Is Use of BMP-2 Associated with Tumor Growth and Osteoblastic Differentiation in Murine Models of Osteosarcoma?

BackgroundThe putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent.Questions/purposes(1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma.MethodsIn this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H₂O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation.ResultsConstitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm³ versus 1316 ± 387.4 mm³, mean difference 1009 mm³ [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm³ versus 326.1 ± 72.8 mm³, mean difference 326.1 mm³ [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10⁸ ± 8.3x10⁸ photons/s versus 9.3 x 10⁷ ± 1.5 x 10⁸ photons/s, mean difference 8.6 x 10⁸ photons/s [95% CI 5.1 x 10⁸ to 1.2 x 10⁹]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm³ versus 1335 ± 102.7 mm³, mean difference 840.3 mm³ [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions.ConclusionsAs demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted.Clinical RelevanceOur results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma.     

Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Hyperdiploidy (greater than 50 chromosomes, or a DNA index greater than 1.16) confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) in vitro experience a better event-free survival than those whose lymphoblasts do not (Blood 76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (greater than 50 chromosomes) accumulated high levels of MTX-PGs (1,095 and 571 to 2,346 pmol/10(9) cells [median and 25% to 75% intraquartile range]). These levels were higher than those in B-lineage lymphoblasts from 19 children with other aneuploidy (326 and 159 to 775 pmol/10(9) cells) and 15 children with diploidy (393 and 204 to 571 pmol/10(9) cells) (P = .0015). Chromosomal trisomies in hyperdiploid cases were highly nonrandom. Chromosome 9 was not one of the chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis. The correlation between MTXPG level and percentage of S-phase cells was weak, suggesting that increased levels of MTXPGs could not be attributed to elevated proportions of cells in active DNA synthesis. The ability of hyperdiploid lymphoblasts to accumulate high levels of MTXPGs may increase their sensitivity to MTX cytotoxicity, accounting in part for the improved outlook for hyperdiploid patients treated with regimens that emphasize MTX as a primary component of continuation therapy.     

Public-private partnerships: the Connecticut model for financing long-term care.

Responses to the growing crisis in long-term care financing have included efforts to negotiate partnerships between the private and public sectors for the purpose of developing innovative models for long-term care insurance. One such set of models has been encouraged by support from the Robert Wood Johnson Foundation's "Long Term Care Insurance Program" grants. The Connecticut Partnership for Long Term Care uses a cooperative approach to encourage the development of private sector long-term care insurance products that are integrated with Medicaid eligibility determinations. The Connecticut model is described, accompanied by a history of its development, and a comparison is made with other models currently under consideration by national policy analysts.     

Awareness of and attitudes towards infertility and its treatment： a cross-sectional survey of men in a United States primary care population

Previous studies have described racial and socioeconomic disparities in the treatment of infertility. Patient factors such as attitudes and awareness may be contributing factors. Since primary care is often the setting that serves as an entry into other areas of medicine, we sought to evaluate men＇s attitudes and awareness of male infertility in the primary care setting. To do this, we performed a cross-sectional survey of men＇s attitudes toward men＇s health issues in 210 men from two primary care clinic waiting rooms in Atlanta, Georgia. The survey was self-administered with closed-ended question items and was approximately 20 min in length. Of the 310 men approached, 210 agreed to participate and returned completed surveys. Overall, 52% of men said they were ＂very＂ or ＂somewhat＂ familiar with infertility and 25% were familiar with treatments for infertility. Some men had heard of surgery （21%） and medication （35%） as treatments for male infertility. Awareness and familiarity with the condition was greater in high socioeconomic status men （i.e. college graduates or those with income 〉$100 k per year） but did not differ by race on multivariate analysis. Attitudes toward infertility varied by race with non-Caucasian men being more likely to indicate that infertility is a serious condition, to be concerned about infertility, and to believe it decreases a man＇s quality-of-life. Therefore, a lack of awareness, but not negative attitudes, may contribute to previously-described disparities in the treatment of infertility.     

Minimum depths to essential structures in a UK military population using computed tomography: application to stab-resistant body armour

International Journal of Legal Medicine - Edged weapons are a known domestic threat to the police forces of the UK. This threat is mitigated by wearing stab-resistant body armour that is either...     

Heated humidified high-flow nasal cannula versus low-flow nasal cannula as weaning mode from nasal CPAP in infants ≤28 weeks of gestation

Despite the paucity of evidence, the practice of weaning nasal continuous positive airway pressure (NCPAP) is widespread. However, the most clinically effective non-invasive ventilatory support strategy remains to be determined. We compared the outcome of very premature infants with respiratory distress syndrome treated with a combination of NCPAP and heated humidified high-flow nasal cannula (HHFNC) versus NCPAP and low-flow nasal cannula (LFNC). Between 2004 and 2008, patients ≤28 weeks of gestation and <1,250 g of birth weight were treated with NCPAP + HHFNC or NCPAP + LFNC. Their respiratory and non-respiratory outcome including cost-effectiveness was compared after matching for antenatal steroid doses, mode of delivery, birth plurality, gestational age, birth weight, gender, surfactant doses, length of mechanical ventilation and clinical risk index for babies-II (CRIB-II) score. Thirty-nine infants received HHFNC + NCPAP, and 40 received NCPAP + LFNC. Median gestational age and birth weight were 27 weeks and 930 g and 27 weeks and 980 g, respectively. The total number of NCPAP days was significantly reduced by 50 % in the HHFNC group. Thirteen percent of the patients on NCPAP suffered from nasal bridge lesions compared to none on HHFNC. Respiratory and non-respiratory outcome was not significantly different otherwise. Combination of NCPAP and HHFNC reduced costs by 33 %. Conclusions: HHFNC shortens NCPAP time without increasing overall length of non-invasive respiratory support in very preterm infants. Unlike NCPAP, HHFNC does not seem to increase the risk of nasal trauma and appears to improve cost-effectiveness whilst producing otherwise equal respiratory and non-respiratory outcomes.