The role of activin A in the human osteoblast cell cycle: a preliminary experimental in vitro study.

Animal studies have previously shown that activin A enhances osteoblast proliferation IN VITRO and increases bone formation and bone mechanical strength IN VIVO. For the further understanding of its action in human osteoblast, we studied the pattern of a cell cycle response to the treatment with activin A. We hypothesize that activin A alters the cell cycle pattern of human osteoblast. Primary cultures of human osteoblast-like cells were treated by activin A in a biologically effective concentration (100 ng/mL). The cells in cultured samples were counted, assayed for cellular alkaline phosphatase activity and calcitonin expression, LDH activity in the medium, cellular BrdU incorporation, cell cycle cytometry and compared to untreated controls. The treated by activin A cells responded by a significant shift toward the G1 phase of the cell cycle with parallel decrease in cell death rate (lower LDH activity and less necrotic cells in cytometric analysis). The treated cells also showed a lower alkaline phosphatase activity and calcitonin expression, indicating their undifferentiated state, and didn't change their proliferation rate. The number of cells in culture increased following treatment with activin A. We show that activin A increases the net osteoblast number in culture by reducing the cell death rate without affecting the cell proliferation. These findings should be part of cellular pathways that are involved in the initial stages of bone tissue generation.

Breakdown limits on Gigavolt-per-meter electron-beam-driven wakefields in dielectric structures.

First measurements of the breakdown threshold in a dielectric subjected to GV/m wakefields produced by short (30-330 fs), 28.5 GeV electron bunches have been made. Fused silica tubes of 100 microm inner diameter were exposed to a range of bunch lengths, allowing surface dielectric fields up to 27 GV/m to be generated. The onset of breakdown, detected through light emission from the tube ends, is observed to occur when the peak electric field at the dielectric surface reaches 13.8+/-0.7 GV/m. The correlation of structure damage to beam-induced breakdown is established using an array of postexposure inspection techniques.

Ionization-induced electron trapping in ultrarelativistic plasma wakes.

The onset of trapping of electrons born inside a highly relativistic, 3D beam-driven plasma wake is investigated. Trapping occurs in the transition regions of a Li plasma confined by He gas. Li plasma electrons support the wake, and higher ionization potential He atoms are ionized as the beam is focused by Li ions and can be trapped. As the wake amplitude is increased, the onset of trapping is observed. Some electrons gain up to 7.6 GeV in a 30.5 cm plasma. The experimentally inferred trapping threshold is at a wake amplitude of 36 GV/m, in good agreement with an analytical model and PIC simulations.

Hosing instability in the blow-out regime for plasma-wakefield acceleration.

The electron hosing instability in the blow-out regime of plasma-wakefield acceleration is investigated using a linear perturbation theory about the electron blow-out trajectory in Lu et al. [in Phys. Rev. Lett. 96, 165002 (2006)10.1103/PhysRevLett.96.165002]. The growth of the instability is found to be affected by the beam parameters unlike in the standard theory Whittum et al. [Phys. Rev. Lett. 67, 991 (1991)10.1103/PhysRevLett.67.991] which is strictly valid for preformed channels. Particle-in-cell simulations agree with this new theory, which predicts less hosing growth than found by the hosing theory of Whittum et al.

Positron production by x rays emitted by betatron motion in a plasma wiggler.

Positrons in the energy range of 3-30 MeV, produced by x rays emitted by betatron motion in a plasma wiggler of 28.5 GeV electrons from the SLAC accelerator, have been measured. The extremely high-strength plasma wiggler is an ion column induced by the electron beam as it propagates through and ionizes dense lithium vapor. X rays in the range of 1-50 MeV in a forward cone angle of 0.1 mrad collide with a 1.7 mm thick tungsten target to produce electron-positron pairs. The positron spectra are found to be strongly influenced by the plasma density and length as well as the electron bunch length. By characterizing the beam propagation in the ion column these influences are quantified and result in excellent agreement between the measured and calculated positron spectra.

Bone defect repair in rat tibia by TGF-beta1 and IGF-1 released from hydrogel scaffold.

Bone repair is one of the major challenges facing reconstructive surgery. Bone regeneration is needed for the repair of large defects and fractures. The ability of TGF-beta1 and IGF-1 incorporated into hydrogel scaffold to induce bone regeneration was evaluated in a rat tibia segmental defect model. External fixation was performed prior to the induction of the segmental bone defect in order to stabilize the defect site. Hydrogel scaffold containing either TGF-beta, IGF-1, TGF-beta + IGF-1, hydrogel containing saline or saline, were inserted in the defect. Calcified material was observed in the defects treated with TGF-beta 2 weeks following the start of treatment. Bone defects treated with TGF-beta, IGF-1 or TGF-beta + IGF-1 revealed significant bone formation after 4 and 6 weeks when compared to the control specimens. X-ray images showed that solid bone was present at the defect site after 6 weeks of treatment with TGF-beta or TGF-beta + IGF-1. A less pronounced bone induction was observed in the control specimens and bones treated with IGF-1. Percent closure ratio of bone defects after 6 weeks were 40, 80, 89, and 97% for saline, hydrogel, IGF-1, TGF-beta and IGF-1 + TGF-beta groups, respectively. It is concluded that hydrogel scaffold can serve as a good osteoconductive matrix for growth factors, and that it provides a site for bone regeneration and enhances bone defect healing and could be used as alternative graft material.

Enhancement of bone defect healing in old rats by TGF-beta and IGF-1.

Bone defects are often created in order to repair bone pathologies. In the aging population, the healing of such defects is very limited. Bone healing in aging depends on the availability of various hormone and growth factors. The ability of growth factors to enhance bone formation in femoral defects in old rats was tested. Bone defects were induced in femurs of old rats. A single dose of transforming growth factor-beta (TGF-beta), IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were tested after 2 and 4 weeks. Radiology revealed that mineralization appeared in the 2 weeks group in defects treated with TGF-beta and in defects treated with TGF-beta, TGF-beta+IGF-1 in the 4 weeks groups. Computerized tomography (CT) coronal and axial images revealed that 4 weeks after treatment with TGF-beta+IGF-1, a complete bone bridge was observed. Morphology revealed that these defects were filled with trabecular bone. A less pronounced bone healing was observed after TGF-beta or IGF-1, while control specimens revealed partial healing of the bone defect. Biomechanical tests indicated that treatment with TGF-beta, IGF-1 or TGF-beta+IGF-1 resulted in a significant increase of bone bending rigidity compared to control in the 4 weeks group and that TGF-beta+IGF-1 was the most inductive in this respect. The ability to induce bone healing in aging by TGF-beta+IGF-1 is of a great clinical importance for restoration of bone strength and biomechanical properties of bone defects in aging.

Metalloproteinases (MMPs -2, -3) are involved in TGF-beta and IGF-1-induced bone defect healing in 20-month-old female rats.

Matrix metalloproteinases are important in the physiological and pathological degradation of extracellular matrix including that of bone and cartilage. The process of bone defect healing is associated with formation of cartilage callus and cancelous bone. With maturation and aging, the response of skeletal tissues to injury is limited. The ability of growth factors to enhance bone defect healing in aged rats was studied. Partial bone defects were induced in femurs of aged rats. A single dose of IGF-1, TGF-beta+IGF-1 or saline was inserted in the defect and bones were examined after 2 and 4 weeks. Morphology revealed that after 2 weeks of treatment with TGF-beta the defects were filled with mesenchyme-like tissue and delicate bone trabeculae. Positive staining for metalloproteinase-2 (MMP-2) was shown at the sites of new bone formation. In defects treated with IGF-1 or TGF-beta+IGF-1 nodules of cartilage and fine bone trabeculae along with positive staining for both MMP-2 and MMP-3 were demonstrated in the healing defects. After 4 weeks radiology revealed mineralization in defects treated with TGF-beta and less pronounced mineralization after treatment with IGF-1, or with TGF-beta+IGF-1, whereas only partial healing of the defects was observed in control specimens. MMP-2 and MMP-3 were detected at sites of new bone formation after treatment with TGF-beta, IGF-1, and TGF-beta+IGF-1. It is concluded that TGF-beta and IGF-1 induced bone defect healing in aged rats. TGF-beta induced bone formation while IGF-1 induced cartilage and than bone formation via endochondral ossification. The localization of MMP-2 and MMP-3 in the healing defects reflected the synthesis of bone or cartilage matrices in the defect, reflecting the involvement of MMPs in the process of bone formation and endochondral ossification. The ability to induce bone defect healing in aging is of great clinical importance and understanding the involvement of MMPs in this process can contribute to future treatment with growth factors to enhance bone defect healing in 20-month-old female rats.

[Vertical alveolar ridge augmentation using distraction osteogenesis].

Alveolar ridge atrophy can be acquired or congenital. Alveolar ridge augmentation can be performed using bone grafting but a donor site morbidity is unavoidable and some resorption of the grafted bone occurs. Distraction osteogenesis offers an alternative method for bone reconstruction without donor site morbidity and sufficient stability of the final results. In order to place implants, vertical alveolar augmentation using Distraction Osteogenesis has been applied to 14 patients aged 17 to 55 years old with edentulous alveolar ridge, 8 in the mandible and 6 in the maxilla. Vertical alveolar bone distraction was started by performing a segmental alveolar osteotomy followed by insertion of a central distraction device--Lead System (Endosseous alveolar distraction system, Leibinger). After three days of latency period, the distraction was started in a rate of 0.8 mm per day for 9 to 16 days as needed. The retention period for the new bone maturation was twice as long as the active distraction period. After removal of the device, 23 cylindrical threaded implants were inserted. The results revealed increase of the alveolar bone height with new bone formation that was observed radiographically and clinically at the removal of the distraction device. The amount of elevation was 7-13 mm. In follow-up of 6-14 months, failure of only one implant was noted, due to inadequate transported bone stability. The advantages of alveolar bone distraction are: Increase of the alveolar bone height with new bone formation, minimal resorption of bone without the bone graft disadvantages. A long term follow up after the bone height achieved and implant anchorage should be performed in the future.

Differential metabolic responses to local administration of TGF-beta and IGF-1 in temporomandibular joint cartilage of aged mice.

Osteoarthritis is a degenerative joint disease characterized by destruction of the articular cartilage in aging and senescence. The aim of this study was to study the possible treatment of this disease by intraarticular injection of growth factors to osteoarthritic joints of aged animals. 20-month-old female ICR mice were injected with insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF-beta) or TGF-beta+IGF-1 on days 1, 4, and 7. On day 9 the joints were dissected and cultured in the presence of 35S-sulfate and 3H-thymidine. Combined treatment of TGF-beta and IGF-1 resulted in elevated 3H-thymidine incorporation and DNA and protein contents, reduction of 35S-sulfate incorporation and alkaline phosphatase activity, with no significant change in the activity of acid phosphatase. Following injections of TGF-beta, contents of DNA and protein, and incorporations of 3H-thymidine were induced, and 35S-sulfate and alkaline phosphatase activity were reduced. Treatment with IGF-1 resulted in reduced incorporation of 3H-thymidine with no significant changes in the activity of acid phosphatase. Atypically hypertrophic chondrocytes were observed along the articular surface and the endogenous production of TGF-beta and of IGF-1, as revealed by immunohistochemistry, was reduced. It is concluded that although 3H-thymidine incorporation and alkaline phosphatase activity appeared to be induced by TGF-beta and IGF-1, the overall responsiveness of cartilage from aged mice to these growth factors appeared to be inhibitory. Moreover, their effects appeared to be limited to specific cell populations in the cartilage itself.

Enhancement of toluidine blue staining by transforming growth factor-beta, insulin-like growth factor and growth hormone in the temporomandibular joint of aged mice.

Osteoarthritic lesions appear in the articular cartilage of the temporomandibular joint of mice aged 7 months and older. Reduced rate of proteoglycan (PG) synthesis leading to destruction of the articular cartilage was observed in this joint. The purpose of the present study was to test the ability of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-1 (IGF-1) and growth hormone (GH) to induce PG synthesis in joint cartilage of aged animals and to compare it with the effect of interleukin-1alpha (IL-1alpha). Mandibular condyle explants from 18-month-old mice were cultured up to 72 h in serum-free medium, supplemented with IL-1alpha (TGF-beta1 (0.1-5.0 ng/ml), TGF-beta1 (1.0 ng/ml) + IGF-1 (2 ng/ml) or GH (10 ng/ml). The incorporation of (35)S-SO(4) into sulfated PG was tested. Cartilage samples were processed for histomorphometry using sections stained with 0.1% toluidine blue (TB), pH 1.8. Results indicated that in cultures supplemented (48 h) with either TGF-beta, TGF-beta + IGF-1 or with GH, an increased height and area of TB-positive staining as well as increased incorporation of (35)S-SO(4) into sulfated PG were observed. In contrast, the cytokine IL-1alpha exerted an inhibitory effect on TB staining and on (35)S-SO(4) incorporation. The present study demonstrated that in vitro supplementation of IL-1alpha to mandibular condyle cartilage reduced the height and area of TB staining and incorporation of (35)S-SO(4), whereas TGF-beta1, TGF-beta1 + IGF-1 or GH increased the height and area of TB staining and increased incorporation of (35)S-SO(4). The two parameters used to identify increased PG synthesis were shown to reveal similar results and were useful for studying the dynamic events taking place in cartilage destruction and repair in osteoarthritis.

Prenatal sonographic diagnosis of nasal malformations.

The aim of this study was to describe the prenatal detection of fetal nasal abnormalities by ultrasound. Transvaginal and transabdominal sonography was used for the detection of fetal malformations in 25, 114 cases. The fetal nose was visualized in all the fetuses in an oblique section and in the lateral profile of the face. Fifteen cases of fetal nasal malformations were detected between the 12th and 27th week of gestation. All but two, who had dilatation and curettage, were confirmed by postabortal or postnatal examination. Dyskaryosis (trisomies 18 and 21, triploidy, and tetrasomy 12 p) was diagnosed in six cases (40 per cent). The nasal malformations were isolated in four cases, one of them being associated with tetrasomy 12 p. The fetal nose should also be a target in the prenatal sonographic screening for malformations in the early mid-trimester. The incidence of fetal nasal anomalies is 1:1674 fetuses (15:25, 114). The fetal nose should be visualized by ultrasound in both an oblique plane and a lateral profile to identify its abnormalities.

Comparison of in vitro response to growth hormone by chondrocytes from mandibular condyle cartilage of young and old mice.

In several aspects the features of aging resemble those of the state of growth hormone (GH) deficiency. Alterations of bone density and increased incidence of osteoporosis are some of the characteristics of aging that are similar to the findings in GH-deficient adults. Osteoarthritis (OA), a nonfatal condition predominating in old age, is characterized by the slowly progressive destruction of articular cartilage of joints. OA lesions are observed in the temporomandibular joint of ICR mice aged 7 months and older. The aim of the present study was to compare the response of chondrocytes from mandibular condyle cartilage of young and old mice to GH and to ascertain whether chondrocytes of old animals could still be stimulated to proliferate and to synthesize matrix components under the direct effect of GH in vitro. Mandibular condyles from young (3-month-old) and old (20-month-old) female ICR mice were cultured up to 72 hours in the presence of recombinant rat GH (0.1-100 ng/ml). 3H-Thymidine and 35S-sulfate were introduced during the last 24 hours of culture. Administration of GH appeared to increase only slightly the incorporation of 3H-thymidine in cartilage from young compared with the response in cartilage from old animals which was much more significant at 24 hours and 48 hours in culture. The same pattern was seen for the effect of GH on the incorporation of 35S-sulfate. Cartilage from young animals responded only slightly to GH, whereas a significant response was observed in cartilage from old animals after 24 and 48 hours in vitro. DNA contents were significantly elevated at all time intervals in both old and young animals, yet more significantly in cultures from old animals. In contrast, the activities of both alkaline and acid phosphatases were elevated at all time intervals in cultures from young animals, whereas no induction was observed in cultures from old animals. Tissue sections from cultures of old animals treated with GH (10 ng/ml, 48 hours) revealed atypical hypertrophic cells along the articular surface and also dark staining along the cartilage-bone interface. Exposure to tetracycline (10 mg/ml, 24 hours) resulted in an induced fluorescence, indicating enhanced mineralization in this region. Results of this study indicate that mandibular condyle cartilage from OA old mice responds in vitro to the addition of GH via anabolic activities of cell proliferation, sulfated proteoglycan synthesis, and possibly enhanced mineralization.

Osteoarthritis in the temporo-mandibular joint (TMJ) of aged mice and the in vitro effect of TGF-beta 1 on cell proliferation, matrix synthesis, and alkaline phosphatase activity.

The temporo-mandibular joint of aged mice develops osteoarthritic (OA) degenerative lesions. Adult chondrocytes have a low rate of cell replication, and cartilage repair potential is very limited. One of the major problems in OA is the low rate of matrix synthesis and the inability of the chondrocytes to exceed the rate of matrix degradation. These combined factors lead to the overall destruction of the cartilage as seen in OA. Cartilage degradation is mediated by elevated proteolytic activity of enzymes. Among the enzymes degrading cartilage are the metalloproteinases, stromelysin and collagenase. Other proteinases that may potentially participate in matrix degradation are the lysosomal enzymes cathepsin B, D, and L, and acid phosphatase. On the other hand, alkaline phosphatase (ALP) is an enzyme that has been shown to be a marker for anabolic activity in skeletal tissues such as bone and cartilage. The cartilage of the mandibular condyle in the T-M-J from aged mice reveals OA lesions. An overall reduction of cell proliferation and sulfated proteoglycan synthesis has been also shown in this joint. In the present study the effects of hTGF-beta on the stimulation of DNA and sulfated GAG synthesis and ALP activity were studied. Mandibular condyle cartilage obtained from 12-month-old ICR male mice were cultured in BGJb serum-free medium for 24-72 hours, supplemented with 0.1-10 ng/ml hTGF-beta 1. 3H-thymidine and 35S-sulfate were added for the last 24 hours of the culture and their incorporation into DNA and sulfated GAGs respectively, as well as the activity of ALP, were determined. Results indicated that hTGF-beta 1 enhanced the incorporation of both 3H-thymidine and of 35S-sulfate into cartilage cultures of aged mice, and also induced ALP activity. It thus appeared that in OA degenerating articular cartilage, the chondrocytes could be stimulated in vitro to proliferate and to synthesize new matrix, thus indicating induced anabolic activity in the tissue.

Effects of transforming growth factor-beta 1 and interleukin-1 alpha on matrix synthesis in osteoarthritic cartilage of the temporo-mandibular joint in aged mice.

Osteoarthritic lesions were observed in the mandibular condyle cartilage of mice aged 7 months and older. These lesions appeared as fibrillations along the articular surface and were accompanied by reduced extracellular matrix synthesis and chondrocyte proliferation. Explants of mandibular condyle cartilage were cultured in serum-free BGJb medium supplemented with ascorbic acid (300 micrograms/ml), penicillin (100 U/ml) and streptomycin (100 micrograms/ml) for up to 72 h. Cultures were further supplemented with either hTGF-beta 1 (0.1-5.0 ng/ml) or human IL-1 alpha (40 U/ml). [3H]thymidine (2 microCi/ml) and [35S]SO4 (10 microCi/ml) were added to the culture medium for the last 24 h of culture and incorporation into DNA and sulfated proteoglycans, respectively, studied. The results indicated that protein and DNA contents, [3H]thymidine and [35S]SO4 incorporation, as well as the specific activity of alkaline phosphatase, were increased by TGF-beta 1. Addition of 1.0 or 5.0 ng/ml hTGF-beta 1 to the cultures for 48 h, had the most stimulatory effect on matrix synthesis and cell proliferation, whereas 0.1 ng/ml hTGF-beta 1 appeared to be inhibitory when compared to controls. Increased [35S]SO4 labeling of chondrocyte clusters was observed by autoradiography in tissue sections from cultures treated with TGF-beta 1 (1.0 and 5.0 ng/ml). In contrast, IL-1 alpha exerted inhibitory effects on cell proliferation and matrix synthesis. However, it induced the activity of acid phosphatase in these cultures. The results of the present study show that IL-1 alpha had catabolic effect on his tissue, while TGF-beta 1 enhanced proliferation and induced synthetic activity of the cartilage cells. Such action by TGF-beta suggests the existance of a possible repair process in osteoarthritic cartilage of the temporo-mandibular joint of aged mice.

Differential response of articular cartilage from young growing and mature old mice to IL-1 and TGF-beta.

Osteoarthritic lesions are observed in temporomandibular joint cartilage of ICR mice aged 7 months and older, accompanied by reduced proliferation and matrix synthesis. Transforming growth factor-beta (TGF-beta), is a multifactorial growth factor affecting matrix synthesis and cell proliferation in bone and cartilage, whereas interleukin-1 (IL-1) is involved in cartilage degradation. In order to establish the repair capacity of cartilage in aging, the response of cartilage from young and old animals to TGF-beta and IL-1 was studied. Mandibular condyles from young (1-month-old) and old (18-month-old) mice were cultured for up to 72 h in medium supplemented with TGF-beta1 or IL-1alpha. TGF-beta increased protein (+9.26%) and DNA (+36.0%) contents in young animals and DNA content (+19.49%) in old animals. Incorporations of [(3)H]thymidine and [(35)S]sulfate were enhanced in young (+254% and +116%, respectively) and in old (+22.6% and +6.88%, respectively) and animals and activity of alkaline phosphatase was induced in old animals. Treatment with IL-1 resulted in reduced DNA content in young (-35.76%) and old (-33.33%) animals, but acid phosphatase activity was induced in old animals. It is concluded that TGF-beta can induce anabolic activity even in cartilage from old animals indicating repair response in articular cartilage in aging.

A crossover study comparing the efficacy of continuous positive airway pressure with anterior mandibular positioning devices on patients with obstructive sleep apnea.

OBJECTIVE: This study compared the efficacy of a removable anterior mandibular positioning (AMP) device to continuous positive airway pressure (CPAP) in patients with obstructive sleep apnea (OSA) using a fully balanced crossover design. DESIGN: Twenty-three male subjects with confirmed OSA were recruited from the Technion Sleep Laboratory in Haifa, Israel, from February 18, 1991 to December 17, 1992. Twenty-one of the 23 subjects enrolled completed all aspects of the study. RESULTS: The mean apnea-hypopnea index (AHI) before treatment was 33.86 +/- 14.30. The mean AHI decreased with CPAP to 59.50%, but decreased only 38.91% with the AMP device. The lowest mean recorded oxygen saturation level for the 21 subjects was 84.30 before treatment, 91.10 after CPAP treatment, and 90.20 after AMP treatment. Sleep data revealed a significant decrease in stage 1 and 2 (p=0.0088) and an increase in rapid eye movement percent (p=0.0066) for both treatments when compared with baseline. Three- to 10-month posttreatment phone interviews showed that 1 subject was not using either device, 1 subject was using CPAP, and 2 subjects were using the AMP device intermittently due to occasional temporomandibular joint pain symptoms. The remaining 17 subjects were all using the AMP device nightly. The symptoms of excessive daytime sleepiness also decreased significantly by both AMP and CPAP. CONCLUSIONS: The AMP device achieved substantial success in most cases, but was less effective than CPAP, especially for the more severe cases. In general, the AMP device was strongly preferred over the CPAP by the subjects of this study.