressed the LPS‐induced inflammation. Subsequent RNA‐seq analysis identified the signaling pathways of PAE‐mediated osteoclastogenesis suppression. Our study indicated that PAE has inhibitive effects on osteoclastic differentiation and may be a potential therapeutic Chinese medicine for bone diseases.     

Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet factor 4 regulates osteoclastic bone resorption in vitro

Platelet factor 4, which inhibits collagenases derived from both human skin and granulocytic extracts, was found to block reversibly parathyroid hormone-stimulated ⁴⁵Ca release from fetal rat bone in vitro. The inhibitory property was equally effective in both actively resorbing bones and in bones stimulated to initiate resorption.     

Transforming growth factor‐β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.     

In vitro and in vivo bioactivity of CoBlast hydroxyapatite coating and the effect of impaction on its osteoconductivity

The novel non-thermal CoBlast process has been used recently to create a hydroxyapatite coating on metallic substrates with improved biological response compared to an uncoated implant. In this study, we compared the biological effect of coatings deposited by this process and the industrial standard technique - plasma-spray. Physicochemical properties of these two coatings have been found to be significantly different in that CoBlast HA is less rough but more hydrophilic than the plasma-spray HA as evidenced by data obtained from profilometry and goniometry. Mesenchymal stem cell attachment and adhesion are enhanced on CoBlast HA. Analysis by a combination of EDX and ICP suggests that the higher crystallinity retained by the CoBlast HA result in slower coating dissolution. Detailed in vitro evaluation reveals that plasma-spray HA might induce slightly faster cell proliferation and earlier osteogenic differentiation, but CoBlast HA becomes equivalent to it by the late osteogenic stage. PCR array facilitated the identification of differentially regulated genes involved in various functional aspects of in vitro osteogenesis by the CoBlast HA coating. The expression level of the functional protein products of these genes are in agreement with the PCR data. Coating metallic screws with HA significantly improves the in vivo osseointegration. By measuring of removal force using torque measurement instrument and analyzing the patterns found in X-ray images it is demonstrated that the two HA coatings elicit comparable osseointegration. Using simulated impaction model, CoBlast HA is shown to maintain better performance in cell attachment and mineralization than plasma-spray HA, especially following significant impactions. This might indicate a potentially greater osteoconductivity of CoBlast HA coating in shear-stress associated surgical applications. Collectively, it was demonstrated that CoBlast HA is an effective alternative to plasma-spray HA coating and a promising replacement for specialized surgical applications.     

Inhibition of human glioma U251 cells growth in vitro and in vivo by hydroxyapatite nanoparticle-assisted delivery of short hairpin RNAs against SATB1

Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be over-expressed in many human tumors and knockdown of SATB1 can inhibit tumor growth. The present study was designed to determine the role of SATB1 in the growth of human glioma U251 cells using the plasmid-based SATB1 short hairpin RNA (shRNA) delivered by hydroxyapatite nanoparticles in vitro and in vivo. The in vitro growth, invasion and angiogenesis assays of human glioma U251 cells were done. U251 cells tumor blocks were transplanted into the nude mice. CaCl₂-modified hydroxyapatite nanoparticles carrying shRNA-SATB1 plasmids were injected into the tumors. The apoptosis of the tumor U251 cells was examined with TUNEL assay and flow cytometer (FCM). The tumor growth and immunohistochemistry were measured. The expression level of SATB1 mRNA was investigated by RT-PCR. The expression levels of SATB1, Cyclin D1, MMP-2, VEGF, Bax and Caspase-9 protein were determined by western blot analysis. The results showed that hydroxyapatite nanoparticles-delivered shRNA-SATB1 could significantly inhibit the growth, invasion and angiogenesis of U251 cells in vitro and the growth of U251 cells in vivo. FCM results showed that Nano HAP-shRNA-SATB1-induced apoptosis (up to 67.8 %). SATB1 expression was strongly down-regulated in the tumor U251 cells. Cyclin D1, MMP-2 and VEGF were also down-regulated in the tumor tissues that also displayed significant increased in Bax expression and Caspase-9 activity. These results show that Nano HAP-shRNA-SATB1 can inhibit the growth of human glioma U251 cells in vitro and in vivo, and hydroxyapatite nanoparticles can be used for the in vitro and in vivo delivery of plasmid-based shRNAs into U251 cells.     

SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.     

Centaurin-alpha 1 associates in vitro and in vivo with nucleolin

Centaurin-alpha(1) was originally described as a binding partner for phosphoinositides. In spite of the presence of a putative ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain, no ARF-GAP activity has been attributed to centaurin-alpha(1) so far. Thus the function of this protein remains to be determined. In order to better understand its intracellular role, we aimed to identify centaurin-alpha(1) partners. Using affinity chromatography followed by mass spectrometry analysis, we identified several potential centaurin-alpha(1) protein partners. Nucleolin, a nucleolar protein involved in ribosome biosynthesis, was the main centaurin-alpha(1) interacting protein. The interaction between centaurin-alpha(1) and nucleolin was confirmed by Western blot analysis and GST pull down assays. Moreover, we have shown that ectopically expressed centaurin-alpha(1) associates in vivo with endogenous nucleolin in human embryonic kidney 293 cells. In addition, the association between nucleolin and centaurin-alpha(1) was disrupted by RNAse treatment, indicating that RNA integrity was necessary for their binding. This suggested that centaurin-alpha(1) was part of a ribonucleoprotein complex.     

Angiopoietin-1 requires p190 RhoGAP to protect against vascular leakage in vivo

Angiopoietin-1 (Ang-1), a ligand of the endothelium-specific receptor Tie-2, inhibits permeability in the mature vasculature, but the mechanism remains unknown. Here we show that Ang-1 signals Rho family GTPases to organize the cytoskeleton into a junction-fortifying arrangement that enhances the permeability barrier function of the endothelium. Ang-1 phosphorylates Tie-2 and its downstream effector phosphatidylinositol 3-kinase. This induces activation of one endogenous GTPase, Rac1, and inhibition of another, RhoA. Loss of either part of this dual effect abrogates the cytoskeletal and anti-permeability actions of Ang-1, suggesting that coordinated GTPase regulation is necessary for the vessel-sealing effects of Ang-1. p190 RhoGAP, a GTPase regulatory protein, provides this coordinating function as it is phosphorylated by Ang-1 treatment, requires Rac1 activation, and is necessary for RhoA inhibition. Ang-1 prevents the cytoskeletal and pro-permeability effects of endotoxin but requires p190 RhoGAP to do so. Treatment with p190 RhoGAP small interfering RNA completely abolishes the ability of Ang-1 to rescue endotoxemia-induced pulmonary vascular leak and inflammation in mice. We conclude that Ang-1 prevents vascular permeability by regulating the endothelial cytoskeleton through coordinated and opposite effects on the Rho GTPases Rac1 and RhoA. By linking Rac1 activation and RhoA inhibition, p190 RhoGAP is critical to the protective effects of Ang-1 against endotoxin. These results provide mechanistic evidence that targeting the endothelium through Tie-2 may offer specific therapeutic strategies in life-threatening endotoxemic conditions such as sepsis and acute respiratory distress syndrome.     

Effect of aflatoxin B1 in vitro and in vivo]

The direct and transplacental action of aflatoxin B1 was studied on organic cultures of the embryonic pulmonary tissue of mice of the A line, BD-IX rats and golden hamsters (Cricetus auratus W.). Its toxic action on the cultures and the absence of any blastomogenic effect was demonstrated. In experiments on mice the transplacental penetration of aflatoxin B1 led to an increase in the incidence of the breast tumours in the progeny.     

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.