Impact of a grant program to spur advances in sickle cell disease research.

More than 20 years ago, clinical trials and federal grant support for sickle cell disease (SCD) research were not on par with support for other genetic diseases. Faced with the opportunity to spur research and advance treatments for SCD, and at the recommendation of advisors, the Doris Duke Charitable Foundation (DDCF) offered an SCD research funding opportunity starting in 2009 through its Innovations in Clinical Research Awards (ICRA) program. Twenty-eight new grants of $450 000 for direct costs over 3 years and 7 renewals were awarded, for a total investment of $17 million. Only about half the research teams garnered follow-on funding directly related to their ICRA projects, but the financial return on the research investment was substantial (∼4 times the original $17 million or 300%). All but 1 of the ICRA investigative teams published original research reports that acknowledged DDCF as a source of funding; the median number of publications per team was 3. Major innovations in the diagnosis and treatment of SCD included but were not limited to a demonstration that genetic modification of BCL11A enhancer is a potentially important treatment modality, establishment that plerixafor mobilization is safe and effective for those with SCD, development and validation of a new diagnostic called SCD BioChip, and evidence that hydroxyurea treatment is safe and efficacious in African children. These outcomes show that relatively small research grants can have a substantial return on investment and result in significant advances for a disease such as SCD.

The Doris Duke Clinical Scientist Development Award: implications for early-career physician scientists.

PURPOSE: The Doris Duke Charitable Foundation Clinical Scientist Development Award (CSDA) supports early-career physician scientists in their transition to independent research funding. The authors aimed to analyze the characteristics associated with success in CSDA competitions, determine whether attainment of a CSDA is associated with receiving subsequent research funding, and assess whether alumni remain in research. METHOD: In 2011, the authors tested for associations between gender, age, race/ethnicity, academic degree, National Institutes of Health (NIH) funding rank of the applicant's institution, and success in CSDA competitions. They compared NIH R01 grant attainment, defined as the percentage of individuals who received at least one R01 grant, between CSDA alumni and highly ranked but unsuccessful CSDA applicants (1998-2007). Finally, the authors surveyed alumni to learn more about their professional activities. RESULTS: Demographic factors were not predictors of success in CSDA competitions; academic degree and funding rank of the applicant's institution, however, were. A greater percentage of CSDA alumni than nonalumni received at least one R01 grant (62% [74/120] versus 42% [44/105]). For CSDA alumni who were 10 or more years from the start of their award, their median percent effort toward research activities was 68%. CONCLUSIONS: The factors associated with success in a CSDA competition included a combined clinical and doctoral research degree and affiliation with a well-funded institution. More alumni received NIH independent research funding than those who applied but did not receive the award. Thus, the CSDA is associated with physicians establishing independent and recognized research careers.

Similarities and differences in philanthropic and federal support for medical research in the United States: an analysis of funding by nonprofits in 2006-2008.

PURPOSE: The medical community currently has no detailed source of information on philanthropic research funding. The authors sought to identify trends in research funding by members of the Health Research Alliance (HRA), a consortium of nonprofit funders of biomedical research, and compare findings with research support from the federal government. METHOD: Thirty-two HRA members uploaded information about grants with start dates in 2006, 2007, and 2008. Data were collected about each grant, investigator, and recipient institution. Disease categorization codes were assigned by a computer process similar to that used by the National Institutes of Health (NIH). RESULTS: In the three years under study, HRA members awarded 9,934 grants, totaling $2,712,418,254 in research and training support. Grant funding increased by 26% between 2006 and 2008. In contrast, NIH research spending increased by only 3% over the same time. Fifty-six percent of HRA grant dollars supported research projects, whereas 30% supported career development and training. During the same period, more than two-thirds of NIH grant dollars supported research projects, although NIH invested proportionally less in career development and training (7%). The largest proportion of HRA grant dollars addressed cancer, followed by diabetes and genetics. Sixty-three percent of HRA-supported investigators were men and 36% were women; 66% of investigators were white, 32% Asian, and fewer than 2% black. CONCLUSIONS: These results indicate that nonprofit organizations play an important role in developing careers and advancing research in significant disease areas such as cancer and diabetes, and in basic science areas such as genetics.

Bone mass is preserved and cancellous architecture altered due to cyclic loading of the mouse tibia after orchidectomy.

INTRODUCTION: The study of adaptation to mechanical loading under osteopenic conditions is relevant to the development of osteoporotic fracture prevention strategies. We previously showed that loading increased cancellous bone volume fraction and trabecular thickness in normal male mice. In this study, we tested the hypothesis that cyclic mechanical loading of the mouse tibia inhibits orchidectomy (ORX)-associated cancellous bone loss. MATERIALS AND METHODS: Ten-week-old male C57BL/6 mice had in vivo cyclic axial compressive loads applied to one tibia every day, 5 d/wk, for 6 wk after ORX or sham operation. Adaptation of proximal cancellous and diaphyseal cortical bone was characterized by muCT and dynamic histomorphometry. Comparisons were made between loaded and nonloaded contralateral limbs and between the limbs of ORX (n = 10), sham (n = 11), and basal (n = 12) groups and tested by two-factor ANOVA with interaction. RESULTS: Cyclic loading inhibited bone loss after ORX, maintaining absolute bone mass at age-matched sham levels. Relative to sham, ORX resulted in significant loss of cancellous bone volume fraction (-78%) and trabecular number (-35%), increased trabecular separation (67%), no change in trabecular thickness, and smaller loss of diaphyseal cortical properties, consistent with other studies. Proximal cancellous bone volume fraction was greater with loading (ORX: 290%, sham: 68%) than in contralateral nonloaded tibias. Furthermore, trabeculae thickened with loading (ORX: 108%, sham: 48%). Dynamic cancellous bone histomorphometry indicated that loading was associated with greater mineral apposition rates (ORX: 32%, sham: 12%) and smaller percent mineralizing surfaces (ORX: -47%, sham: -39%) in the final week. Loading resulted in greater BMC (ORX: 21%, sham: 15%) and maximum moment of inertia (ORX: 39%, sham: 24%) at the cortical midshaft. CONCLUSIONS: This study shows that cancellous bone mass loss can be prevented by mechanical loading after hormonal compromise and supports further exploration of nonpharmacologic measures to prevent rapid-onset osteopenia and associated fractures.

The effects of RANKL inhibition on fracture healing and bone strength in a mouse model of osteogenesis imperfecta.

Currently, the standard treatment for osteogenesis imperfecta (OI) is bisphosphonate therapy. Recent studies, however, have shown delayed healing of osteotomies in a subset of OI patients treated with such agents. The current study sought to determine the effects of another therapy, RANKL inhibition, on bone healing and bone strength in the growing oim/oim mouse, a model of moderate to severe OI. Mice [73 oim/oim and 69 wild-type (WT)] were injected twice weekly with either soluble murine RANK (RANK-Fc) (1.5 mg/kg) or saline beginning at 6 weeks of age. At 8 weeks of age, the animals underwent transverse mid-diaphyseal osteotomies of the right femur. Therapy was continued until sacrifice at 2, 3, 4, or 6 weeks postfracture. At 6 weeks post-fracture, greater callus area (6.59 +/- 3.78 mm(2) vs. 2.67 +/- 2.05 mm(2), p = 0.003) and increased radiographic intensity (mineral density) (0.48 +/- 0.14 vs. 0.30 +/- 0.80, p = 0.005) were found in the RANK-Fc versus saline oim/oim group, indicating a delay in callus remodeling. Despite this delay, mechanical tests at 6 weeks postfracture revealed no significant differences in whole bone properties of stiffness and failure moment. Further, RANKL inhibition resulted in a greater failure moment and greater work to failure for the nonfractured contralateral WT bones compared to the nonfractured saline WT bones. Together, these results demonstrate that RANKL inhibition does not adversely affect the mechanical properties of healing bone in the oim/oim mice, and is associated with increased strength in intact bone in the WT mice.

Dynamics of an intervertebral disc prosthesis in human cadaveric spines.

Low-back pain is a common, disabling medical condition, and one of the major causes is disc degeneration. Total disc replacements are intended to treat back pain by restoring disc height and re-establishing functional motion and stability at the index level. The objective of this study was to determine the effect on range of motion (ROM) and stiffness after implantation of the ProDisc-L device in comparison to the intact state. Twelve L5-S1 lumbar spine segments were tested in flexion/extension, lateral bending, and axial rotation with axial compressive loads of 600 N and 1,200 N. Specimens were tested in the intact state and after implantation with the ProDisc-L device. ROM was not significantly different in the implanted spines when compared to their intact state in flexion/extension and axial rotation but increased in lateral bending. Increased compressive load did not affect ROM in flexion/extension or axial rotation but did result in decreased ROM in lateral bending and increased stiffness in both intact and implanted spine segments. The ProDisc-L successfully restored or maintained normal spine segment motion.

In vivo cyclic axial compression affects bone healing in the mouse tibia.

Abundant evidence exists that fracture healing can be influenced by mechanical loading. However, the specific loading parameters that are osteogenic remain unknown. We hypothesized that the bone healing response in mouse tibial osteotomies would be different with a short delay before loading compared to immediate load application, as well as with higher and lower load magnitudes applied. Eighty 12-week-old mice underwent osteotomy of the left tibia followed by intramedullary nailing. Mice were divided into six groups based on days delayed until application of load (0 days or 4 days) and amplitude of cyclic load (0.5N, 1N, or 2N). Loading regimens were applied at 1 Hz for 100 cycles per day, 5 days per week for 2 weeks, using an external device that applied axial compression to the tibia. Bone healing was assessed by both microcomputed tomography (CT) and four-point bend testing. A short delay followed by cyclic application of a relatively low load led to improved fracture healing, as determined by increased callus strength, but this enhancement disappeared as load amplitudes increased. Load initiation immediately following fracture inhibited healing, regardless of the magnitude of load applied. MicroCT measurements of calluses in the early healing stage did not predict the mechanical strength of the fractures. These findings confirm that controlled, noninvasive cyclic loading can improve the strength of healing callus. However, application of load immediately after fracture appears to be detrimental to healing. Load magnitude also plays a critical role, and must be taken into account in future studies and clinical applications. As the loading parameters necessary to enhance fracture healing become refined, external compression may be used as a potent stimulus for treating fractures with decreased biological capacity.

Cancellous bone adaptation to in vivo loading in a rabbit model.

Biophysical stimuli are important to the development and maintenance of cancellous bone, but the regulatory mechanisms need to be understood. We investigated the effects of mechanical loading applied in vivo to native cancellous bone in the rabbit on bone formation and trabecular realignment. A novel device was developed to apply controlled compressive loads to cancellous bone in situ. The effect of loading on cancellous bone volume fraction and architecture was quantified. A 4-week experiment was performed in rabbits with devices implanted bilaterally. Cyclic 1 MPa pressures were applied daily to the right limb for 10, 25, or 50 cycles at 0.5 Hz, and the left limb served as the control without any applied loading. Microcomputed tomography and histomorphometry were used to characterize the cancellous tissue within a 4-mm spherical volume located below the loading core. In vivo cyclic loading significantly increased the bone volume fraction, direct trabecular thickness, mean intercept length, and mineral apposition rate in the loaded limbs compared with contralateral limbs. Insufficient evidence was found to demonstrate an effect of number of cycles on the cancellous adaptation between loaded and control limbs. Using a rabbit model, we demonstrated that mechanical loading applied to cancellous bone in situ increased bone formation and altered trabecular morphology. This in vivo model will allow further investigation of cancellous functional adaptation to controlled mechanical stimuli and the influence of mechanical loading parameters, metabolic status, and therapeutic agents.

Assessing the stiffness of spinal fusion in animal models.

The clinical goal of spinal fusion is to reduce motion and the associated pain. Therefore, measuring motion under loading is critical. The purpose of this study was to validate four-point bending as a means to mechanically evaluate simulated fusions in dog and rabbit spines. We hypothesized that this method would be more sensitive than manual palpation and would be able to distinguish unilateral vs bilateral fusion. Spines from four mixed breed dogs and four New Zealand white rabbits were used to simulate posterolateral fusion with polymethyl methacrylate as the fusion mass. We performed manual palpation and nondestructive mechanical testing in four-point bending in four planes of motion: flexion, extension, and right and left bending. This testing protocol was used for each specimen in three fusion modes: intact, unilateral, and bilateral fusion. Under manual palpation, all intact spines were rated as not fused, and all unilateral and bilateral simulated fusions were rated as fused. In four-point bending, dog spines were significantly stiffer after unilateral fusion compared with intact in all directions. Additionally, rabbit spines were stiffer in flexion and left bending after unilateral fusion. All specimens exhibited significant differences between intact and bilateral fusion except the rabbit in extension. For unilateral vs bilateral fusion, significant differences were present for right bending in the dog model and for flexion in the rabbit. Unilateral fusion can provide enough stability to constitute a fused grade by manual palpation but may not provide structural stiffness comparable to bilateral fusion.

DMP1 depletion decreases bone mineralization in vivo: an FTIR imaging analysis.

UNLABELLED: The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype. INTRODUCTION: Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls. MATERIALS AND METHODS: Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (N(total) = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by muCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined. RESULTS: The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on muCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and muCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values. CONCLUSIONS: The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca x P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.

Bone density accumulation is not affected by brace treatment of idiopathic scoliosis in adolescent girls.

Bracing for adolescent scoliosis has been postulated to cause permanent loss of bone mass and to predispose to adult osteoporosis. To determine whether brace use affects the rate of bone accretion with growth, the authors conducted a prospective study of 52 girls with adolescent idiopathic scoliosis. Dual-energy x-ray absorptiometry (DEXA) showed a significant increase in spinal bone mineral density (BMD) over a 1-year period of brace wear. BMD correlated with measures of growth and pubertal status, but not average daily brace wear or severity of scoliosis. The annual rate of bone density accumulation was similar to reported normal values. The annual rate of change of volumetric bone density increased only slightly during the study period, suggesting that most of the change in BMD with time reflects growth in the dimensions of the spine. Brace treatment does not appear to inhibit bone density accumulation in girls with adolescent idiopathic scoliosis.

Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model.

UNLABELLED: To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer was performed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precursors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transfer enhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer. INTRODUCTION: Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 gene transfer was performed to test whether this approach improves osteoblastic differentiation and bone formation compared with single BMP gene transfer. MATERIALS AND METHODS: A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2 (AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus. Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7 was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeks after surgery for radiographic and mechanical fusion, bone formation, and mineralization. RESULTS: BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 and AdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relatively low doses (7-140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12 and MC3T3-E1 cells, that were up to 6- and 40-fold higher, respectively, than levels induced by maximal doses (200-1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 and AdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineralization rate and bone volume in the fusion mass versus single BMP gene transfer (p < 0.02, all comparisons). CONCLUSION: Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.

High-dose alendronate uncouples osteoclast and osteoblast function: a study in a rat spine pseudarthrosis model.

The effect of alendronate on osteoclast and osteoblast function was studied in a novel spine pseudarthrosis model in rats. Sixty-three Sprague-Dawley rats were divided into three groups: control group (saline), therapeutic dose group (1 microg/kg/week), and one-log overdose group (10 microg/kg/week). Animals had L4-L5 posterior intertransverse process fusion with limited bone graft and were sacrificed at 2, 4, and 6 weeks. Manual palpation showed no notable differences among groups. Treatment group radiographic scores were equal to or better than control group scores and were higher than the overdose group at 2 and 6 weeks. Qualitatively, limited histologic remodeling and poor osteoclastic and osteoblastic function were noted in the alendronate treated groups. Quantitative histologic analysis showed fewer osteoclasts in the therapeutic and high-dose groups (p < 0.001). The percent osteoblasts per bone surface area was lower in the high-dose group (p < 0.05). The results suggest that the effect of alendronate was dose dependent and animal model dependent and that supranormal doses of alendronate had a deleterious effect on osteoclastic and osteoblastic function in this model.