Joseph A. Spadaro

Clinical experiences with low intensity direct current stimulation of bone growth.

Low intensity direct current stimulation of bone growth involves the continuous application of cathodic currents in the nanoampere range. The technique has been applied to 13 patients with a variety of non-unions and pseudarthroses with a success rate of 77 per cent. Preliminary data indicate that a range of total energy, from 0.6 to 2.5 Joules, is maximally effective. The technique has been combined with anodic control of local bacterial infection with promising results. Both the osteogenic stimulation and the bacterial suppression techniques as described in this paper, appear to be safe and effective.

Cross-sectional study of bone mineral density in adult survivors of solid pediatric cancers.

To investigate the hypothesis that survivors of pediatric solid cancer have low bone mineral density, a cross-sectional study was done of subjects who had received treatment for pediatric solid tumors before 16 years of age and were less than 40 years old at follow-up. Excluded were subjects treated for acute lymphoblastic leukemia or those who had received cranial irradiation, total body radiation, or nonautologous bone marrow transplant. The study group consisted of 38 subjects, with the most common diagnoses being lymphoma (n = 17), sarcoma (n = 8), Wilms tumor (n = 5), and neuroblastoma (n = 4). Median age was 22 years (range 12-32). Time from diagnosis of underlying cancer averaged 12.6 years (range 5.5-20.3). Using criteria of osteopenia (Z-score ≤−1.0 and >−2.0) and osteoporosis (Z-score ≤ −2.0) for any one or more areas including total body, lumbar spine, total hip, or femoral neck density, 13 of the 38 subjects (34%) had osteopenia or osteoporosis. A further six subjects (16%) had isolated upper extremity osteopenia or osteoporosis. Multivariate analysis showed a direct relationship between the number of chemotherapy drugs administered and the presence of osteopenia or osteoporosis in the lower extremities (P = 0.03). Young survivors of childhood solid tumors are at increased risk of developing premature osteopenia or osteoporosis, and screening evaluations and follow-up are warranted.

Sparing of radiation-induced damage to the physis: fractionation alone compared to amifostine pretreatment

PURPOSE The purpose of this study was to determine the relative benefits of sparing longitudinal bone growth by fractionation alone compared to pretreatment with amifostine, a chemical that provides differential radioprotection of normal tissues. METHODS AND MATERIALS Twenty-four weanling 4-week-old male Sprague-Dawley rats were randomized into 2 overall treatment groups: fractionation alone (n = 12) and amifostine pretreatment (n = 12). The distal femur and proximal tibia in the right leg of each animal were exposed to a therapeutic X-irradiation dose (17.5 Gy total in 3 or 5 fractions) with the contralateral left leg as control. In 12 of the animals, amifostine (100 mg/kg) was administered intraperitoneally 20 min before radiation exposure. Six weeks later, growth was calculated based upon measurement of the bone lengths. RESULTS Fractionated radiation resulted in a mean percent overall limb growth loss of 21. 1 +/- 7.0%. The addition of amifostine brought the mean percent overall limb growth loss to 16.3% +/- 4.6%, which showed a strong trend toward significance compared to fractionation alone (p = 0. 061). The addition of radioprotection with amifostine to 5 fractions irradiation significantly reduced the femoral and overall percentage growth arrest and limb length discrepancy compared to 5 fractions alone. CONCLUSIONS These results support further investigation of amifostine and other radioprotectants in combination with fractionation for use in growing children requiring radiotherapy to the extremity for malignant tumors.

In vivo and in vitro effects of a pulsed electromagnetic field on net calcium flux in rat calvarial bone.

Although PEMFs have been found to promote fracture healing and to modulate the activity of bone cells in vitro, effects on bone metabolism are largely unexplored. A bioassay using neonatal rat calvarial bone was used to determine the early effects of a pulsing electromagnetic field (PEMF) exposure in vivo and in vitro on bone metabolic calcium exchange. Bone discs taken from whole body exposed animals (0-4 hours) show a log exposure time-dependent average increase in net Ca uptake in the 0-50% range (r2 = 0.83). This increase could be detected immediately after exposure and also after 24 hours, but not 48 hours later. Animals given whole body PEMF exposure also showed a decrease in serum calcium and did not elevate serum Ca after administration of exogenous parathyroid hormone (PTH). Bone discs from untreated rats, exposed to PEMF for 15 minutes in vitro and then assayed, showed net Ca uptake increases of a similar magnitude and also were refractory to the Ca-releasing effect of PTH. Unexposed discs responded normally to PTH by decreasing net Ca uptake. Treatment of calvarial discs with calcitonin or acetazolamide, both of which inactivate osteoclasts, made the bone refractory to further increases in Ca uptake by PEMF. These results suggest that PEMF exposure produces PTH-refractory osteoclastics and has a relatively rapid effect on increasing net bone Ca uptake, putatively due to a decrease in PTH/paracrine-mediated bone resorption.

Amifostine before fractionated irradiation protects bone growth in rats better than fractionation alone.

The aim of this study was to determine the independent and combined effects of 100 mg/kg and 200 mg/kg doses of the radioprotectant amifostine and radiotherapy dose fractionation in preserving the integrity of or minimizing damage to the physis during high-dose radiation exposure in an animal model. Thirty-six weanling four-week-old male Sprague-Dawley rats were randomized into six study groups of six animals each. The distal femur and proximal tibia in the right leg of each animal was exposed to X-irradiation, with the contralateral left leg serving as the nonirradiated control. Three groups received a single 25 Gy radiotherapy dose: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Three groups received a total of 25 Gy in three equal fractions: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Fractionation of the 25 Gy radiation dose reduced the mean percent overall limb growth loss to 44.8%, a statistically significant reduction compared to a mean 58.8% reduced growth with the single 25 Gy dose. Addition of amifostine at 100 and 200 mg/kg before each of the three fractions of radiotherapy further decreased the mean percent overall limb growth loss to 35.2% and 28.5%, respectively, both statistically significant reductions beyond that achieved by fractionation alone.

Sequential Histomorphometric Analysis of the Growth Plate Following Irradiation with and without Radioprotection

Background: The availability of radioprotectant drugs that selectively protect normal cells but not tumor cells has rekindled interest in the effects of irradiation on the growth plate. The purpose of the present study was to quantitatively examine the sequential histomorphometric effects of irradiation and pretreatment with a free radical scavenger radioprotectant, amifostine, on the growth plate over time. Methods: Sixty four-week-old male Sprague-Dawley rats were randomized into five groups of twelve animals that were to be killed at 0.5, one, two, three, or four weeks after irradiation. One-half of the animals also received amifostine (100 mg/kg) prior to irradiation. In all animals, the right knee was treated with a single 17.5-Gy dose of radiation. End points were assessed with quantitative histomorphometric analysis of the growth plate, BrdU labeling for evidence of proliferation, evaluation of chondroclast cellularity, and determination of growth rates by means of oxytetracycline labeling. Results: The mean lengths of the femur, tibia, and hind limb continued to increase at each time-interval following treatment, but by one week the mean limb length was 4% less on the irradiated side than on the control side, and this difference remained significant for four weeks (p < 0.05). The proximal tibial growth rate decreased during the first week to 18% of the control level. Nevertheless, growth continued even at the earliest time-periods, began to return toward normal at two weeks, and ultimately returned to at least 80% of normal by four weeks after irradiation. The area fraction of matrix in the hypertrophic zone increased initially and returned to control levels at three and four weeks. The administration of the radioprotectant resulted in significant increases in growth, growth rate, growth plate height, hypertrophic zonal height, and chondroclast profiles compared with the values for limbs in which irradiation had not been preceded by treatment with amifostine. Conclusions: We found an initially profound but transient direct inhibitory effect of irradiation on growth plate chondrocytes. Recovery of growth plate function after irradiation corresponded temporally with the appearance of newly formed islands of proliferating chondrocytes. Accumulation of matrix led to a transient increase in overall growth plate height, which was most pronounced in the hypertrophic zone. This was due, in part, to the sensitivity of chondroclasts to irradiation. The radioprotectant amifostine reduced these effects on growth rate, growth plate height, matrix accumulation, and limb length. Clinical Relevance: The transient effects of irradiation on the growth plate are reduced by a clinically utilized radioprotectant drug. Use of radioprotectants may have potential for reducing the damaging effects of irradiation on the growth plate while preserving the desirable effects of irradiation on tumors.

Temporal Changes in PTHrP, Bcl-2, Bax, Caspase, TGF-β, and FGF-2 Expression Following Growth Plate Irradiation With or Without Radioprotectant

This study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-β was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-β, Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.

Transiently increased bone density after irradiation and the radioprotectant drug amifostine in a rat model.

At therapeutic levels in pediatric patients, radiation causes damage to the growth plate and contributes to growth deformity and fractures. The purpose of this project was to examine the effects of x-ray irradiation on regional bone mineral density (BMD) and osteoclast histology of rat bone with and without radioprotectant amifostine (AMF) pretreatment. Seventy-two weanling rats had their right knee irradiated with single fraction 17.5 Gy, whereas the left leg was used as an internal control. Twelve animals were euthanized at each of 6 time periods (0.5–6 wk) after irradiation, half having received 100 mg/kg amifostine. BMD (g/cm3) was determined for both the right and left femurs using peripheral quantitative computed tomography (CT) (pQCT). Tibial sections were stained for osteoclasts/chondroclasts with tartrate-resistant acid phosphatase. Statistically significant increases in BMD within the radiation field were seen in the treatment groups’ right irradiated legs over the control unirradiated left legs at all time points from 0.5 through 6 weeks. Anatomically, a peak in BMD occurs in the region immediately adjacent to the chondro-osseous junction at 2 weeks after irradiation and then moves proximally within the adjacent metaphysis after 3 weeks. Corresponding to these findings, histologically a 2-week nadir occurs after irradiation in osteoclasts/chondroclast numbers adjacent to the chondro-osseous junction with a 71.9% decrease compared with controls (p <0.05). At 3 weeks, the numbers of osteoclasts/chondroclasts in this region have increased to 47.4% greater than the control legs (p <0.03) The animals receiving amifostine had BMD that was consistently closer to controls only adjacent to the chondro-osseous junction at 0.5, 2, and 3 weeks and osteoclast/chondroclast numbers that were closer to controls only at 4 weeks.

Novel radioprotectant drugs for sparing radiation-induced damage to the physis.

Purpose: To determine if pentoxifylline, interleukin 1α, selenium and misoprostol can minimize damage to physeal longitudinal growth during single radiation dose exposure in an animal model. Materials and methods: Eighty‐seven weanling Sprague–Dawley rats were randomized into 15 drug/dose groups. All groups received a single 17.5‐Gy gamma‐irradiation exposure to the right knee, the left limb serving as an internal control. Pentoxifylline was injected 30 min before exposure, sodium selenite and interleukin 1α 24 h before exposure and misoprostol 2 h before exposure. Positive controls received 17.5 Gy. At 6 weeks, animals were sacrificed, the hind limb lengths were measured and detailed histomorphometric analysis was performed. Results: Statistically significant reductions (p≤0.03) in mean limb length discrepancy compared with irradiation alone were seen following administration of pentoxifylline (50 mg kg−1), interleukin 1α (15 mcg kg−1), selenium (5 mg kg−1) and misoprostol (20 mg kg−1). Histomorphometric endpoints and growth rate remained altered at 6 weeks despite treatment, but length discrepancy reduction was highly correlated with the appearance of regenerative clones. Conclusions: Each drug reduced the amount of anticipated growth arrest in the animal model and some compared favourably in magnitude with that previously demonstrated for the established radioprotectant drug amifostine. Restoration of growth appears related to appearance of regenerative clones.

Precision and Accuracy of DXA and pQCT for Densitometry of the Rat Femur

Measurements of bone mineral density and bone mineral content are key data in the study of osteoporosis and pathologic skeletal disease. Dual-energy X-ray absorptiometry and peripheral quantitative computed tomography are used in human and small animal studies. The purpose of this study was to evaluate the precision, accuracy, and systematic bias of measurement of the rat femur. Comparing machine-measured parameters with standard, nonradiographic measurements, we assessed validation of relative and absolute accuracy. Regression analysis and calculations of percent difference from standard values were used to determine the accuracy of each densitometry technique. Machine-specific and subject-specific precision was evaluated for each densitometer using repeated scans to calculate coefficients of variation. Each of the methods of densitometry examined in this study produced comparable results and was sensitive to small changes following experimental stimuli. Further, our assessment of the precision and accuracy observed between methods of scanning excised rat femurs validates our data acquisition method and serves as a foundation for future densitometry studies.