Substrate modulation of osteoblast adhesion strength, focal adhesion kinase activation, and responsiveness to mechanical stimuli.

Osteoblast interactions with extracellular matrix (ECM) proteins are known to influence many cell functions, which may ultimately affect osseointegration of implants with the host bone tissue. Some adhesion-mediated events include activation of focal adhesion kinase, and subsequent changes in the cytoskeleton and cell morphology, which may lead to changes in adhesion strength and cell responsiveness to mechanical stimuli. In this study we examined focal adhesion kinase activation (FAK), F-actin cytoskeleton reorganization, adhesion strength, and osteoblast responsiveness to fluid shear when adhered to type I collagen (ColI), glass, poly-L-lysine (PLL), fibronectin (FN), vitronectin (VN), and serum (FBS). In general, surfaces that bind cells through integrins (FN, VN, FBS) elicited the highest adhesion strength, FAK activation, and F-actin stress fiber formation after both 15 and 60 minutes of adhesion. In contrast, cells attached through non-integrin mediated means (PLL, glass) showed the lowest FAK activation, adhesion strength, and little F-actin stress fiber formation. When subjected to steady fluid shear using a parallel plate flow chamber, osteoblasts plated on FN released significantly more PGE2 compared to those on glass. In contrast, PGE2 release of osteoblasts attached to FN or glass was not different in the absence of fluid shear, suggesting that differences in binding alone are insufficient to alter PGE2 secretion. The increased adhesion strength as well as PGE2 secretion of osteoblasts adhered via integrins may be due to increased F-actin fiber formation, which leads to increased cell stiffness.

Paracrine activation of extracellular signal-regulated kinase in a simple in vitro model of wounded osteoblasts.

The immediate signal-transduction response of osteoblasts to acute trauma is poorly characterized. We have developed a simple in vitro model for osteoblast trauma to investigate aspects of the molecular mechanisms of wound healing in bone. Herein we report the specific, rapid, and transient phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2 in osteoblasts as a response to disruption ("wounding") of a confluent monolayer. The mitogen-activated protein kinase (MAPK) cascades of p38 and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) were not activated by this perturbation. The response to wounding was equivalent to the activation of ERK by the addition of exogenous growth factors, and the perturbation-dependent phosphorylation of ERK can be suppressed by an inhibitor of heparin-binding growth factors. Conditioned media from wounded monolayers can induce the phosphorylation of ERK in unperturbed monolayers. Using immunohistochemistry, it was demonstrated that the cells with increased levels of phosphorylated ERK were not localized to the wound edges. These results indicate that ERK activation is the result of an autocrine/paracrine response by osteoblasts to trauma. We speculate that osteoblasts respond to trauma with the release of soluble factors as part of an autocrine/paracrine modulation of the wound-healing process in bone.

Degradative pathways in tissues of the temporomandibular joint. Use of in vitro and in vivo models to characterize matrix metalloproteinase and cytokine activity.

Identification of a small animal model that undergoes pathological temporomandibular joint (TMJ) degeneration would represent a significant research tool. To date however, no such model has been described. We therefore have investigated the pathological and immunohistochemical features of the TMJ of a transgenic mouse that over expresses the human form of TNFalpha. The TMJ of this animal appears to undergo changes that resemble arthriditics of temporomandibular dysfunction. Furthermore, the disc and articular cells express MMP9 and IL-1. Future work should validate this animal model as one that would have utility for the study of TMJ disorders. Maintenance of connective tissues in joints such as the TMJ is a normal process that allows for the reconstitution of important anatomic features. This maintenance involves both the removal and re-synthesis of structural proteins such as collagens, elastins and proteoglycans. An imbalance in the pathways for degradation and synthesis can lead to the degeneration of joint tissues. We describe the presence of a matrix metalloproteinase, MMP9 (92-kD gelatinase), in TMJ disc and articular cells that likely function in the degradative process. Additionally, we show that this enzyme is under the control of pro-inflammatory cytokines whereby TGFbeta and IL-1 stimulate and PGE(2) inhibits its activity.

Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.

PURPOSE: This study compared two methods of preparing platelet-rich plasma (PRP) gel and the levels of PDGF and TGFbeta in each preparation. MATERIALS AND METHODS: Platelet-rich plasma gel was prepared by centrifugation and clotted using the ITA gelling agent (Natrex Technologies Inc, Greenville, NC) or by the addition of thrombin and calcium chloride. The levels of platelet-derived growth factor (PDGF) and transforming growth factor beta (TGFbeta) generated by clot formation were assayed by enzyme-linked immunoassay (ELISA). RESULTS: Both methods of preparation yielded PRP gel in less than 30 minutes. However, the ITA preparation did not require thrombin to achieve adequate gel formation. The levels of PDGF and TGFbeta were similar regardless of which method was used for initiation of clot formation. CONCLUSION: Use of ITA for gel preparation is equivalent to using calcium chloride and thrombin, without the need for special equipment and the risk of coagulopathy.

Differential activation by cytokines of mitogen-activated protein kinases in bovine temporomandibular-joint disc cells.

Temporomandibular disorders affect a significant proportion of the population. While their aetiology is not well defined, recent histological studies suggest that the majority are similar to the osteoarthritis seen in other joints. Inflammatory cytokines such as interleukin-1 and tumour necrosis factor-alpha appear to be important in the cascade of events leading to joint destruction in osteoarthritis. Here, cells from the disc of bovine temporomandibular joint were used to examine the response to various cytokines in vitro. Disc cells were stimulated with interleukin-1alpha, tumour necrosis factor-alpha, transforming growth factor-beta, platelet-derived growth factor, and basic fibroblast growth factor. Their effects were monitored by assessing the phosphorylation of selected signal-transduction intermediates using western blot. Mitogen-activated protein kinases (Erk 1, Erk 2) were rapidly phosphorylated by exposure to basic fibroblast growth factor, platelet-derived growth factor, and tumour necrosis factor-alpha, while interleukin-1alpha showed a weak response. Transforming growth factor-beta failed to activate these kinases. Examination of the effect of these cytokines on p38 (an intermediate in the stress-activated protein-kinase pathway) showed an increase in phosphorylated p38 when stimulated with tumour necrosis factor-alpha and interleukin-1alpha. The amounts of phosphorylated signal transducer and activator of transcription-3 did not significantly increase when the cells were exposed to any of the cytokines.

Cellular, biochemical and molecular characterization of the bovine temporomandibular joint disc.

The cellular and collagenous components of the bovine temporomandibular joint (TMJ) disc have been isolated and analysed. In the central regions of the disc, significant amounts of type I, II, IX and XII collagen were found. The identity of these molecules was verified with collagenase digestions, Western blot analysis and Northern blot analysis (for type II collagen). Cells isolated from the TMJ disc synthesized alkaline phosphatase, proteoglycans and collagen in culture; however, the basal rate of synthesis for these molecules was lower than that for isolated osteoblasts, articular and growthplate chondrocytes. The TMJ disc cells proliferated more rapidly in culture than osteoblasts or chondrocytes. Transforming growth factor-beta stimulated proliferation by 250%, whereas prostaglandin E2 had no effect.

The mandibular condylar growth center: separation and characterization of the cellular elements.

The developing mandibular condylar growth center consists of a number of histologically distinct cell types. There is an increase in cell volume that takes place from the condylar surface layer through the center of ossification, resulting in a disorganized, irregular cellular pattern. Consequently, the isolation and separation of the different cells from this tissue is difficult using standard methodologies. Countercurrent centrifugal elutriation, whereby cells are separated on the basis of size, was applied to bovine mandibular condylar growth center cells. The cell volume, alkaline phosphatase content, proteoglycan synthesis, and type X collagen synthesis all showed a positive correlation with increasing cell size. The largest cells had characteristics that are consistent with hypertrophic chondrocytes; the smallest cells, on the other hand, had many fibroblastic characteristics.

Alterations of T helper/inducer and T suppressor/inducer cells in patients with recurrent aphthous ulcers.

The peripheral blood lymphocytes of patients with recurrent aphthous ulcers (RAU) were studied during various stages of their disease by two-color immunofluorescence with the use of monoclonal antibodies directed against human T cells (CD3), T helper cells (CD4), T suppressor cells (CD8), T helper/inducer cells (CDw29), and T suppressor/inducer cells (CD45R). All patients with severe RAU showed increased numbers of T helper/inducer cells (CDw29) and decreased numbers of T suppressor/inducer cells (CD45R). One of six patients with RAU showed a decreased T helper/T suppressor ratio (CD4/CD8). These findings suggest that patients with RAU may possess primary immunologic abnormalities.

Autologous mixed lymphocyte reaction in human neonatal lymphocytes.

Human neonatal mononuclear cells were examined to determine their ability to participate in an autologous mixed lymphocyte reaction (AMLR). Stimulator cells were isolated by plastic adherence and nylon wool adherence. The nylon wool-nonadherent cells were used as responder cells. In 10 of 10 neonatal samples and 6 of 7 adult samples, a significant AMLR was present when plastic-adherent cells were used as stimulators. Neonatal blood showed a mean increase in proliferation of 7.6 (3.6-14.9), while adult cultures showed a mean stimulation index of 11.8 (1.0-39.0). When nylon wool-adherent cells were used as stimulator cells, only 2 of 7 neonatal blood samples and 1 of 5 adult blood samples showed a significant AMLR. When recombinant interleukin 2 (IL-2) was added to AMLR cultures of plastic-adherent cells and nylon wool-nonadherent cells, a mean augmentation of 12.0 was seen in the neonatal AMLR, while the adult cultures were augmented by a mean response of 4.1. Addition of IL-2 to nylon wool-nonadherent cells alone produced a 5.9-fold increase in adult cells, while neonatal cells showed an 85.8-fold mean increase in proliferation. The results suggest that autoreactive T cells are present in neonatal blood and that these cells can be activated by plastic-adherent autologous cells. However, neonatal and adult nylon wool-adherent cells do not consistently activate autoreactive T cells.

Bilateral hyperplasia of the mandibular coronoid processes: a report of two cases.

Two cases of bilateral coronoid hyperplasia of the mandible are presented. Consistent with other reported cases, the patients were men whose onset of symptoms correlated with the onset of puberty. One case was unusual in that bilateral coronoidectomies did not relieve the restriction of mandibular movement. Additional masseter muscle and fascial surgery was required to provide unrestricted mandibular motion.

Alterations in helper-inducer and suppressor-inducer T-cell subsets in human neonatal blood.

The distribution of T-helper cell subsets of human umbilical cord lymphocytes was compared with that of adult peripheral blood lymphocytes. Cytofluorometric analysis revealed a similar Leu 3/Leu 2 (helper/suppressor) ratio in neonates compared with adults. Total T-cell numbers were slightly decreased in neonatal blood. The Leu 3-positive-4B4-positive cell (helper-inducer) subset, however, was markedly reduced in neonatal blood, while the Leu 3-positive-2H4-positive cell (suppressor-inducer) subset was increased compared with that of normal adults. These findings may contribute to the poor help and enhanced suppression observed in vitro with neonatal T cells.

Metal-free and metal-substituted cytochromes c. Use in characterization of the cytochrome c binding site.

The luminescent properties of metal-free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivates bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal-free 'porphyrin' cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70 degrees C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.

Interaction of general anesthetics with phospholipid vesicles and biological membranes.

Low concentrations of general anesthetics, including halothane, ethrane, trilene, diethyl ether and chloroform are observed to shift the phase transitions of phospholipid vesicles to lower temperatures, and from these data partition coefficients for the anesthetic between lipid and water can be calculated. In contrast to the anesthetics, high concentrations of ethanol are required to shift the phase transition of lipids and glycerol causes no effect. Above the phase transition general anesthetics alter nuclear magnetic resonance spectra of phospholipid dispersions and increase the rotational and lateral diffusion rates of fluorescent probes located in the hydrocarbon core of the bilayer, indicating that they induce disorder in the structure. In red blood cell membranes and sarcoplasmic reticulum fragments, the rotational diffusion rate of 1-phenyl-6-phenylhexatriene is increased in the presence of general anesthetics. The 220 MHz nuclear magnetic resonance spectra of sarcoplasmic reticulum reveal some resolved lines from the lecithin fatty acid protons; addition of general anesthetic increases the contribution of these peaks. The data from the NMR and fluorescence techniques lead to the conclusion that general anesthetics increase the pool size of melted lipids in the bimolecular phospholipid layers of biological membranes; this would account for the ability of general anesthetics to increase passive diffusion rates of various substances in membranes.