Radiofrequency energy on cortical bone and soft tissue: a pilot study.

BACKGROUND: Radiofrequency-generating energy devices have been used clinically in musculoskeletal procedures to provide hemostasis and capsular shrinkage (thermal capsulorrhaphy). However, the dose-effects are not well known. QUESTIONS/PURPOSES: We therefore determined dosage effects of radiofrequency energy on bone, skin incisions, and joint capsule in sheep. METHODS: Five mature sheep had six 2.5-cm(2) tibial periosteal defects and six 1.0-cm skin incisions assigned to six treatments varying by watts and fluence (f = watts . seconds/cm(2)): (1) untreated control, (2) 50 W for 9.5 seconds (190f; n = 5), (3) 110 W for 4.3 seconds (190f; n = 5), (4) 170 W for 2.8 seconds (190f; n = 5), (5) 170 W for 5.6 seconds (380f; n = 5), or (6) 170 W for 8.4 seconds (570f; n = 5). Outcomes included hemostasis, contraction, healing, and histomorphometry for inflammation and necrosis at 2 weeks. RESULTS: Radiofrequency energy application on skin at 190f or greater had more than 80% hemostasis and dose-dependent contraction, inflammation, and necrosis. Radiofrequency energy application on bone had good (70%) hemostasis at 190f and complete (> 95%) hemostasis at 380f and 570f, without histologic or clinically detectable necrosis. CONCLUSIONS: Hemostasis can be achieved with radiofrequency energy at 190f in skin and bone. Bone necrosis was not detected at up to 570f. Using fluence greater than 190f in skin achieved dose-dependent necrosis and incisional contraction. CLINICAL RELEVANCE: Radiofrequency energy can be used on bone and skin for hemostasis, but potential incisional complications, such as necrosis and an atypical firm and desiccated surface, should be expected.

Non-invasive, quantitative assessment of the morphology of γ-irradiated human mesenchymal stem cells and periosteal cells using digital holographic microscopy.

PURPOSE: To assure the quality of cells to be used in cell therapy, we examined the applicability of digital holographic microscopy (DHM) for non-invasive, quantitative assessment of changes in cell morphology. MATERIALS AND METHODS: Mesenchymal stem cells derived from adipose tissue (MSC-AT) and bone marrow (MSC-BM), in addition to human alveolar periosteal cells (PC) as a reference, were γ-ray irradiated (1 and 4 Gy), and their morphological changes were quantified without fixation using holographic microscopy. After detachment and fixation with ethanol, cell number and surface antigen expression were determined using an automated cell counter kit and flow-cytometry, respectively. RESULTS: Among various indexes, only indexes related to cell size were significantly changed after γ-irradiation. Both BMC-AT and BMC-BM were enlarged and more sensitive to a low dose of γ-irradiation than PC. In contrast to PC, proteins related to DNA damage repair (γ-H2AX, p21waf1, p53 and Rb) were not substantially upregulated or sustained for a week in either MSC-AT or MSC-BM. CONCLUSION: Instead of DNA damage markers, we suggest that cell morphological parameters (e.g. cell volume) that are monitored by DHM could be a useful and more stable marker of MSC quality.

Parathyroid hormone attenuates radiation-induced increases in collagen crosslink ratio at periosteal surfaces of mouse tibia.

As part of our ongoing efforts to understand underlying mechanisms contributing to radiation-associated bone fragility and to identify possible treatments, we evaluated the longitudinal effects of parathyroid hormone (PTH) treatment on bone quality in a murine model of limited field irradiation. We hypothesized PTH would mitigate radiation-induced changes in the chemical composition and structure of bone, as measured by microscope-based Raman spectroscopy. We further hypothesized that collagen crosslinking would be especially responsive to PTH treatment. Raman spectroscopy was performed on retrieved tibiae (6-7/group/time point) to quantify metrics associated with bone quality, including: mineral-to-matrix ratio, carbonate-to-phosphate ratio, mineral crystallinity, collagen crosslink (trivalent:divalent) ratio, and the mineral and matrix depolarization ratios. Irradiation disrupted the molecular structure and orientation of bone collagen, as evidenced by a higher collagen crosslink ratio and lower matrix depolarization ratio (vs. non-irradiated control bones), persisting until 12weeks post-irradiation. Radiation transiently affected the mineral phase, as evidenced by increased mineral crystallinity and mineral-to-matrix ratio at 4weeks compared to controls. Radiation decreased bone mineral depolarization ratios through 12weeks, indicating increased mineral alignment. PTH treatment partially attenuated radiation-induced increases in collagen crosslink ratio, but did not restore collagen or mineral alignment. These post-radiation matrix changes are consistent with our previous studies of radiation damage to bone, and suggest that the initial radiation damage to bone matrix has extensive effects on the quality of tissue deposited thereafter. In addition to maintaining bone quality, preventing initial radiation damage to the bone matrix (i.e. crosslink ratio, matrix orientation) may be critical to preventing late-onset fragility fractures.

Effect of irradiation on autogenous bone transplantation in rat parietal bone.

To determine the appropriate time for bone reconstruction after irradiation, the healing process after autogenous iliac bone transplantation in the irradiated parietal bone was examined by scanning electron microscopy and light microscopy. Bone transplantation was carried out at the second and the fourth weeks after Cobalt-sixty (60Co) irradiation with calculated dose and fractionation. Animals without irradiation were used as control. The results show the appearance of mesenchymal cells and blood vessels around the transplantation to be extremely few one week after transplantation which was carried out at the second week after irradiation. These inhibitions were still seen two weeks after transplantation. Four weeks after transplantation, there were no differences in the bone formation among the experimental groups. Bone formation in the transplantation at the fourth week after irradiation was similar to that of the control group. Microvascularization in the transplantation at the second week after irradiation was inhibited one week after transplantation. The delay in bone healing was responsible for the retardation of revascularization and caused microcirculatory failures as well as the damage of osteogenic cells. It is quite clear that damaged cells and tissues recovered by the elapse of time under the irradiation procedure employed in this study and also that bone formation was carried out in the physiological process. We think that bone transplantation after irradiation should be done after recovery from the radiation damage to the periosteal cells and the blood vessels.

Effects of irradiation on cortical bone and their time-related changes. A biomechanical and histomorphological study.

The effects of high-dose irradiation on the biomechanical and morphological properties of cortical bone and their time-related changes were studied in male Sprague-Dawley rats. A single fraction of 3500 rads was applied to the middle part of the right thigh. Age and sex-matched control rats that did not receive radiation were used for comparison. Two weeks after irradiation, the animals had lost weight and disappearance of bone cells, shrunken osteocytes, and significantly decreased bone-turnover activities were noted. The bone marrow showed reduced hematopoietic elements and sinusoids with increased fat. No change in the torsional strength and geometric properties of bone was noted at this time. Six to ten weeks after irradiation, histopathological abnormalities of the bone persisted, and there was an accelerated resorption process. However, significant increases in the strength and cortical area of bone appeared only in the non-irradiated, contralateral femur. On the irradiated side, decreased cortical area and increased porosity of bone were found at fourteen to eighteen weeks when compared with the control rats. Other histomorphological properties, such as bone porosity, osteocyte count, and periosteal new-bone formation, appeared to recover eighteen weeks after irradiation, as they became similar to the values for the age-matched normal control rats.

Post-irradiation changes in the blood vessels of the adult human mandible.

Changes in the inferior alveolar artery and periosteal vessels of the post-irradiation mandible have been investigated. The inferior alveolar artery underwent comparatively little change, stenosis being noted in just over half the specimens examined. The number of periosteal arteries and veins increased, the changes being most marked on the body where the effective tissue dose of radiation was highest. The increase in periosteal vessels was independent of stenosis of the inferior alveolar artery.

Comparing calvarial transport distraction with and without radiation and fat grafting.

The purpose of this study is to: a) assess transport distraction to reconstruct cranial defects in radiated and non-radiated fields b) examine adipose grafting's effect on the bony regenerate and overlying wound, and c) elucidate sources of bone formation during transport distraction osteogenesis. Twenty-three male New Zealand white rabbits (3 months; 3.5 kg) were used, 10 non-irradiated and 13 irradiated (17 treatment, 6 control) with a one-time fraction of 35 Gy. A 16 × 16 mm defect was abutted by a 10 × 16 mm transport disc 5 weeks after irradiation, and 11 animals were fat grafted at the distraction site. Latency (1 day), distraction (1.5 mm/day), and consolidation (4 weeks) followed. Fluorochromes were injected subcutaneously and microCT, fluorescence, and histology assessed. In distracted animals without fat grafting, bone density measured 701.87 mgHA/ccm and 2271.95 mgHA/ccm in irradiated and non-irradiated animals. In distracted animals with fat grafting, bone density measured 703.23 mgHA/ccm and 2254.27 mgHA/ccm in irradiated and non-irradiated animals. Fluorescence revealed ossification emanating from the dura, periosteum, and transport segment with decreased formation in irradiated animals. Transport distraction is possible for cranial reconstruction in irradiated fields but short-term osseous fill is significantly diminished. Adipose grafting enhances wound healing in previously irradiated fields but does not enhance ossification.

The effects of low-level laser therapy on palatal mucoperiosteal wound healing and oxidative stress status in experimental diabetic rats.

OBJECTIVE: The biostimulation effects of low-level laser therapy (LLLT) have recently been demonstrated. In this study, we aimed to investigate the effects of LLLT on palatal mucoperiostal wound healing and oxidative stress status in experimental diabetic rats. MATERIALS AND METHODS: Forty-two male Wistar rats that weighed 250-300 g were used in this study. Experimental diabetes was induced in all of the rats using streptozotocin. A standardized full thickness wound was made in the mucoperiosteum of the hard palates of the rats using a 3 mm biopsy punch. The rats were divided into groups: 1 (control group, non- irradiated), and 2 (experimental group, irradiated). Treatment using a GaAlAs laser at a wavelength of 940 nm and at dose of 10 J/cm(2) began after surgery, and was repeated on the 2nd, 4th, and 6th days post-surgery. Seven animals from each group were killed on the 7th, 14th, and 21st day after surgery. Biopsies were performed for the histological analysis and blood samples were collected by cardiac puncture for biochemical analysis. RESULTS: The histopathological findings revealed reduced numbers of inflammatory cells, and increased mitotic activity of fibroblasts, collagen synthesis, and vascularization in rats in group 2. The total oxidative status was significantly decreased in the laser-treated group on the 21st day. CONCLUSIONS: LLLT elicits a positive healing effect on palatal mucoperiostal wounds, and modulates the oxidative status in experimental diabetic rats.

Suppurative osteomyelitis, bisphosphonate induced osteonecrosis, osteoradionecrosis: a blinded histopathologic comparison and its implications for the mechanism of each disease.

Statistically, significant numbers of central bone specimens of suppurative osteomyelitis of the jaws (SOJ), bisphosphonate induced osteonecrosis of the jaws (BIONJ), and osteoradionecrosis of the jaws (ORNJ) were compared. All three evidenced the common finding of necrotic bone with empty osteocytic lacunae, Haversian and Volkmann canals, but each showed a distinctive histopathologic pattern indicating a different disease mechanism and treatment options. Suppurative osteomyelitis was characterized by intense marrow inflammation and marrow vessel thrombosis with retention of viable osteoclasts and periosteum. Bisphosphonate induced osteonecrosis was characterized by an empty marrow space with empty Howship's lacunae and an absence of osteoclasts but viable periosteum. Osteoradionecrosis was characterized by a collagenous hypocellular, hypovascular marrow space and nonviable periosteum. Histologic evidence in SOJ indicates a microorganism provoked intense inflammation and marrow vascular thrombosis creating an environment conducive to continual bacterial proliferation. BIONJ is seen as a non-inflammatory drug toxicity to bone by osteoclastic death leading to over suppression of bone renewal, and ORN as another non-inflammatory condition caused by a high linear energy transfer that impairs or kills numerous cell types in the field of radiation including periosteum, bone, and all soft tissue.

Extracorporeal shock wave-induced proliferation of periosteal cells.

The cambium cells of the periosteum are an important cell source for select tissue engineering/regenerative medicine applications due to their osteogenic and chondrogenic potential. However, the cambium layer is only 2-5 cells thick, which complicates its harvest, and the low cell number limits its suitability for certain applications. Extracorporeal shock waves (ESWs) have been reported to cause periosteal osteogenesis following cambium layer thickening. This study quantified the proliferation of cambium cells in the femur and tibia of adult rats following ESW treatment at two different energy flux densities. Four days after application of ESWs, there was a significant (3- to 6-fold) increase in cambium layer thickness and cell number. Proliferation was seen with an energy flux density as low as 0.15 mJ/mm(2). The tibial cambium cells were more proliferative than those of the femur, with the cells closest to the ESW source proliferating the most. Within the thickened periosteum, α-smooth muscle actin and von Willebrand Factor expression were upregulated, suggesting a vascular role in ESW osteogenesis. Bone formation was seen within the stimulated periosteum at day 4. We propose that non-invasive ESWs can be used to rapidly stimulate cambium cell proliferation, providing a larger cell population for use as a progenitor cell source for tissue engineering applications, than can normally be provided by periosteum.

Effects of local and whole body irradiation on the appearance of osteoblasts during wound healing in tooth extraction sockets in rats.

Irradiation before tooth extraction delays wound healing in the alveolar socket. This study examined the influences of local and whole body irradiation before tooth extraction on appearance of osteoblasts in the alveolar bone of rat maxillary first molars because bone formation is observed at the initial phase of wound healing. Several osteoblasts were generated 3 days after tooth extraction, and the number of cells increased day by day. Morphological studies showed there were little differences between local irradiation and non-irradiated controls. In contrast, the extraction wound in the whole body irradiation group showed delayed healing, and there was poor granulation tissue and very few osteoblasts at the bottom of the socket. An ultrastructural study showed that the osteoblasts in the extraction socket of whole body irradiation rats were smaller, and had poorly developed organelles. Injection of bone marrow cells to whole body-irradiated animals immediately after tooth extraction partially restored the number of osteoblasts. New periosteal bone formations outside of sockets showed little delay in the whole body irradiation group. These findings suggest that bone formation in the wound healing of extraction socket requires bone marrow cells from hematopoietic organs such as the bone marrow as well as local sources around the alveolar socket, during the initial phase of wound healing.

Osteogenic effects of low-intensity pulsed ultrasound, extracorporeal shockwaves and their combination - an in vitro comparative study on human periosteal cells.

Our previous studies have shown that on human periosteal cells, low-intensity pulsed ultrasound (LIPUS) has an immediate stimulatory effect whereas extracorporeal shockwaves (ESW) have an delayed stimulatory effect. Therefore, we hypothesized that a combined ESW and LIPUS treatment might provide additive or synergistic effects on periosteal cells, by using ESW to trigger a biological activity while using LIPUS to maintain the stimulated activity. Human periosteal cells were subjected to a single session of ESW treatment on day 0 and/or daily LIPUS treatments or no treatment (control). The cell viability, proliferation, and alkaline phosphatase activity on day 6 and day 18 as well as matrix mineralization on day 35 were measured. Results revealed that LIPUS alone had early positive effects on the activities on day 6 only. In contrast, ESW alone had an early destructive effect but exerted delayed stimulatory effects on the cellular activities on day 18. The combined treatment of ESW plus LIPUS produced effects that were comparable to the ESW treatment alone. Although these findings suggest that ESW and LIPUS stimulate the periosteal cells in two different ways and at different times, their additive or synergistic effects could not be proven.

Radiation-induced craniofacial bone growth inhibition: in vitro cytoprotection in the rabbit orbitozygomatic complex periosteum-derived cell culture.

BACKGROUND: Radiotherapy for the management of head and neck cancer in pediatric patients results in severe inhibition of craniofacial bone growth. Previously, the infant rabbit orbitozygomatic complex was established as an experimental model. Amifostine, a cytoprotective agent, was found effective in preventing radiation-induced bone growth inhibition. This study was designed to investigate the effects radiation on osteogenic cells from infant rabbit orbitozygomatic complex periostea and to assess the effects of cytoprotection in vitro. METHODS: Infant New Zealand White rabbits (n = 18) were randomized into three groups and received radiation (0, 10, or 15 Gy) to both orbitozygomatic complexes. Cell cultures were developed from orbitozygomatic complex periostea, and cell numbers, proliferation, alkaline phosphatase, and collagen type I expression and mineralization were assessed. Subsequently, rabbits (n = 18) were randomized into three groups to receive either radiation at the effective dose, pretreatment with amifostine (300 mg/kg, intravenously, 20 minutes before irradiation) with the effective radiation dose, or no treatment. Cell cultures were developed and tested for proliferation and alkaline phosphatase expression. RESULTS: Irradiation resulted in a significant inhibition of cell numbers (p < 0.001) and proliferation (p < 0.01) at the 15-Gy dose and no statistically significant changes in alkaline phosphatase activity. Collagen type I expression and mineralization were also significantly reduced at the 15-Gy dose. Pretreatment with amifostine significantly (p < 0.05) enhanced the number of surviving cells. CONCLUSIONS: Amifostine is capable of protecting orbitozygomatic complex periosteum-derived osteogenic cells from the deleterious effects of radiation. This study provides the basis for understanding the cellular mechanisms of radiation-induced craniofacial bone growth inhibition and cytoprotection by amifostine.

An in vitro model of radiation-induced craniofacial bone growth inhibition.

Radiation-induced craniofacial bone growth inhibition is a consequence of therapeutic radiation in the survivors of pediatric head and neck cancer. Previously, the infant rabbit orbitozygomatic complex (OZC) was established as a reliable animal model. The purpose of this study was to develop a cell culture model from the rabbit OZC to study the effects of radiation in the craniofacial skeleton. Infant (7-week-old) New Zealand white rabbits were used in this study. Periostea from both OZC were harvested in sterile conditions, introduced into cell culture by way of sequential digestion, and subcultured at confluence. Cultures were analyzed for cellular proliferation (methylthiazoletetrazolium assay), alkaline phosphatase activity, collagen type I expression, and mineralization. Electron microscopy was performed to reveal the in vitro ultrastructure. Subsequently, rabbits were irradiated with sham or 15 Gy radiation, and cell cultures were developed and analyzed for cell numbers. Cell cultures, grown from OZC periostea, expressed osteoblast-like phenotype, with high alkaline phosphatase activity, collagen type 1 expression, and mineralization in an osteogenic environment. Electron microscopy confirmed the characteristic ultrastructural features of osteogenesis in vitro. Finally, significantly (P < 0.01) fewer cells were obtained from animals treated with 15 Gy radiation compared with those from control animals.A primary cell culture with osteoblast-like cellular phenotype was developed from infant rabbit OZC periosteum. This cell culture system responded to in vivo administered radiation by a significant decrease in cell numbers. This in vitro model will be subsequently used to study the cellular mechanisms of radiation and radioprotection in craniofacial osteoblast-like cells.

Delayed stimulatory effect of low-intensity shockwaves on human periosteal cells.

We investigated the effect of shockwaves on cells explanted from normal human periosteum to study the potential mechanisms of their responses and to determine suitable treatment settings. The cells were subjected to one shockwave treatment with systematic combinations of energy intensities (range, 0.05-0.5 mJ/mm) and number of shocks (range, 500-2000) whereas control cells received no treatment. The immediate effect on cell viability and the long-lasting effect on proliferation, viable cell number at Day 18, and mineralization at Day 35 were assessed. We observed an immediate dose-dependent destructive effect of shockwaves. Energy intensity and number of shocks contributed equally to viability. Total energy dose (intensity x number of shocks) was a better reference for determining the shockwave effect. We also found a long-term stimulatory effect on proliferation, viable cell number, and calcium deposition of human periosteal cells. At the same total energy dose, low-intensity shockwaves with more shocks (0.12 mJ/mm at 1250 shocks) were more favorable for enhancing cellular activities than high-intensity waves with fewer shocks (0.5 mJ/mm at 300 shocks). These findings document some of the biochemical changes of periosteal cells during shockwave treatments.

[The influence of microwave monochromatic radiation on bone fracture union in rabbits]. / Wplyw monochromatycznego promieniowania mikrofalowego na zrost zlaman kosci promieniowej u królików.

The influence of 53.57 GHz microwave radiation of 10mW/cm2 power density on fresh radial bone fracture in 16 rabbits has been investigated. After osteotomy, the occipital area was irradiated one hour daily during two weeks. The development of bony union in experimental and control group (12 rabbits) were compared on the basis of radiographic and physical assessments on the 7-th, 14-th, 21-st and 28-th day after osteotomy. Favourable effect of microwave radiation on bone union was found, especially in the first 14 days after osteotomy. An intense periosteal reaction accompanied the healing process in experimental animals. Biochemical and morphological blood tests run simultaneously were standard.

Effect of low level laser therapy on wound healing after palatal surgery in beagle dogs.

The effect of low level laser therapy on wound healing and wound contraction after palatal surgery in Beagle dogs of 12 weeks of age was investigated. A total of 30 Beagle dogs was used and assigned to a control group (Group C; n = 6) and two experimental groups (Group L; n = 12 and group LL; n = 12). In both experimental groups, Von Langenbeck's palatal repair was simulated. Then in the LL group the denuded bony areas were treated with low level laser therapy using a continuous Ga-As-A1 laser beam (830 nm) and energy output set at 30 mW. Per treatment a dosage of 1 J/cm2 wound surface area was used. Therapy was carried out three times a week with a total of ten treatments. Wound healing was observed clinically until wound healing was completed at 4 weeks p.o. and wound areas were measured at regular intervals on standardized intra-oral photographs. Wound contraction was measured as the increments of the distances between tattoo points on the opposite wound margins. No significant differences were found in the quality and rate of wound healing between the two experimental groups. The same held true for the increments of the distances between opposite tattoo points. It was concluded that macroscopically low level laser therapy under conditions used in this study did not have an influence on wound closure or wound contraction.

Effect of irradiation on early enzymatic changes in healing mandibular periosteum and bone. A histochemical study on rats.

The influence of irradiation was studied histochemically in healing mandibular periosteum and bone. After a cut line had been made on both sides of the mandible the rats were exposed to roentgen ray irradiation. The single doses were 15, 20, 30, 35 or 40 Gy. The animals were killed 1, 2, 4, 8, 10, 12, 16 and 24 hours after irradiation, for histochemical analysis. All enzymes, acid phosphatase, cytochrome oxidase, lactate, isocitrate, glucose-6-phosphatase and succinate dehydrogenase, showed a greater increase in enzyme staining in the irradiated cut lines than in the non-irradiated control lines. The intensity of the staining increased with time and dose over 24 hours. The observation time included an inflammatory phase with vascular, enzymatic and cellular responses to periosteal and bone injury. The increase in staining was dependent on the time after surgical trauma and radiation dose. The increase in enzyme staining probably represents the initial cell damage after irradiation.