Biomolecular phases in transverse palatal distraction: A review

Transverse palatal distraction is a biological process of regenerating new bone and enveloping soft tissues in the maxillary palate region. This technique is similar to Osteo-distraction (OD) procedure for bone lengthening in which gradual and controlled traction forces are applied on the osteotomy gaps to produce new bone in between the surgically separated bone segments. This review describes the different phases after osteotomy and the biological process involved during the new bone and soft tissue formation. The mechanical environment formed in the distraction area is due to the traction forces by the distractor appliance. This environment stimulates differentiation of pluripotent cells, neovascularization, osteogenesis and remodeling of newly formed bone. The role of different pro-inflammatory cytokines, interleukins, bone morphogenic proteins, transforming growth factors, fibroblast growth factors-2) and extracellular matrix proteins (osteonectin, osteopontin) during the distraction phases has been described in detail. Also, an important note on the nutritional aspect during Osteo-distraction will benefit the clinicians to guide their patients after osteotomy throughout the distraction process.     

CT characteristics of solitary long bone cysts before and after treatment

Computed tomography was used to study the X-ray morphological characteristics of femoral and humoral bone cysts and the course of a reparative process after surgery in 23 patients with varying activity of solitary bone cysts. Treatment using transosseous osteosynthesis, intraosseous armoring, and therapeutic and diagnostic punctures in patients with solitary bone cysts could yield the most active osteoproductive effect and cause a considerable increase in the density of cyst contents in active and activity-losing stages, which was most evident around the intraosseous wires. Patients with late-stage cysts were found to have an osteogenic effect, mainly in an area of osteotomy.     

A Vital Microscopic Description of the Effects of Electrical Stimulation of Bone Tissue

An implantable, titanium, optical chamber that allows vital microscopic observations of bone tissue during continuous electrical stimulation is described. Tissue reactions to direct current stimulation of a defined bone tissue compartment may be repeatedly observed and recorded on film for indefinite follow-up periods. In this study 5, 20 or 50 μA direct current were applied to chambers inserted into the rabbit tibia. The bone tissue was found to increase in volume after stimulation with 5 and 20 μA, while 50 μA caused bone resorption. Fat-cell concentration was independent of the current levels as used in the present study. Vital microscopy revealed no signs of acute microvascular alterations in the hours following onset of the current. Over an eleven-week follow-up, vessels close to bone borders were found to increase in size from 15–25 microns to 30–50 microns, independent of the level of stimulation. The amount of vessels increased after stimulation with 5 and 20 μA, while in the 50 μA group the vessels decreased to 50% or less of the vascular density observed before start of the stimulation.     

BREAKDOWN OF CREATINE PHOSPHATE AND ATP IN NERVE TERMINALS AND ELECTROPLAQUES OF THE TORPEDO ELECTRIC ORGAN: COMPARISON WITH THE ELECTRICAL ENERGY DISSIPATED

Abstract— The electrical work performed by the electric organ of Torpedo was compared with the energy provided by the net breakdown of ATP and creatine phosphate (CrP). The electrical work was calculated for single impulses and for repetitive stimulations. The content in CrP and ATP was measured at different times in the course of stimulation and during the period of recovery. The chemical expenditure due to activity of the nerve terminals was distinguished from the total expenditure by the use of curare which interrupts synaptic transmission but does not interfere to any great extent with the release of acetylcholine. In the presence of curare the breakdown of phosphagen started only after more than 1 min of stimulation; it represented the loss of about 20-25% of the initial store. In untreated tissue the breakdown of CrP and ATP occurred in two phases and continued within the first minute after the end of the stimulation; as much as 77% of the phosphagen content was utilized under these conditions. The recovery of ATP and CrP was completed only 3-5 h after stimulation, a long time after the restoration of the physical capabilities of the tissue. The electrical energy dissipated during activity was smaller than the chemical energy provided by the net breakdown of phosphagens. This suggests that only a fraction of the chemical energy is utilized directly to compensate for the physical work accomplished, i.e. for the restoration of the ionic electromotive force. The electric organ also requires chemical energy for other purposes, particularly in the nerve endings where the presynaptic machinery seems to utilize an important fraction of the high energy phosphates stored in the tissue.     

Study of thermal effects of ultrasound stimulation on fracture healing

Low intensity ultrasound stimulation has been used as a strategy to promote fracture healing. This study investigated the mechanism of ultrasound stimulation in enhancing fracture healing. Forty-five adult New Zealand White rabbits were divided into control, microwave treated, and ultrasound stimulation groups. After anesthesia, transverse osteotomy was created at midportion of the fibula bone. Intravital staining followed by fluorescence microscopic examination of new bone formation in the osteotomy site and biomechanical tests on torsional stiffness of the osteotomy site were performed. The difference between each examination was evaluated and analyzed. After ultrasound stimulation, new bone formation in the osteotomy site of the stimulated limb was 23.1-35.8% faster than that of the sham treated limb; the torsional stiffness of the stimulated limb was 44.4-80.0% higher than that of the sham treated limb. In the group of microwave hyperthermia treatment, the new bone formation was higher than that of the sham treated limb, but the difference was not statistically significant. The difference in torsional stiffness between the microwave hyperthermia treated limbs and the sham treated limb was not quite statistically significant. We demonstrated that low intensity ultrasound stimulation could increase the new bone formation and torsional stiffness. These effects probably are not mediated via hyperthermia.     

External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts

External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways. The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium. The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min). The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later. During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts. Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled. From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.     

Evolution of marrow regeneration as revealed by transplantation studies

This study is concerned with the regeneration of bone marrow as an organized tissue. It is addressed specifically to the time in the regenerative program when the emerging tissue acquires the potential to reconstitute marrow. The evolution of this potential was investigated in rabbits by making autologous subcutaneous implants of tissue obtained at intervals after evacuation of a femur shaft. Analysis of 140 implant sites (89 of regenerating tissue and 51 of normal marrow) reveals a striking similarity in development of implants of a 2 to 4 day regenerating tissue and of normal marrow. New marrow encapsulated by bone can be seen in each instance 5 weeks after implantation. This property of regenerating tissue is uncovered before there are any obvious hemic elements. Significantly, the likelihood of a take is greatly increased when the implant contains a connective tissue. Marrow was always found in the implantation site when an ossicle was forming. It never occurred in the absence of bone. We conclude from this study that the appearance of modulated mesenchymal elements in the early regenerating tissue imparts the quality of normal marrow.     

The influence of transplanted culture of bone marrow stromal cells on reparative osteohistogenesis in parietal bone defect

Recently the problem connected with transplantation of bone marrow stromal cells for optimization of reparative osteogenesis is very actively studied. However, the objective methods allowing to observe the behavior of transplanted cells in a bone wound and to estimate character of regenerative osteogenesis after cell transplantation are used in an insufficient measure in both experimental and clinical researches. The aim of this study is to clarify the fate of stromal cells in a bone wound and to investigate the influence of bone marrow stromal cells on the process ofposttraumatic osteogenesis after cell transplantation in parietal bone defect. The experiments were carried out on 38 rabbits with artificially made parietal bone defect (diameter 1.0 cmm). The rabbits were divided into three groups: the first group was a control one; the rabbits of the second group were injected autogenic cultivated bone marrow stromal cells (10(6)); the rabbits of the third group were injected with autogenic cultivated bone marrow stromal cells (10(6)) in collagn gel. The methods of light and fluorescent microscopy, histomorphometry and statistical treatment of the data were used to estimate the results. The obtained data showed that transplanted cells were viable at least during 18 days after transplantation and efficiently took part in the reparative process. The transplantation of cultivated bone marrow stromal cells in collagen gel caused 30% increase in the part of bone tissue in the bone regenerate tissue in comparison with control after 120 days.     

Mineral and matrix contributions to rigidity in fracture healing

The purpose of this study was to investigate the relationships among selected properties of fracture callus: bending rigidity, tissue density, mineral density, matrix density and mineral-to-matrix ratio. The experimental model was an osteotomized canine radius in which the development of the fracture callus was modified by electrical stimulation with various levels of direct current. This resulted in a range of values for the selected properties of the callus, determined post mortem at 7 weeks after osteotomy. We found that the rigidity (R) of the bone-callus combination obeyed relationships of the form R = axb, where x is the tissue density, mineral density, matrix density or the mineral-to-matrix ratio of the repair tissue. These are analogous to power-law relationships found in studies of compact and cancellous bone. The results suggest that fracture callus at 7 weeks after osteotomy in canine radius behaves more like immature compact bone than cancellous bone in its mechanical and physicochemical properties. The present study demonstrates the feasibility of developing non-invasive in vivo densitometric methods to monitor fracture healing, since models may be developed that can predict mechanical properties from densitometric data. Further studies are needed to develop a refined model based on experimental data on the mechanical and physicochemical properties and microstructure of fracture callus at different stages of healing.     

Metabolic interactions of glucose, acetoacetate and adrenaline in rat submaxillary gland in vitro.

1. The metabolic interactions between glucose, acetoacetate and adrenaline were studied in submaxillary-gland slices. 2. Acetoacetate (2.5 mM) inhibited glucose removal by 22% and entry of glucose carbon into the tricarboxylic acid cycle by 54%. 3. Acetoacetate caused an increase in (glucose 6-phosphate) together with an increase in (citrate), a finding that suggests that the phosphofructokinase step might be inhibited by the elevated (citrate). Support for this suggestion was obtained in experiments in which fluoracetate was used to elevate (citrate). 4. A further site of action of acetoacetate at the pyruvate dehydrogenase step was suggested by an increase in the lactate+pyruvate pool, and the finding that pyruvate removal and (3-14C)pyruvate oxidation were inhibited by acetoacetate. 5. Adrenaline, a stimulator of secretion by this tissue, increased glucose removal by 25%. Adrenaline increased glucose removal to the same extent when acetoacetate was also present in the incubation medium. In both cases the increase was accompanied by a fall in (glucose 6-phosphate). 6. Adrenaline also overcame the inhibition of pyruvate removal caused by acetoacetate. 7. The tissue (ATP) decreased by about 50% on addition of adrenaline, and a similar fall was observed in vivo after adrenergic stimulation by isoproterenol. 8. Omission of Ca-2+ from the medium prevented the fall in (glucose 6-phosphate) and (ATP) caused by adrenaline, although adrenaline was still able to stimulate glucose removal. The inhibitory effect of acetoacetate on gluocse removal was reversed by adrenaline, but there was no stimulation above the control rates. Inhibition of pyruvate removal by acetoacetate was not overcome by adrenaline in the absence of Ca-2+. 9. Dibutyryl cyclic AMP had no effect on glucose removal or on (ATP). 10. Possible mechanisms by which adrenaline can bring about its metabolic effects are discussed.     

Regulation of Cell Cytoskeleton and Membrane Mechanics by Electric Field: Role of Linker Proteins

Cellular mechanics is known to play an important role in the cell homeostasis including proliferation, motility, and differentiation. Significant variation in the mechanical properties between different cell types suggests that control of the cell metabolism is feasible through manipulation of the cell mechanical parameters using external physical stimuli. We investigated the electrocoupling mechanisms of cellular biomechanics modulation by an electrical stimulation in two mechanically distinct cell types—human mesenchymal stem cells and osteoblasts. Application of a 2 V/cm direct current electric field resulted in approximately a twofold decrease in the cell elasticity and depleted intracellular ATP. Reduction in the ATP level led to inhibition of the linker proteins that are known to physically couple the cell membrane and cytoskeleton. The membrane separation from the cytoskeleton was confirmed by up to a twofold increase in the membrane tether length that was extracted from the cell membrane after an electrical stimulation. In comparison to human mesenchymal stem cells, the membrane-cytoskeleton attachment in osteoblasts was much stronger but, in response to the same electrical stimulation, the membrane detachment from the cytoskeleton was found to be more pronounced. The observed effects mediated by an electric field are cell type- and serum-dependent and can potentially be used for electrically assisted cell manipulation. An in-depth understanding and control of the mechanisms to regulate cell mechanics by external physical stimulus (e.g., electric field) may have great implications for stem cell-based tissue engineering and regenerative medicine.     

Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities

Electric stimulation has been used successfully to treat a wide range of bone disorders. However, the mechanism by which the electric fields can influence the bone cells behavior remains poorly understood. The purpose of this research was to assess the possible mechanism of the stimulatory effect of pulsed electromagnetic field (PEMF) on bone cells. A PEMF with a frequency of 15 Hz (1 G [0.1 mT]; electric field strength 2 mV/cm) were applied to neonatal mouse calvarial bone cell cultures for 14 days. The temporal effects of PEMF on the osteoblasts were evaluated by the status of proliferation, differentiation, mineralization, and gene expression on the 3rd, 5th, 7th, and 14th days of culture. Our results demonstrated that PEMF stimulation significantly increased the osteoblasts' proliferation by 34.0, 11.5, and 13.3% over the control group after 3, 5, and 7 days' culture. Although the alkaline phosphatase (ALP) staining and the mineralization nodules formation did not change, the ALP activity of the bone cells decreased significantly after PEMF stimulation. Under the PEMF stimulation, there was no effect on the extracellular matrix synthesis, while the osteoprotegerin (OPG) mRNA expression was up regulated and the receptor activator of NF-kappaB ligand (RANKL) mRNA expression were down regulated, compared to the control. In conclusion, the treatment by PEMF of osteoblasts may accelerate cellular proliferation, but did not affect the cellular differentiation. The effect of PEMF stimulation on the bone tissue formation was most likely associated with the increase in the number of cells, but not with the enhancement of the osteoblasts' differentiation.     

Humoral mechanisms of regulating reparative osteogenesis

The effect of the blood serum of animals with active osteogenesis on the biosynthesis of nucleic acids, protein, and on the mineralization of the regenerating bone tissue was studied in experiments in vivo and in vitro. Incorporation of DNA and protein labeled precursors (3H-thymidine and 14 C-proline, respectively) was increased and the mineralization of the bone callus (85Sr incorporation) was accelerated in the recipients. Comparison of nucleic acids and protein biosynthesis stimulation sequency allows to suppose that the active serum principle promotes the increased cell proliferation in the fracture area.     

Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair.     

Assessment of a mechano-regulation theory of skeletal tissue differentiation in an in vivo model of mechanically induced cartilage formation

Mechanical cues are known to regulate tissue differentiation during skeletal healing. Quantitative characterization of this mechano-regulatory effect has great therapeutic potential. This study tested an existing theory that shear strain and interstitial fluid flow govern skeletal tissue differentiation by applying this theory to a scenario in which a bending motion applied to a healing transverse osteotomy results in cartilage, rather than bone, formation. A 3-D finite element mesh was created from micro-computed tomography images of a bending-stimulated callus and was used to estimate the mechanical conditions present in the callus during the mechanical stimulation. Predictions regarding the patterns of tissues—cartilage, fibrous tissue, and bone—that formed were made based on the distributions of fluid velocity and octahedral shear strain. These predictions were compared to histological sections obtained from a previous study. The mechano-regulation theory correctly predicted formation of large volumes of cartilage within the osteotomy gap and many, though not all patterns of tissue formation observed throughout the callus. The results support the concept that interstitial fluid velocity and tissue shear strain are key mec- hanical stimuli for the differentiation of skeletal tissues.     

The fate of mechanically induced cartilage in an unloaded environment.

According to mechanobiologic theories, persistent intermittent mechanical stimulation is required to maintain differentiated cartilage. In a rat model for bone repair, we studied the fate of mechanically induced cartilage after unloading. In three groups of rats, regenerating mesenchymal tissue was submitted to different loading conditions in bone chambers. Two groups were immediately killed after loading periods of 3 or 6 weeks (the 3-group and the 6-group). The third group was loaded for 3 weeks and then kept unloaded for another 3 weeks (the (3 + 3)-group). Cartilage was found in all loaded groups. Without loading, cartilage does not appear in this model. In the 3-group there was no clear ongoing endochondral ossification, the 6-group showed ossification in 2 out of 5 cartilage containing specimens, and in the (3 + 3)-group all cartilage was undergoing ossification. These results suggest a tendency of the cartilage to be maintained also under unloaded conditions until it is reached by bone that can replace it through endochondral ossification.Additional measurements showed less amount of new bone in the loaded specimens. In most of the loaded specimens in the 3-group, necrotic bone fragments were seen embedded in the fibrous tissue layer close to the loading piston, indicating that bone tissue had been resorbed due to the hydrostatic compressive load. In some specimens, a continuous cartilage layer covered the end of the specimen and seemed to protect the underlying bone from pressure-induced resorption. We suggest that one of the functions of the cartilage forming in the compressive loaded parts of a bone callus is to protect the surrounding bone callus from pressure-induced fluid flow leading to resorption.     

The spatio-temporal arrangement of different tissues during bone healing as a result of simple mechanobiological rules

During secondary bone healing, different tissue types are formed within the fracture callus depending on the local mechanical and biological environment. Our aim was to understand the temporal succession of these tissue patterns for a normal bone healing progression by means of a basic mechanobiological model. The experimental data stemmed from an extensive, previously published animal experiment on sheep with a 3?mm tibial osteotomy. Using recent experimental data, the development of the hard callus was modelled as a porous material with increasing stiffness and decreasing porosity. A basic phenomenological model was employed with a small number of simulation parameters, which allowed comprehensive parameter studies. The model distinguished between the formation of new bone via endochondral and intramembranous ossification. To evaluate the outcome of the computer simulations, the tissue images of the simulations were compared with experimentally derived tissue images for a normal healing progression in sheep. Parameter studies of the threshold values for the regulation of tissue formation were performed, and the source of the biological stimulation (comprising e.g. stem cells) was varied. It was found that the formation of the hard callus could be reproduced in silico for a wide range of threshold values. However, the bridging of the fracture gap by cartilage on the periosteal side was observed only (i) for a rather specific choice of the threshold values for tissue differentiation and (ii) when assuming a strong source of biological stimulation at the periosteum.     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

Electron radioautographic study of osteogenesis during distraction osteosynthesis

Electron microscopic radioautography was used to study the osteogenesis during the replacement of bone tissue defects by graded distraction osteosynthesis in 12 patients with open fractures of the tibia. In the early stages of reparative tissue formation, the cells that intensely synthesized DNA and DNA were located exceptionally in the vascular walls or near them. The authors assume that vascular cells, most probably pericytes, are a basic source of the origin of the reparative tissue, turning firstly to fibroblast-like cells and then to osteoblasts.     

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re-established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re-established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.