Negative pressure therapy stimulates healing of critical-size calvarial defects in rabbits.

Negative pressure therapy (NPT) is the controlled application of subatmospheric pressure to wounds. It has been shown to stimulate healing across a broad spectrum of soft-tissue wounds, at least in part from the application of mechanical stress on cells and tissues in the wound environment. This study tests the hypothesis that application of NPT to cranial critical-size defects (CSD) in skeletally mature rabbits leads to osseous healing. NPT was delivered 1, 4, 6 or 10 days over CSD-containing calcium phosphate scaffolds placed in contact with intact dura. At 12 weeks after defect creation, NPT groups exhibited significantly greater defect bridging and bone within the scaffolds (P<0.01). Increasing duration of NPT did not result in a greater amount of bone within the scaffolds, but did increase the amount of bone distributed in the upper half of the scaffolds. Appearance of tissue within defects immediately following the removal of NPT at day 6 suggests alternating regions of dural compression and distention indicative of cell stretching. Dura and adjacent tissue were composed of multiple cell layers that extended up into the scaffolds, lining struts and populating pore spaces. An extracellular matrix densely populated with cells and capillaries, as well as larger vessels, infiltrated pores of NPT-treated scaffolds, while scattered spindle-shaped cells and sparse stroma are present within pores of control scaffolds. This rabbit model data suggest that NPT activates within mature dura a natural healing cascade that results in osseous tissue formation without the addition of exogenous factors or progenitor cells.

Angiogenic activity of an enamel matrix derivative (EMD) and EMD-derived proteins: an experimental study in mice.

OBJECTIVES: To determine whether all or only certain proteins in an enamel matrix derivative (EMD) are angiogenic. MATERIALS AND METHODS: The angiogenic effect was analysed using an in vivo angiogenesis assay. Silicon tubes were filled with or without potential and known angiogenic-modulating factors: (i) an EMD parent, (ii) nine pools of EMD proteins, (iii) fibroblast growth factor/vascular endothelial growth factor and (iv) amelogenin. Silicon tubes were implanted subcutaneously in mice. Dextran-fluorescein isothiocyanate (FITC) was injected via the tail vein, mice were euthanized and tubes were retrieved. Neovascularization was determined by measuring the amount of dextran-FITC within the tubes. RESULTS: The greatest angiogenic potential of the EMD parent was at a weight of 125 ng, resulting in a 4.3-fold increase compared with the negative control. Five pools of EMD proteins showed a stronger angiogenic activity than the EMD parent. Pool 5 showed the greatest angiogenic activity, when compared with the negative control (8.1-fold increase) and with 125 ng of the EMD parent (4.2-fold increase). Amelogenin demonstrated a significantly higher angiogenic activity than the negative control (increase up to 4.0-fold) and the EMD parent (increase up to 1.6-fold). CONCLUSIONS: EMD parent, recombinant porcine amelogenin and certain pools of EMD proteins induced significant angiogenesis compared with the controls using a standardized in vivo assay.

In vivo angiogenic activity of enamel matrix derivative.

BACKGROUND: Porcine enamel matrix derivative (EMD) has a clinical use in facilitating periodontal healing by enhancing the regeneration of alveolar bone, cementum, and periodontal ligament. The mechanism of clinical use has not been elucidated, but in vitro studies suggest that EMD may enhance healing, in part, by stimulating angiogenesis. This study analyzes the effect of EMD on the production of blood vessels in the chorioallantoic membrane (CAM) of the developing chicken egg. METHODS: Various amounts of EMD ranging from 15 to 125 ng/5 microl were pipetted onto a 3-mm diameter x 2-mm thick bioabsorbable hemostatic gelatin and placed onto the surface of the CAM. Recombinant human amelogenin and a purified 5-kDa protein fraction derived from EMD were also tested at various amounts ranging from 15 to 62 ng/5 microl. A mixture of fibroblast growth factor and vascular endothelial growth factor served as the positive control. The negative control was 0.9% saline. A histologic examination of the interface of the gelatin and CAM was performed, evaluating for new blood-vessel formation on an ordinal scale of 0 to 3. Non-parametric statistical analyses were applied to compare test groups with the negative controls. RESULTS: CAM treated with EMD displayed moderate vascularity as indicated by a maximal score of 3.0 +/- 0.05 (mean +/- SEM). This compared favorably to the degree of vascularity stimulated by the mixture of fibroblast growth factor and vascular endothelial growth factor, which had a score of 3.0 +/- 0.05. Interestingly, the stimulation of angiogenesis by EMD was significant only at the lowest amounts tested. At the higher amounts, vascularity was reduced and not significantly different from the negative control. Vascularity was also increased by recombinant human amelogenin as indicated by a maximal score of 2.9 +/- 0.14. By contrast, there was only mild vascularity in sections treated with the negative control as indicated by a score of 1.7 +/- 0.4. The vascularity of the 5-kDa EMD-protein fraction was not different from the negative-control group (2.5 +/- 0.5 versus 1.7 +/- 0.4, respectively). CONCLUSIONS: EMD stimulates angiogenesis in the CAM model. As a heterogeneous mixture of extracellular matrix components, EMD may have multiple biologic functions, but it is likely that at least part of the explanation for its observed positive clinical effects may be the stimulation of angiogenesis. The fact that vascularity was also increased by recombinant human amelogenin raises the possibility that this 28.9-kDa protein may be the source of the angiogenic activity because it is the predominant protein of the EMD mixture. These results, taken together with results from previously reported in vitro studies, suggest that EMD may increase angiogenesis directly and/or indirectly at the wound-healing site.

Osteogenesis in an in vitro coculture of human periodontal ligament fibroblasts and human microvascular endothelial cells.

BACKGROUND: Periodontal bone healing is a complex process involving many cells and processes that must function flawlessly for proper healing to occur. The exact progenitor cells that contribute to this process are not fully characterized. Periodontal fibroblasts and pericytes were postulated to be potential osteoprogenitor cells. This study describes a viable coculture model for the in vitro study of osteogenesis. METHODS: Human microvascular endothelial cells (HMVEC) and human periodontal ligament (HPDL) fibroblasts were cocultured in a layered model and monitored for the development of runt-related transcription factor 2 (runx2) and desmin expression by real-time polymerase chain reaction. Conditions shown to be osteogenic (bone morphogenetic protein [BMP]-2 and enamel matrix derivative [EMD]) were compared to a control coculture that was unstimulated. RESULTS: The HMVEC migrated into a layer of collagen containing only HPDL cells as monitored by fluorescent labeling. runx2 and desmin expressions were increased in stimulated cocultures in week 2 compared to controls. At week 3, the unstimulated control cocultures developed the expression of runx2 and desmin, and the cocultures that were stimulated with EMD and BMP-2 achieved significantly higher levels of these factors than any of the other conditions. CONCLUSIONS: Signs of osteogenesis were present in the cocultures in unstimulated and stimulated conditions. However, in the stimulated condition, osteogenic markers were increased at earlier time points. As such, this model may provide a good method for the study of specific cellular processes that may lead to osteogenesis and eventually for understanding the regeneration of periodontal bone in vivo.

Cellular effects of enamel matrix derivative are associated with different molecular weight fractions following separation by size-exclusion chromatography.

BACKGROUND: Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography. METHODS: Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction. RESULTS: EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak I was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD. CONCLUSIONS: The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metalloproteinase-20.

Levels of endothelial nitric oxide synthase and calcitonin gene-related peptide in the Charcot foot: a pilot study.

UNLABELLED: The pathogenesis of Charcot neuroarthropathy is unclear. To investigate the possibility that decreased levels of calcitonin gene-related peptide and endothelial nitric oxide synthase are involved in the process, we studied bone samples from healthy subjects (n = 4), subjects with diabetic neuropathy (n = 4), and subjects with Charcot neuroarthropathy (n = 4). A statistically significant difference was found in endothelial nitric oxide synthase expression between bone specimens in patients with diabetic neuropathy, Charcot neuroarthropathy, and normal bone (P = .008). A trend toward calcitonin gene-related peptide intensification was observed in normal bone as compared to diabetic neuropathy and Charcot neuroarthropathy bone specimens, but it did not reached statistical significance (P = .23). This pilot study suggests that abnormal calcitonin gene-related peptide and endothelial nitric oxide synthase activity may play a role in the development of Charcot neuroarthropathy. LEVEL OF CLINICAL EVIDENCE: 4.

Effects of trabecular calcium phosphate scaffolds on stress signaling in osteoblast precursor cells.

The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an osteoblast precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and beta tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of osteoblast precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that osteoblast precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.

In vitro effects of enamel matrix derivative on microvascular cells.

BACKGROUND: Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment. METHODS: The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using microporous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: EMD at concentrations <50 microg/ml resulted in significant (P <0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P <0.05). All doses of EMD tested (25, 50, and 100 microg/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 microg/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media. CONCLUSIONS: It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.

Human periodontal fibroblast response to enamel matrix derivative, amelogenin, and platelet-derived growth factor-BB.

BACKGROUND: The ideal goal of clinical therapy in periodontal defects is regeneration of all lost structures. For regeneration to occur, cell proliferation, migration, and extracellular matrix synthesis are prerequisites. Attempts at regeneration of periodontal defects by guided tissue regeneration using bone grafts and membranes have not always yielded predictable results. Recently, attempts at engineering the defects using various materials have shown promising results. Two such approaches have been used to regenerate periodontal defects, one using extracellular matrix such as enamel matrix proteins and the other using growth factors. However, to our knowledge, no study has looked at combining these two approaches to achieve potentially even greater regeneration. METHODS: Primary human periodontal ligament (PDL) fibroblasts were explanted, and alkaline phosphatase (ALK PHOS) activity was determined. Phenotypically different cell lines were incubated for 1, 3, 6, and 10 days in 0.2% fetal bovine serum (FBS) media containing different concentrations of either enamel matrix derivative (EMD), amelogenin, platelet-derived growth factor-BB (PDGF-BB), EMD+PDGF-BB, or amelogenin+PDGF-BB. A culture of 0.2% FBS alone served as a negative control, and a culture of 10% FBS served as a positive control. Cell proliferation was measured using a Coulter counter to determine the cell number. The effects on a wound-fill model were evaluated by scraping a 3-mm wide cell-free zone in PDL monolayers across the diameter of the tissue-culture plate and determining PDL cell migration into the cell-free zone using computer assisted histomorphometry. RESULTS: Compared to the control, only EMD+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (-) cell line (P<0.001), and EMD alone, EMD+PDGF-BB, and amelogenin+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (+) cell line (P<0.001) with EMD+PDGF-BB showing a trend for greater proliferation than either PDGF or EMD alone. Individually, EMD and amelogenin had no significant effect on PDL cell proliferation. In the wound-fill experiment, all factors and their combinations except amelogenin significantly enhanced cell migration compared to the control (P<0.05) at the wound edge. In addition, EMD+PDGF-BB had additive effects on the ALK PHOS (-) cell line at the wound edge. At the center of the wound, neither EMD nor amelogenin had a significant wound-fill effect. However, the combination of EMD+PDGF-BB additively increased wound fill for both ALK PHOS (+) and ALK PHOS (-) cells. CONCLUSIONS: The combination of EMD and PDGF-BB produces greater proliferative and wound-fill effects on PDL cells than each by themselves. If these combined effects can be translated clinically, one may see greater regeneration in periodontal defects with this combination. However, amelogenin does not have significant effects on PDL cell proliferation or migration by itself. This may suggest that either another enamel matrix component in EMD may be responsible for some of its clinical effects, or that amelogenin alone may not trigger the regenerative potential of periodontal tissues and that it requires a combined interaction with other enamel matrix components of EMD to direct the regenerative process.

In vitro cytotoxicity of a low-shrinkage polymerizable liquid crystal resin monomer.

The objective of this study was to determine the in vitro cytotoxicity of novel, polymerizable liquid crystal resin monomers when placed in direct contact with dental and nondental cell lines. One common dimethacrylate and three liquid crystal compounds, Bis-glycidyl methacrylate (Bis-GMA), 2-(t-butyl)-1,4-bis-{4-(6-acryloxy-hexane-1-oxy)-benzoyloxy}-benzene (C6), 2-(t-butyl), 1-[6-(3-acryloxy-propionoxy)-hexane-1-oxy-benzoyloxy], 4-[4-(6-acryloxy-hexane-1-oxy)-benzoyloxy]-benxene (by-product), and a 3:2 mixture of C6 and by-product, respectively, were tested for relative cytotoxicity in vitro. Cultured dental and nondental cells were treated for 24 h with test compound dissolved in media over a fourfold range of concentration (10(-4) -10(-7) mol/L). Cytotoxicity was measured using the WST-1 reagent as an indicator of remaining cell numbers based on the reduction of WST-1 substrate by mitochondrial dehydrogenases in viable cells. Bis-GMA ID(50) was found to be consistent with ID(50) values reported in the literature. A small but significant difference in the sensitivity of the dental and nondental cells in regard to their response to this dimethacrylate was noted. The liquid crystal resin monomers were significantly less cytotoxic to all cell lines tested. ID(50) values of >1 x 10(-4) mol/L were registered for the C6 and by-product monomers alone. The 3:2 mixture of C6 and by-product had a slightly higher cytotoxicity (ID(50) = 1 x 10(-4) mol/L); however, this remained significantly less than that of Bis-GMA. The results demonstrate that the newly synthesized low-shrinkage, polymerizable liquid crystal resin monomers demonstrate a minimal cytotoxic effect on both dental and nondental cells. These data suggest that the low-shrinkage liquid crystal resin monomers will not elicit a response by oral tissues (pulp tissue) when used to repair carious lesions in posterior teeth.

In vitro cytotoxicity of a remineralizing resin-based calcium phosphate cement.

UNLABELLED: Recently, a resin-based calcium phosphate cement (RCPC) has been reported as a remineralizing pulp-capping or lining cement. RCPC consists mainly of tetracalcium and dicalcium phosphates, ethoxylated bisphenol A dimethacrylate and pyromellitic glycerol dimethacrylate monomers and photo- and chemical initiators. OBJECTIVES: Here, the cytotoxic effects of RCPC were evaluated. The hypothesis was that RCPC induced only minor cytotoxic response in immortalized murine odontoblast and pulp cells, comparable to that produced by similar dimethacrylates due to unpolymerized dimethacrylate monomer present after curing. METHODS: Cytotoxicity was determined following the changes in cell succinate dehydrogenase activity after 24 h exposure to the cement components and after a 24 h recovery period. A fourfold range of concentrations was tested of the monomers, the eluate of cured RCPC leached in Dulbecco's modified Eagle's medium, and crushed cured cement in dimethyl sulfoxide. RESULTS: The monomers themselves had cytotoxicities similar to those reported for other dimethacrylates, although they are significantly less toxic than Bis-GMA. Differential cell sensitivity was demonstrated, with the pulp cells having greater sensitivity to the unpolymerized monomer than the odontoblast-like cells. The leached components have cytotoxicity similar to that of the free monomers. The crushed material demonstrated no apparent cytotoxicity at the dilutions tested. SIGNIFICANCE: These data demonstrate that RCPC has an in vitro cytotoxicity that is comparable to other materials containing dimethacrylate monomers and suggest that the material may be suitable for use in dental restorations. The data also indicate that the pulp cells appear more sensitive to dimethacrylates than the odontoblasts.

In vivo evaluation of hydroxyapatite coatings of different crystallinities.

PURPOSE: The influence of calcium phosphate (CaP) and hydroxyapatite (HA) crystallinity on bone-implant osseointegration is not well established. In this study, the effect of HA crystallinity and coating method on bone-implant osseointegration was investigated using a rat tibia model. MATERIALS AND METHODS: HA coatings 1 to 5 microm thick were produced using a supersonic particle acceleration (SPA) technology. The HA crystallinities used for this study were weight ratios of 30%, 50%, 70%, and 90%. A total of 128 HA-coated implants were placed into the tibiae of 64 male Sprague-Dawley rats. Bone-implant interfaces were evaluated using histology and push-out strength testing at 3 and 9 weeks after implantation. RESULTS: The 70% crystalline coatings exhibited significantly greater interfacial strength (5 implants/time point/treatment) than the 30%, 50%, and 90% crystalline coatings at 3 and 9 weeks following implantation. The implants with coatings of 70% crystallinity also had the greatest bone contact length. In addition, the HA coatings produced with SPA demonstrated greater interfacial strength and bone contact length than plasma-sprayed HA coatings (except for the HA coating with 30% crystallinity). DISCUSSION: HA coatings of different crystallinities exhibited different dissolution and re-precipitation properties which may enhance early bone formation and bone bonding. CONCLUSIONS: This study suggested that coating crystallinity and coating methods can influence the bone-implant interface.

The effect of sputtered calcium phosphate coatings of different crystallinity on osteoblast differentiation.

BACKGROUND: Coating titanium implants with hydroxyapatite (HA) has been suggested to increase osseointegration by stimulating early osteoblast function. The goal of this study was to determine the extent to which the crystalline content of the HA surface affected osteoblast function in vitro. METHODS: Osteoblasts were isolated from fetal rat calvaria. Titanium coupons were sputter coated and analyzed. Mineralized nodule formation on plastic using von Kossa staining was compared to tetracycline and procion dye labeling. Cell proliferation, adhesion, alkaline phosphatase activity, morphology and spreading, and cytoskeletal arrangement were analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of mRNA for specific proteins. RESULTS: The percent crystallinity of coatings was 0% (HA1), 1.9% +/- 0.4% (HA2), and 66.4% +/- 2.8% (HA3). The nodule formation and cell number were greatest on titanium and HA3 compared to HA1 and HA2 (P < 0.01). At weeks 2 to 4, all samples showed strong alkaline phosphatase, osteocalcin, monocyte-colony stimulating factor (M-CSF), and receptor activator of nuclear factor kappa B ligand (RANKL) expression, but the specific activity of alkaline phosphatase decreased. Cell adherence was greater than 60% of applied cells for all surfaces except HA3. The cells were significantly more elongated on titanium, with no difference on the HA-coated surfaces. Actin filaments were arranged peripherally at 5 hours but arranged parallel to the long axis of the cell at 20 hours. CONCLUSIONS: Procion labeling is a valid method for evaluating mineralized nodule formation on opaque surfaces. There were no major differences in osteoblast function using titanium or high-crystalline coatings, and most functions were decreased on amorphous or low-crystalline coatings.

Tetracycline at subcytotoxic levels inhibits matrix metalloproteinase-2 and -9 but does not remove the smear layer.

BACKGROUND: The antibacterial and anticollagenolytic properties of tetracycline (TCN) are valuable in periodontal therapy, and TCN treatment can remove the smear layer following root instrumentation. However, recent reports pointing to cytotoxic effects of several acids prompted this study to define TCN concentrations that are anticollagenolytic and remove the smear layer, but have low cytotoxicity. METHODS: Human gingival (hGF) and periodontal ligament (hPDL) cells were treated short- (3 minutes) or long-term (24 hours) with TCN to determine concentrations yielding 50% (TD(50)) and 90% (TD(10)) cell survival. Activity assays measured TCN concentrations with half-maximal inhibition (IC(50)) of matrix metalloproteinase- 2 and -9 (MMP-2 and -9). Finally, we analyzed the effects of TCN with high (75 mg/ml) or low (1 mg/ml) cytotoxicity on the smear layer by scanning electron microscopy (SEM). RESULTS: The TD(50) for TCN after short-term treatment was 4 mg/ml for both hGF and hPDL. Ninety percent of the cells survived 0.2 mg/ml. With long-term treatment, the TD(50) for hGF and hPDL was 70 and 30 microg/ml, respectively, and the TD(10) was 20 and 5 microg/ml. HGF and hPDL recovered from the 3-minute treatment with 1 mg/ml, but not from concentrations exceeding 3 and 9 mg/ml, respectively. The IC(50) was 25 microg/ml for both MMP-2 and MMP-9. Whereas 75 mg/ml TCN removed the smear layer, 1 mg/ml TCN had no effects. CONCLUSIONS: Tetracycline has significant cytotoxicity on periodontal cells. Since non-cytotoxic concentrations of TCN inhibited MMP-2 and -9 but had no effects on the smear layer, TCN is not recommended for root surface treatment.

Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP).

Matrix Extracellular Phospho-glycoprotEin (MEPE) and proteases are elevated and PHEX is defective in HYP. PHEX prevents proteolysis of MEPE and release of a protease-resistant MEPE-ASARM peptide, an inhibitor of mineralization (minhibin). Thus, in HYP, mutated PHEX may contribute to increased ASARM peptide release. Moreover, binding of MEPE by PHEX may regulate this process in normal subjects. The nature of the PHEX-MEPE nonproteolytic interaction(s) (direct or indirect) is/are unknown. Our aims were to determine (1) whether PHEX binds specifically to MEPE, (2) whether the binding involves the ASARM motif region, and (3) whether free ASARM peptide affects mineralization in vivo in mice. Protein interactions between MEPE and recombinant soluble PHEX (secPHEX) were measured using surface plasmon resonance (SPR). Briefly, secPHEX, MEPE, and control protein (IgG) were immobilized on a Biacore CM5 sensor chip, and SPR experiments were performed on a Biacore 3000 high-performance research system. Pure secPHEX was then injected at different concentrations, and interactions with immobilized proteins were measured. To determine MEPE sequences interacting with secPHEX, the inhibitory effects of MEPE-ASARM peptides (phosphorylated and nonphosphorylated), control peptides, and MEPE midregion RGD peptides on secPHEX binding to chip-immobilized MEPE were measured. ASARM peptide and etidronate-mediated mineralization inhibition in vivo and in vitro were determined by quenched calcein fluorescence in hind limbs and calvariae in mice and by histological Sanderson stain. A specific, dose-dependent and Zn-dependent protein interaction between secPHEX and immobilized MEPE occurs (EC50 of 553 nM). Synthetic MEPE PO4-ASARM peptide inhibits the PHEX-MEPE interaction (K(D(app)) = 15 uM and B(max/inhib) = 68%). In contrast, control and MEPE-RGD peptides had no effect. Subcutaneous administration of ASARM peptide resulted in marked quenching of fluorescence in calvariae and hind limbs relative to vehicle controls indicating impaired mineralization. Similar results were obtained with etidronate. Sanderson-stained calvariae also indicated a marked increase in unmineralized osteoid with ASARM peptide and etidronate groups. We conclude that PHEX and MEPE form a nonproteolytic protein interaction via the MEPE carboxy-terminal ASARM motif, and the ASARM peptide inhibits mineralization in vivo. The binding of MEPE and ASARM peptide by PHEX may explain why loss of functional osteoblast-expressed PHEX results in defective mineralization in HYP.

Evaluation of titanium plasma-sprayed and plasma-sprayed hydroxyapatite implants in vivo.

In this study, bone interfacial strength and bone contact length at the plasma-sprayed hydroxyapatite (HA) and titanium plasma-sprayed (TPS) implants were evaluated in vivo. Non-coated titanium (Ti) implants were used as controls. Cylindrical coated or non-coated implants (4.0mm diameter by 8mm long) were implanted in the dogs' mandibles. Loading of the implants was performed at 12 weeks after implantation. At 12 weeks after implantation (prior to loading) and 1 year after loading, implants were evaluated for interfacial bone-implant strength and bone-implant contact length. No significant differences in interfacial bone-implant strength for all groups at 12 weeks after implantation and after 1 year loading in normal bone were found. However, bone contact length for HA implants was significantly higher than the TPS and Ti implants for both periods tested (12 weeks after implantation and 1 year after loading). It was concluded that TPS implants exhibited similar pull-out strength compared to the HA implants. In addition, the lower bone contact length on the TPS surface compared to HA surfaces did not affect the interfacial bone-implant strength for both implants.

Platelet-derived growth factor-BB stimulated cell migration mediated through p38 signal transduction pathway in periodontal cells.

BACKGROUND: Intracellular signaling pathways mediate specific responses to growth factors. The manipulation of these pathways ultimately may be used to control the clinical outcomes of periodontal regenerative therapy. The purpose of this study was to examine the role of the p38 signal transduction pathway in the responses of periodontal cells to platelet-derived growth factor-BB (PDGF). METHODS: Primary cultures of human periodontal ligament cells (PDLs) and gingival fibroblasts (GFs) were used for all experiments. Cell numbers, 3H-thymidine incorporation, and Boyden chamber assays were used to characterize the effects of SB 203580 (SB), a specific inhibitor of the p38 signaling pathway, on cell proliferation and migration. An in vitro wound model also was used to assess the effects of SB. For the in vitro wound assay, triplicate wells were incubated for 1, 3, 5, and 7 days using 0.1% fetal bovine serum (FBS), 10% FBS +/- 10 microM SB, or 20 ng/ml PDGF +/- 10 microM SB. Digital histomorphometric analysis assessed cellular fill within the wound area. RESULTS: SB specifically inhibited PDGF-induced migration in the Boyden chamber assays without affecting cell proliferation. The wound model data showed similar levels of wound fill for PDLs and GFs in 10% FBS. Relative to 10% FBS, PDLs stimulated with PDGF showed significantly (P < 0.01, analysis of variance) greater wound fill (74%) than GFs (12%). SB inhibited the PDGF-induced wound fill of PDLs and GFs by 64% and 57%, respectively. This inhibition was significant (P < 0.01, ANOVA) only for PDLs. The addition of SB to 10% FBS did not significantly affect the wound fill response of either cell type compared to 10% FBS alone. CONCLUSIONS: These results demonstrate that periodontal cells possess distinct responses to PDGF that may be altered at the signal transduction level. The manipulation of these responses through the use of inhibitors to specific signaling pathways may enhance our control of periodontal regeneration in the future.

Cytotoxicity of dentin conditioners and primers on human periodontal ligament cells in vitro.

PURPOSE: To examine the cytotoxicity of dentin conditioners and primers on human periodontal ligament (PDL) cells in vitro. MATERIALS AND METHODS: Primary PDL cells were plated in 96 well culture plates and exposed to 100 microL of test solutions. Undiluted Cavity Conditioner (CC), Vitremer Primer (VP), Uni-Etch (UE), All-Bond 2 (AB), Gluma conditioner (GC), and Gluma primer (GP) were examined at full strength and at 1/100 and 1/1000 dilutions in culture medium. Cytotoxicity of the undiluted material was determined immediately following exposure of the cells to the test substance. Cytotoxicity of the diluted materials was determined immediately following a 300-second exposure of the cells to the test solution, as well as 24 hours after removal of the test solution. Cytotoxicity was expressed as lactate dehydrogenase activity retained within the cells following exposure divided by the activity in unexposed control cells. RESULTS: Exposure to each undiluted test substance resulted in severe damage to the cells (78.2-100%). At 1/100 dilution, only exposure to UE resulted in significant cytotoxicity (72.9%) immediately following removal of the solution. But significant cytotoxicity (21-100%) was evident in cells 24 hours after removal of each of the materials. At 1/1000 dilution, exposure to UE (14.8%) and GP (27.2%) resulted in mild cytotoxicity. Twenty-four hours after removal of the solutions, there was a mild but significant cytotoxic effect of each of the test substances (18.5-49.4%).

Bone response to plasma-sprayed hydroxyapatite and radiofrequency-sputtered calcium phosphate implants in vivo.

PURPOSE: The objective of this study was to evaluate the effect of radiofrequency- (RF) sputtered calcium phosphate (CaP) coating of titanium implants on bond strength at the bone-implant interface and percent bone contact length. MATERIALS AND METHODS: Cylindric sputtered CaP-coated and plasma-sprayed hydroxyapatite- (HA) coated implants (4.0 mm diameter and 8 mm length) were implanted in dog mandibles. Half the sputtered CaP-coated implants were heat-treated. RESULTS: Twelve weeks after implant placement, no statistical differences in the mean ultimate interfacial strengths were observed between as-sputtered CaP-coated, sputtered CaP-coated heat-treated, and control plasma-sprayed HA-coated implants. Histomorphometric evaluation indicated that the percent bone contact lengths for the plasma-sprayed HA-coated implants and the as-sputtered CaP-coated implants were similar and significantly greater than that for the sputtered CaP-coated heat-treated implants. Differences in the ultimate interfacial strength and percent bone contact length between different implant sites in the mandible were not observed. DISCUSSION: The results of this study, considered together with the results of previous studies, suggest that once early osseointegration is achieved, biodegradation of the thin CaP coatings is not detrimental to bone-coating-implant fixation, and does not compromise bone responses to the coated implant surfaces. CONCLUSION: The interfacial strength and histomorphometric data suggest that the CaP coatings applied using the sputtering process produce bone responses similar to those of HA coatings applied using plasma spraying.