Gayle E. Lester

Increased Rate of Fractures and Severe Kyphosis: Sequelae of Living into Adulthood with Cystic Fibrosis

Cystic fibrosis is the most common fatal autosomal recessive genetic disease in white persons; it affects approximately 30 000 Americans and a similar number of Europeans [1]. Cystic fibrosis mutations occur in approximately 1 of every 2500 live births in the white population and lead to death or lung transplantation in more than 500 persons annually in the United States alone [2]. Although respiratory disease is the greatest cause of illness and death in patients with cystic fibrosis, improved therapy for chronic pulmonary infection has markedly extended life expectancy and has led to the discovery of myriad other problems that afflict these patients [3]. Osteoporosis, in particular, increases pain and debilitation in patients with cystic fibrosis as they live into adulthood. Patients with cystic fibrosis have increased risk for osteoporosis as a result of multiple factors [4]. Poor nutrition, pancreatic insufficiency, reduced absorption of calcium and vitamin D, reduced physical activity, delayed and reduced production of sex steroids, use of corticosteroids, and increased circulating concentrations of osteoclast-activating factors (such as tumor necrosis factor- and interleukin-1) may all cause reduced bone mineral density in patients with cystic fibrosis. Young patients with cystic fibrosis invariably fail to reach peak bone mass [5-10], and this contributes to low bone mineral density in adulthood. Unbalanced bone formation and resorption [11], caused by accelerated bone loss, may lead to further bone demineralization in early adulthood. Both increased bone resorption and decreased bone formation probably play a role in osteoporosis in cystic fibrosis [8, 12, 13]. Osteoporosis in patients with cystic fibrosis is well documented [5-10, 12-16]. Hahn and colleagues [5] were the first to show low bone mineral density (mean reduction, 15%) in the distal radii of these patients. In the past decade, reductions in bone mineral density for the femur (mean decrease, 11.1%), lumbar spine (mean decreases, 12.5% to 35%), and total body (mean decrease, 10%) [6, 12, 14] have been reported in adults with cystic fibrosis. Henderson and Madsen [7] recently found that patients with cystic fibrosis had low bone mineral density in childhood and that it worsened with age. The average z-scores for the lumbar spine were 0.39 for children aged 5 to 8 years, 0.99 for children aged 8 to 12 years, and 1.69 for children aged 12 to 18 years. Taken together, these results show that osteopenia may occur as early as the first decade of life in patients with cystic fibrosis and that bone loss accelerates during adolescence and early adulthood. Although low bone mineral density in cystic fibrosis has attracted considerable attention, data on the complications of osteoporosis, including fractures and kyphosis, are limited. Despite low bone mineral density and anecdotal reports of increased fracture rates [9, 14, 15, 17-20], no reports have documented significant increases in fracture rates in adults with cystic fibrosis. It has been suggested that the decreased activity level that accompanies progressive cystic fibrosis offers a protective influence with respect to the occurrence of fractures. Our main goal was to quantitate the clinical sequelae of osteoporosis, namely, fractures and kyphosis, in a large group of adults with cystic fibrosis to test the hypothesis that fracture rates and kyphosis angles are greater in patients with cystic fibrosis than in the general population. A second goal was to investigate the role of potentially important clinical variables in the pathogenesis of osteoporosis in cystic fibrosis. Methods Patients The study sample consisted of 70 adults (older than 18 years of age) with advanced cystic fibrosis who were referred for lung transplantation at the University of North Carolina between January 1994 and December 1996. The Committee on Human Research (IRB #96-Med-336) approved this retrospective cohort study and required verbal consent from participating patients. Cystic fibrosis was diagnosed by elevated sweat chloride concentrations and an appropriate clinical picture. All patients had end-stage lung disease and an anticipated survival of less than 2 to 3 years [21]. Fracture History The history, date, and mechanism of fracture were determined by personal interviews done using methods similar to those of the National Health Interview Study [22]. Fractures were required to have patient report of radiographic confirmation at the time of the fracture, but we did not review these radiographs. Confirmation of long-bone fractures required patient report of treatment with casting. Fractures occurring between birth and 6 years of age were not assessed because most patients were unable to provide accurate histories for those years. The number of years that each patient was at risk for fracture was summed by age interval to determine the total number of patient-years for this cohort. All patients were asked to give their date of puberty; to give a detailed history of corticosteroid use (expressed as cumulative dose of prednisone in grams); to state whether they had received therapy for osteoporosis; and, if they were female, to state whether and when they had had oligomenorrhea. Bone Densitometry Bone mineral density was measured in all patients by a single, registered radiologic technologist using dual-energy x-ray absorptiometry (Hologic QDR 1000/W, Waltham, Massachusetts) [23]. Lumbar spine (L1-L4), nondominant femoral neck, and total-body bone mineral densities were measured; measurements were expressed in grams of bone mineral per cm2 of bone. Quality control was maintained by daily scanning of an anthropomorphic spine phantom. The coefficient of variation for our Hologic QDR 1000/W densitometer is 0.3%, and the reference limits for variation are 1.5%. Results were expressed as T-scores, which are the number of SDs that the bone mineral density measurement is above (positive value) or below (negative value) expected peak bone mass. The age at which peak bone mass is achieved differs slightly for each site but is usually between 20 and 30 years. Using World Health Organization guidelines, we defined osteopenia as a T-score greater than 2.5 and 1.0 or less. Osteoporosis was defined as a T-score of 2.5 or less [24]. Normal bone mineral density was defined as a T-score of 0 1. Laboratory Measurements Serum calcium, phosphorus, alkaline phosphatase, and creatinine concentrations were measured with an automatic analyzer (Hitachi 911, Boehringer Mannheim, Indianapolis, Indiana). Vitamin D metabolites were extracted from serum specimens (obtained while patients were fasting) with column chromatography and were measured with radiobinding assays (Nichols Institute, Raleigh, North Carolina). The 1,25-dihydroxyvitamin D and 25-hydroxyvitamin D assays had sensitivities of 5 pg/mL and 5 ng/mL, respectively. Testosterone levels were measured by using a competitive radioimmunoassay (Diagnostics Product Corp., Los Angeles, California). Genotype analysis for the 15 most common CFTR mutations was done by using published methods [25]. Radiographic Analysis Screening of the most recent posteroanterior and lateral chest radiographs for the extent of thoracic kyphosis as well as rib and vertebral body fractures was done by a single musculoskeletal radiologist on 65 patients (5 patients did not undergo radiography at the University of North Carolina). Thoracic kyphosis was measured from the second or third to the twelfth thoracic vertebral body by using a modification of the method of Cobb [26]. The number of vertebral compression fractures was also determined from the lateral radiograph by measuring anterior and posterior vertebral body height and expressing the difference as a percentage [27]. The posteroanterior chest radiograph was examined for the presence of rib fractures. Nonacute fractures appeared as focal deformities in the rib contour with varying degrees of reparative bone formation. Statistical Analysis Analyses were done with SAS software (version 6.12, SAS Institute, Cary, North Carolina) [28], and significance was based on two-tailed tests with a P value less than 0.05. Discrete variables are summarized as percentages, and continuous variables are summarized as means SD. Nonparametric methods were used when the distribution of the continuous variable was skewed. Student t-tests were used to compare differences in all clinical and anthropomorphic variables except fracture rates [29]. Relationships between spine and femur T-scores and seven continuous variables-age, age at puberty, cumulative steroid dose, body mass index, FEV1, FVC, and vitamin D levels-were assessed with either the Pearson or Kendall correlation coefficients [30]. Backward stepwise regression analysis was used to determine the multivariate relation between spine and femur T-scores and the aforementioned predictors. Clinical predictors and T-scores were compared, using the Kruskal-Wallis test, by dividing the patients into three groups: those without fractures, those with one fracture, and those with more than one fracture. This was done to determine whether differences existed between groups. Using spine or femur z-scores (the number of SDs that a bone mineral density measurement was above or below that of age- and sex-matched normal controls) rather than T-scores gave similar results (data not shown) because most patients were in the age range at which peak bone mass is expected. For the analysis of fractures and kyphosis angles, patients were grouped by age to conform with published databases (normal databases are based on age) [22, 31]. Two separate fracture rate analyses were done: In one, all fractures were used as the numerator; in the other, all persons with any fracture were used as the numerator. The latter analysis was done to eliminate the possibility that fracture clustering had affected the results. We used person-years as the denominator fo

Effect of cefazolin and vancomycin on osteoblasts in vitro.

The effect of cefazolin and vancomycin on osteoblast-like cells was studied. Cells from the MG-63 human osteosarcoma cell line were grown in antibiotic free media and exposed to concentrations of cefazolin and vancomycin at order of magnitude intervals between 0 and 10,000 Ug/ml. For cefazolin, a second interval was performed between 100 and 1000 ug/ml to define toxic levels more accurately. Cell number and 3H-thymidine incorporation at 0, 24, and 72 hours were determined. The results of this study show that local levels of vancomycin of 1000 μg/ml and less have little or no effect on osteoblast replication, and concentrations of 10,000 μg/ml cause cell death. Concentrations of cefazolin of 100 μg/ml and less have little or no effect on osteoblast replication, 200 μg/ml significantly decrease cell replication, and 10,000 μg/ml cause cell death. The authors conclude that vancomycin is less toxic than is cefazolin to osteoblasts at higher concentrations and may be a better antibiotic for local administration in the treatment of similarly sensitive bacterial infections.

Association between low levels of 1,25-dihydroxyvitamin D and breast cancer risk

OBJECTIVE To determine if blood levels of 25-hydroxyvitamin D (25-D) or its active metabolite, 1,25-dihydroxyvitamin D (1,25-D), are lower in women at the time of first diagnosis of breast cancer than in comparable women without breast cancer. DESIGN This was a clinic-based case-control study with controls frequency-matched to cases on race, age, clinic and month of blood drawing. SETTING University-based breast referral clinics. SUBJECTS One hundred and fifty-six women with histologically documented adenocarcinoma of the breast and 184 breast clinic controls. RESULTS There were significant mean differences in 1,25-D levels (pmol ml(-1)) between breast cancer cases and controls; white cases had lower 1,25-D levels than white controls (mean difference +/-SE: -11.08+/-0.76), and black cases had higher 1.25-D levels than black controls (mean difference +/-SE: 4.54+/-2.14), although the number of black women in the study was small. After adjustment for age, assay batch, month of blood draw, clinic and sample storage time, the odds ratio (95% confidence interval, CI) for lowest relative to highest quartile was 5.2 (95% CI 2.1, 12.8) for white cases and controls. The association in white women was stronger in women above the median age of 54 than in younger women, 4.7 (95% CI 2.1, 10.2) vs. 1.5 (95% CI 0.7, 3.0). There were no case-control differences in 25-D levels in either group. CONCLUSIONS These data are consistent with a protective effect of 1,25-D for breast cancer in white women.

Low frequency EMF regulates chondrocyte differentiation and expression of matrix proteins

This study describes the enhancement of chondrogenic differentiation in endochondral ossification by extremely low frequency pulsed electric/magnetic fields (EMFs). The demineralized bone matrix (DBM)‐induced endochondral ossification model was used to examine the effects of EMF stimulation. [35S]‐Sulfate and [3H]‐thymidine incorporation and glycosaminoglycan (GAG) content were determined by standard methods. Proteoglycan (PG) and GAG molecular size and composition were determined by gel chromatography and sequential enzyme digestion. Immunohistochemical and Western blot analysis of PGs were done with antibodies 2B6, 3B3, 2D3 and 5D4. Northern analysis of total RNA extracts was performed for aggrecan, and type II collagen. All data was compared for significance by Students t‐ or analysis of variance (ANOVA)‐tests.

An in vivo model of degenerative disc disease.

Although the precise etiology of low back pain is disputed, degeneration of the intervertebral disc is believed to play an important role. Many animal models have been described which reproduce the changes found in degenerative disc disease, but none allow for efficient, large‐scale testing of purported therapeutic agents. The purpose of this study was to develop a simple animal model resembling degenerative disc disease using the intervertebral discs found in the tails of rats. The proximal two intervertebral discs in the tails of 20 rats were injected with either chondroitinase ABC or control (phosphate buffered saline, PBS). The tails were harvested at 2 weeks, and measurements were made of intervertebral disc height (measured radiographically and histologically), biomechanics (stiffness, hysteresis, and residual deformation), and histologic appearance. Treatment with chondroitinase ABC resulted in a significant loss in intervertebral disc height (radiographic intervertebral disc height, p < 0.001; histologic intervertebral disc height, p < 0.001) and significant increases in all biomechanical parameters (stiffness, p < 0.001; hysteresis, p < 0.006; residual deformation, p < 0.004) compared to PBS controls. Intervertebral discs treated with chondroitinase ABC had significantly lower histologic grades for each grading category (nucleus pulposus (NP), annulus fibrosus, and proteoglycan staining) compared to controls. The results of injury with chondroitinase ABC were similar to the findings in degenerative disc disease: reduced intervertebral disc height, diminished proteoglycan content, loss of NP cells, and increased stiffness of the disc. Thus, the model appears to be a reasonable tool for the preliminary in vivo evaluation of proposed treatments for degenerative disc disease.

The effect of a nonsteroidal antiinflammatory drug on the healing of ligaments

Because of the increasing number of ligament sprains being treated with nonsteroidal antiinflammatory drugs (NSAIDs), this study was undertaken to document the effects of one such drug on ligament healing in an experimental setting. Male Sprague-Dawley rats weigh ing between 400 and 500 g were used to evaluate the effect of the NSAID piroxicam on the healing of an experimental injury to the medial collateral ligament (MCL). The following factors were varied in the experi ments: dosage, days of treatment, and the day postin jury when treatment was begun. Piroxicam-treated rats were compared to placebo-treated rats in terms of the drugs effect on the mechanical strength of the healing ligament. The ligaments were mechanically tested in tension to failure at a constant deformation rate of 0.25 mm/sec on a materials testing machine. Administration of piroxicam on Days 1 to 6 postinjury resulted in a 42% increase in strength at Day 14 postinjury for the piroxicam-treated ligaments (P < 0.01) when compared with the placebo-treated controls. Neither doubling nor halving the standard piroxicam dose significantly al tered this increased healing strength. Biochemical analysis of collagen synthesis demonstrated a sugges tive, although not statistically significant, increase in collagen synthesis and collagen content in the piroxi cam-treated healing ligament. In separate experiments, piroxicam had no effect on the healed ligament at 21 days or on the strength of uninjured ligaments. In conclusion, piroxicam increased the early strength of healing ligaments in the rat when the drug was admin istered for short periods of time after injury. It did not affect the final strength when healing was complete, nor did it alter the strength of uninjured ligaments.

Abnormal bone turnover in cystic fibrosis adults.

Abstract: Cystic fibrosis (CF) patients often have low bone mineral density (BMD) and may suffer from fractures and kyphosis. The pathogenesis of low BMD in CF is multifactorial. To study bone metabolism, we collected fasting serum and urine from 50 clinically stable CF adults (mean age 28 years) and 53 matched controls to measure markers of bone formation and bone resorption. The CF subjects had moderate lung disease (FEV1: 46.1 ± 18.6% predicted) and malnutrition (BMI: 20.0 ± 3.3 kg/m2). Only 3 subjects had normal BMD. CF subjects had higher urinary N-telopeptides of type I collagen (81.0 ± 60.0 vs 49.0 ± 24.2 nm BCE/mmol creatinine, p= 0.0006) and free deoxypyridinoline (7.3 ± 5.0 vs 5.3 ± 1.9 nM/mM, p= 0.004) levels than controls. Serum osteocalcin levels were similar in the two groups, a result confirmed by two immunoassays that recognize different epitopes on osteocalcin. Serum bone-specific alkaline phosphatase levels were elevated in CF patients (32.0 ± 11.3 vs 21.8 ± 7.0 U/l, p<0.0001), but were much more closely associated with serum total alkaline phosphatase levels (r = 0.51, p = 0.001) than with age or gender. Parathyroid hormone levels were elevated (p= 0.007) and 25-hydroxyvitamin D levels were depressed (p= 0.0002) in the CF patients in comparison with controls. These results indicate that adults with CF have increased bone resorption with little change in bone formation. Medications that decrease bone resorption or improve calcium homeostasis may be effective therapies for CF bone disease.

Histomorphometric evaluation of stress shielding in mandibular continuity defects treated with rigid fixation plates and bone grafts

Histomorphometric techniques were used to evaluate the stress shielding effect in bilateral bone grafts in 4 Macaca fascicularis monkeys. Bilateral continuity defects were created and grafted by replacing the resected portion of the mandible and iliac crest bone into the defect area. Both sides were plated with rigid internal fixation plates. Three months after bone grafting, the plate was removed from one side while the other plate was left in place. The animals were sacrificed at 1, 4, 6, and 8 months after plate removal and the grafted areas removed for histomorphometric analysis. This study documents decreased bone volume as well as smaller interlabel width on the plated side, suggesting a stress shielding effect as a result of the rigid internal fixation plate.

Altered calcium homeostasis in adults with cystic fibrosis.

Abstract: Bone mineral density (BMD) in cystic fibrosis (CF) patients falls progressively below normal with advancing age, in part due to steroid administration, low levels of sex hormones, chronic inflammatory disease, physical inactivity, and chronic malabsorption of calcium and/or vitamin D. The purpose of this study was to compare the fractional absorption of 45Ca and urinary excretion of calcium in CF subjects and normal controls following a high-calcium breakfast containing 45Ca. Seven young men and 5 young women with CF with pancreatic insufficiency were studied on two separate occasions, with and without administration of pancreatic enzymes. Eleven healthy young adults with normal BMD measurements served as controls. Mean T-scores at the lumbar spine and femur were significantly lower in the CF subjects (p<0.002). Following baseline, fasting collections, timed serum and urine samples were obtained for 5 h after the meal. Fractional absorption (FA) of 45Ca was estimated by the method of Marshall and Nordin. At baseline, CF subjects had lower mean serum 25-hydroxyvitamin D, calcium and albumin values (p<0.03 for each), slightly, but not significantly (p= 0.12), lower albumin-corrected calcium values, equivalent serum 1,25-dihydroxyvitamin D values and a trend toward a higher mean serum parathyroid hormone (PTH) value (p= 0.10). Without pancreatic enzymes, CF subjects showed significantly impaired calcium absorption (5 h FA: 11.8 ± 0.5 for controls vs 8.9 ± 0.2 for CF subjects, p= 0.02) and excretion (4 h excretion: 0.20 ± 0.08 mg Ca/mg creatinine for controls vs 0.16 ± 0.09 mg Ca/mg for CF subjects, p= 0.025). Addition of pancreatic enzymes did not fully compensate for this deficiency. In addition, CF patients had higher serum PTH values after a high-calcium meal (p= 0.03), suggesting mild secondary hyperparathyroidism. Altered calcium homeostasis is likely to be a factor in the development of bone disease in CF patients.

Differential Distribution of Lumican and Fibromodulin in Tooth Cementum

The objectives of this study were to isolate and characterize the major proteoglycans of tooth cementum in relation to the tissues mineralization. Cementum was collected from the root apex region of bovine molars and pulverized. It was first extracted with 6M guanidine-HCI, pH 7.4 (G-extract, mineral-unassociated) and then demineralized and extracted with 0.5M EDTA (E-extract, mineral-associated). Both extracts were applied to anion exchange and then molecular sieve chromatography to isolate proteoglycans. The fractions collected were assayed for chondroitin-(CS) and keratan sulfate (KS) containing proteoglycans using the monoclonal antibodies 2-B-6 and 5-D-4, respectively. It was found that the KS was the major glycosaminoglycan and was enriched in the G-extract fraction. The major KS fraction was then applied to 7.5% SDS-PAGE. The major broad band (69 kDa) was 5-D-4 positive in Western blot analysis and separated into two bands (46 kDa and 50 kDa) after treatment with keratanase II and endo-beta-galactosidase. These two proteins were transfered to PVDF membrane and analyzed for amino acid sequence. The results showed the major band (46 kDa) to be lumican and the minor (50 kDa) fibromodulin. In addition, based on the immunohistochemical study using a number of mono- and polyclonal antibodies including 5-D-4, anti-lumican core protein as well as anti-fibromodulin core protein antibodies, the KSPGs were found to be located almost exclusively in nonmineralized portions of cementum such as precementum and the pericementocyte area. These biochemical as well as immunohistochemical data suggest that the major KSPGs of cementum, lumican and fibromodulin, have a specific tissue distribution and may have regulatory roles in cementum mineralization.