Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative.

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.

Enamel matrix derivative promotes reparative processes in the dental pulp.

During odontogenesis, amelogenins from the preameloblasts are translocated to differentiating odontoblasts in the dental papilla, suggesting that amelogenins may be associated with odontoblast changes during development. In the present study, we have explored the effects of enamel matrix derivative (EMD) on the healing of a pulpal wound. Coronal pulp tissue of permanent maxillary premolars of miniature swine were exposed through buccal class V cavities. The exposed pulp was capped with EMD. The contralateral teeth served as controls and were capped with a calcium hydroxide paste (Dycal). The cavities were sealed with glass-ionomer cement. After 2 and 4 weeks, the histology of the teeth was analyzed. In the EMD-treated teeth, large amounts of newly formed dentin-like hard tissue with associated formative cells outlined the pulpal wound separating the cavity area from the remaining pulp tissue. Inflammatory cells were present in the wound area but not subjacent to the newly formed hard tissue. Morphometric analysis showed that the amount of hard tissue formed in EMD-treated teeth was more than twice that of the calcium-hydroxide-treated control teeth (p < 0.001), suggesting that EMD is capable of promoting reparative processes in the wounded pulp more strongly than is calcium hydroxide.

Emdogain--periodontal regeneration based on biomimicry.

Biomimicry has been introduced as a term for innovations inspired by nature [1]. Such innovations may appear in almost every part of modern society. This review on the effects of enamel matrix proteins on the formation of cementum and the development of emdogain for regeneration of periodontal tissues lost due to periodontitis shows an example of biomimicry in dentistry. Findings from clinical and laboratory investigations are summarized and the biological basis for enamel matrix-induced periodontal regeneration is discussed.

Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins.

There is increasing evidence that cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in the formation of acellular cementum, the key tissue in the development of a functional periodontium. The purpose of the present study was to explore the effect of locally applied enamel matrix and different protein fractions of the matrix on periodontal regeneration in a buccal dehiscence model in monkeys. Buccal, mucoperiosteal flaps were raised from the canine to the 1st molar on each side of the maxilla. The buccal alveolar bone plate, the exposed periodontal ligament and cementum were removed. Various preparations of porcine enamel matrix with or without vehicles were applied before the flaps were repositioned and sutured. After 8 weeks, the healing was evaluated in the light microscope, and morphometric comparisons were made. Application of homogenized enamel matrix or an acidic extract of the matrix containing the hydrophobic, low molecular weight proteins, amelogenins, resulted in an almost complete regeneration of acellular cementum, firmly attached to the dentin and with collagenous fibers extending over to newly formed alveolar bone. After application of fractions obtained by neutral EDTA extraction containing the acidic, high molecular weight proteins of the enamel matrix, very little new cementum was formed and hardly any new bone. The results of the controls in which no test substance was applied before the repositioning of the flap, were very similar to those obtained with the EDTA extracted material. Propylene glycol alginate (PGA), hydroxyethyl cellulose and dextran were tried as vehicles for the enamel matrix preparations. Only PGA in combination with the amelogenin fraction resulted in significant regeneration of the periodontal tissues.

In vitro studies on periodontal ligament cells and enamel matrix derivative.

The recognition that periodontal regeneration can be achieved has resulted in increased efforts focused on understanding the mechanisms and factors required for restoring periodontal tissues so that clinical outcomes of such therapies are more predictable than those currently being used. In vitro models provide an excellent procedure for providing clues as to the mechanisms that may be required for regeneration of tissues. The investigations here were targeted at determining the ability of enamel matrix derivative (EMD) to influence specific properties of periodontal ligament cells in vitro. Properties of cells examined included migration, attachment, proliferation, biosynthetic activity and mineral nodule formation. Immunoassays were done to determine whether or not EMD retained known polypeptide factors. Results demonstrated that EMD under in vitro conditions formed protein aggregates, thereby providing a unique environment for cell-matrix interaction. Under these conditions, EMD: (a) enhanced proliferation of PDL cells, but not of epithelial cells; (b) increased total protein production by PDL cells; (c) promoted mineral nodule formation of PDL cells, as assayed by von Kossa staining; (d) had no significant effect on migration or attachment and spreading of cells within the limits of the assay systems used here. Next, EMD was screened for possible presence of specific molecules including: GM-CSF, calbindin D, EGF, fibronectin, bFGF, gamma-interferon, IL-1 beta, 2, 3, 6; IGF-1,2; NGF, PDGF, TNF, TGF beta. With immunoassays used, none of these molecules were identified in EMD. These in vitro studies support the concept that EMD can act as a positive matrix for cells at a regenerative site.

Formulation of enamel matrix derivative for surface coating. Kinetics and cell colonization.

Enamel Matrix Derivative (EMD) contains a protein complex belonging to the amelogenin family. Enamel matrix as well as EMD have been found to promote periodontal regeneration when applied onto denuded root surfaces in dehiscence models. In the present studies it is shown that propylene glycol alginate (PGA) is a suitable vehicle for EMD for its local application. EMD can be dissolved in PGA at an acidic pH, resulting in a highly viscous solution. At neutral pH and body temperature the viscosity decreases and EMD precipitates. Multilayers of EMD on mineral or protein surfaces have been analysed using ellipsometry, total internal reflection fluorescence (TIRF) and biospecific interaction analysis (BIA). The studies show that EMD adsorbs both to hydroxyapatite and collagen and to denuded dental roots. It forms insoluble spherical complexes, and detectable amounts remain at the site of application on the root surface for two weeks, as shown with radiolabelled protein in rats and pigs. Scanning electron micrograph (SEM) studies on monkey teeth further indicate that EMD in PGA may promote repopulation of fibroblast-like cells during the first weeks after application.

Clinical safety of enamel matrix derivative (EMDOGAIN) in the treatment of periodontal defects.

The aim of the present clinical trial was to test tolerability during 2 treatments with EMDOGAIN in a large number of patients. An open, controlled study design in 10 Swedish specialist clinics was chosen, with a test group of 107 patients treated with EMDOGAIN in connection with periodontal surgery at 2 surgical test sites per patient. The procedures were performed 2 to 6 weeks apart on one-rooted teeth with at least 4 mm deep intraosseous lesions. A control group of 33 patients underwent flap surgery without EMDOGAIN at 1 comparable site. In total, 214 test and 33 control surgeries were performed. Serum samples were obtained from test patients for analysis of total and specific antibody levels. 10 of the patients had samples taken before and after the first surgery, 56 other samples were taken after one treatment with EMDOGAIN, and 63 after 2 treatments. None of the samples, not even from allergy-prone patients after 2 treatments, indicated deviations from established baseline ranges. This indicates that the immunogenic potential of EMDOGAIN is extremely low when applied in conjunction with periodontal surgery. Comparison between the test and control groups demonstrated the same type and frequency of postsurgical experiences, i.e., reactions caused by the surgical procedure itself. Clinical probing and radiographic evaluation was performed at baseline and 8 months postsurgery. About half of the patients (44 test and 21 control) were also evaluated after 3 years. There was a significant difference between the test and control results at 8 months postsurgery, and this difference had increased further at the 3 year follow-up. The 2.5-3 mm increase in attachment and bone level after treatment with EMDOGAIN was of the same magnitude as seen in the studies with split-mouth design aiming for test of effectiveness of EMDOGAIN.

Integrated myogenic and metabolic control of vascular tone in skeletal muscle during autoregulation of blood flow.

The hypothesis that autoregulation of blood flow in cat skeletal muscle is due to metabolic factors related to blood flow that interact with force-sensitive myogenic mechanisms is tested by means of a mathematical model. The vascular bed is assumed to consist of the reactive series-coupled proximal arterial and microvascular sections and the passive large vein section. Myogenic mechanisms are described by a slowly adapting force-receptor that determines the activation level of smooth muscle. The contractile machinery is represented by our recently developed mathematical model of smooth muscle mechanics. The metabolic control is described by the vasodilator theory whereby changes in the interstitial concentration of a dilator substance(s) alter the excitation-contraction coupling by shifting the [Ca2+]-force relationship. Experimental results indicate that the model correctly simulates vascular resistance responses to a variety of pressure stimuli, as well as autoregulation of blood flow and capillary pressure. Our results show that autoregulation of blood flow requires myogenic mechanisms which act synergistically with metabolic factors related to blood flow. It is also shown that the autoregulation of capillary pressure can be attributed to passive pressure-induced changes of postcapillary resistance, and that in autoregulation of blood flow the concentration of the mediating vasodilator metabolite(s) is a controlled variable being kept virtually constant in the range where blood flow is autoregulated.

Analysis of gated membrane currents and mechanisms of firing control in the rapidly adapting lobster stretch receptor neurone.

1. The gated membrane currents (a tetrodotoxin-sensitive Na+ current and a tetraethylammonium- and 4-aminopyridine-sensitive K+ current) of the rapidly adapting stretch receptor neurone of lobster were investigated with respect to their kinetic properties using electrophysiological, pharmacological, and mathematical techniques. 2. The currents were found to be controlled by slow inactivations as well as by fast Hodgkin-Huxley (1952) gating processes. They could be described by kinetic expressions which differed from those inferred for the slowly adapting receptor (Gestrelius & Grampp, 1983a; Gestrelius, Grampp & Sjölin, 1983) only with respect to some of the parameter values. 3. With these expressions, and additional equations for the cell's pump and leak current components (Edman, Gestrelius & Grampp, 1986), a mathematical receptor model was formulated which accurately predicts the impulse activity of the living preparation in different functional circumstances and which, therefore, was adopted as an appropriate theory of firing regulation. 4. From a model analysis it appeared (a) that the 'rapid' adaptation of the receptor's impulse activity is mainly an effect of slow Na+ current inactivation starting a regenerative process of accommodation which, basically, is due to a small ratio of subthreshold Na+ to K+ currents; (b) that, because of the transmembrane Na+ influx being limited by accommodation, impulse firing is only little affected by a Na+-dependent pump current activation; and (c) that the phenomenon of increased firing frequency initially during prolonged stimulation ('negative adaptation') is an effect of the slow K+ current inactivation being faster than the slow Na+ current inactivation at comparable degrees of membrane polarization. 5. From further model studies it also appeared that, during depolarizations between successive action potentials evoked by constant stimulation, the membrane behaves like a high-resistance constant-current generator feeding into a short-circuiting capacitor. In consequence, the cell's stimulus sensitivity (change in firing frequency with stimulation strength) is, at functionally relevant stimulation intensities, mainly determined by the membrane capacitance and by the amplitude of the interspike membrane depolarization while, at higher stimulation intensities and firing frequencies, it becomes more and more a function of the spike duration itself.

Current activation by membrane hyperpolarization in the slowly adapting lobster stretch receptor neurone.

1. A polarization-induced membrane current, IQ, was investigated in the slowly adapting lobster stretch receptor neurone using conventional electrophysiological techniques including intracellular ion measurements. 2. The current was readily blocked by Cs+ in a voltage-dependent manner, but proved to be unaffected by tetrodotoxin, tetraethylammonium and 4-aminopyridine. 3. From an analysis of the ionic basis of IQ, it appeared that the current is carried by both Na+ and K+ through a membrane channel whose permeability for K+ is about six times larger than that for Na+ in a normal ionic environment. In the presence of reduced external Na+ concentration the Q-channel increases its permeability for both Na+ and K+, but more so for Na+ than for K+. 4. Kinetically, IQ was found to be characterized by a steep sigmoidal relationship between membrane voltage and steady-state current activation, and by a bell-shaped relationship between membrane voltage and the time constant of the exponential phase of current activation or deactivation. A significant feature of the latter relationship is a tendency to level off at finite time-constant values in both strongly hyperpolarizing and strongly depolarizing voltage regions. 5. From the experiments a mathematical IQ model was inferred. This model was based on constant-field and channel-gating kinetics involving a voltage-dependent reaction step in series with a voltage-independent reaction step. The model was found to successfully reproduce IQ behaviour in the living preparation. 6. Functionally, the activation of IQ was found to play a role in setting the cell's resting polarization and membrane excitability. This function was inferred from experiments on unimpaled cells in which it was possible to demonstrate some overlap between the voltage ranges of IQ activation and impulse initiation. In addition, in impaled cells the activation of IQ was found to cause some shortening of post-tetanic membrane hyperpolarization and to accelerate, thereby, the post-tetanic restoration of membrane excitability to control levels.

Transmembrane ion balance in slowly and rapidly adapting lobster stretch receptor neurones.

The transmembrane exchange of Na+, K+, and Cl- in slowly and rapidly adapting lobster stretch receptor neurones was studied using ion-sensitive microelectrodes in combination with conventional electrophysiological techniques. The investigation was founded on the assumption that the transmembrane ion exchange is accomplished by active and passive transports which add up to zero in steady state for each ion involved. The active transports are assumed to include Na+ and K+ transports driven by an electrogenic Na-K pump. To these transports are also added equimolar fluxes of K+ and Cl- leaking from the impaling micro-electrode. The passive transports are assumed to pass through membrane channels in accordance with constant field kinetics. For a quantitative evaluation of the transmembrane ion exchange in resting conditions measurements were made of the resting concentrations of Na+, K+ and Cl-; the voltage dependence of the ungated leak current; and ouabain-induced changes in resting membrane current and intracellular ion concentrations. From the results it follows that both the resting pump current and the leak permeabilities for the ions investigated have values which do not seem to differ between slowly and rapidly adapting receptor neurones. For a quantitative evaluation of the relation between internal Na+ and pump current production, measurements were made of the outward membrane current as a function of internal Na+ and K+ following a shift of these ions by means of prolonged repetitive impulse activation. It was found that the investigated relation is compatible with Garay-Garrahan kinetics (Garay & Garrahan, 1973) in both receptor neurones, but the results imply a larger maximum Na+-extrusion capacity in slowly than in rapidly adapting cells. From recordings of the time course of post-tetanic normalization of both the membrane current and intracellular Na+ concentration, cell volume values could be deduced which were closely similar in slowly and rapidly adapting receptors. A corresponding similarity was also found for the cell area which was derived from membrane capacitance measurements.

A dynamic model of smooth muscle contraction.

A dynamic model of smooth muscle contraction is presented and is compared with the mechanical properties of vascular smooth muscle in the rat portal vein. The model is based on the sliding filament theory and the assumption that force is produced by cross-bridges extending from the myosin to the actin filaments. Thus, the fundamental aspects of the model are also potentially applicable to skeletal muscle. The main concept of the model is that the transfer of energy via the cross-bridges can be described as a 'friction clutch' mechanism. It is shown that a mathematical formulation of this concept gives rise to a model that agrees well with experimental observations on smooth muscle mechanics under isotonic as well as isometric conditions. It is noted that the model, without any ad hoc assumptions, displays a nonhyperbolic force-velocity relationship in its high-force portion and that it is able to maintain isometric force in conditions of reduced maximum contraction velocity. Both these findings are consistent with new experimental observations on smooth muscle mechanics cannot be accounted for by the classical Hill model.

Impulse firing in the slowly adapting stretch receptor neurone of lobster and its numerical simulation.

A mathematical model of the electrical activity of the slowly adapting lobster stretch receptor neurone is presented. The model is based on constant field and state transition theory and employs measurements of the kinetics of membrane currents in sub- and near-threshold voltage regions (Gestrelius, Grammp & Sjölin 1981, Gestrelius & Grampp 1983, Gestrelius, Grampp & Sjölin 1983, Edman, Gestrelius & Grampp 1983). In addition to the classical action potential generating mechanisms (Hodgkin & Huxley 1952) the model also includes the processes of slow Na and K inactivation, ion flux dependent changes of the intracellular Na+ and K+ concentrations, and the activity of an electrogenic Na-K pump sensitive to intracellular Na+ accumulation. The model is able to correctly simulate recorded action potentials as well as repetitive firing both with respect to stimulus dependence (sensitivity) and time dependence (adaptation) during prolonged electrical stimulation. In the living cell firing adaptation is found to consist of an initial phase with a relatively high, and a later phase with a lower rate of adaptation. From the model properties it can be concluded that the initial phase is mainly caused by the slow Na inactivation, while the later phase is due to a slow Na+ influx dependent pump current activation.

Intracellular ion control in lobster stretch receptor neurone.

The control of intracellular ion concentrations by means of passive and active transmembrane ion transports was investigated in the lobster stretch neurone using electrophysiological and pharmacological techniques in combination with recording with ion-sensitive microelectrodes. In resting conditions [Na+]i, [K+]i, and [Cl-]i were, in both slowly and rapidly adapting cells, found to be in the order of 20, 155, and 50 mM, respectively. In the slowly adapting cell impulse firing at stationary frequencies of 7-10 Hz caused an increase in [Na+]i and a decrease in [K+]i of 20-30 mM; [Cl-]i was only little affected, the rise in [Na+]i led to an enhanced Na-K pump activity noticeable as an increase in pump current production. In stationary conditions the quotient between pump current and Na+ influx increments was about 0.3, which is compatible with 3:2 Na-K pumping ratio in the present preparation. From measurements of the pump current activation during stationary firing at maximum tolerable frequencies an estimate was made of the cell's maximum pump current production. The measurements were used in the formulation of a mathematical model of the intracellular ion control in which expressions of active and passive transmembrane ion transports are incorporated into the continuity equation for the ion fluxes involved.

Kinetics of the TEA and 4-AP sensitive K+ current in the slowly adapting lobster stretch receptor neurone.

The kinetics of the TEA and 4-AP sensitive K+ current (IK) in the slowly adapting lobster stretch receptor neurone were investigated in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. In dynamic conditions IK was found to display both fast and slow reactions. These were attributed to a Hodgkin-Huxley type of K activation, and a slow type of K inactivation, respectively. The slow K inactivation could be shown to be unrelated to K+ flux dependent changes in intra- and pericellular K+ concentrations. Its stationary voltage dependence was however shifted in a depolarizing direction by increasing, and in hyperpolarizing direction by decreasing the extracellular Ca++ concentration. In view of these findings, and of its kinetic properties, the slow K inactivation was classified as a genuine channel gating process. The process of K activation was too fast for a dynamic analysis with the recording technique available. An estimate of its stationary voltage dependence could however be obtained in a voltage range from about -100 to about -40 mV. The experimental observations were utilized in the formulation of a mathematical model describing the kinetic behaviour of IK in the present preparation based on constant field and state transition theories.

Kinetics of the TTX sensitive Na+ current in the slowly adapting lobster stretch receptor neurone.

The kinetics of the TTX sensitive Na+ current (INa) in the slowly adapting lobster stretch receptor neurone were investigated in sub- and near-threshold voltage regions using electrophysiological and pharmacological techniques. In dynamic conditions INa was found to display both fast and slow reactions. These were attributed to a fast Hodgkin-Huxley type of Na activation and inactivation, and a slow type of Na inactivation, respectively. In stationary conditions the voltage dependence of the slow Na inactivation was shifted in a depolarizing direction by increasing, and in a hyperpolarizing direction by decreasing the extracellular Ca++ concentration. From this finding as well as from its kinetic properties the slow Na inactivation was classified as a genuine gating process. The processes of fast Na activation and inactivation were too fast for a dynamic analysis with the recording technique available. An estimate of their stationary voltage dependence could however be obtained in a voltage range from about -80 to about -50 mV. The experimental findings were used for the formulation of a mathematical description of INa in the present preparation based on constant field and state transition theories.