PERSPECTIVE: Critical Cooling and Warming Rates as a Function of CPA Concentration.

Cryoprotective agents (CPAs) are routinely applied in cryopreservation protocols to achieve the vitrified state thereby avoiding the damaging effects of ice crystals. Once the CPA has been added, the system needs to cool at a rate ≥ critical cooling rate (CCR) to avoid ice crystallization and successfully enter the vitrified state. Subsequently, upon warming the system needs to meet or exceed a critical warming rate (CWR), often one to two orders of magnitude higher than the CCR, to avoid ice formation and return the system to physiological temperatures for use. Many experimental and theoretical studies have been published on CCRs and CWRs, and correlation for these rates as a function of concentration has been explored for some single component CPAs, but not the CPA cocktails which are commonly used in tissue and organ cryopreservation. In this paper, we summarize the available data of CCRs and CWRs for a variety of CPAs, and suggest a convenient mathematical expression for CCR and CWR that can guide general use for cryoprotective protocol, but also highlights the critical need for further study on CPA cocktails and tissue systems in which CPAs may behave differently and/or may not be fully equilibrated to the loaded CPA.

Clonal chondroprogenitors maintain telomerase activity and Sox9 expression during extended monolayer culture and retain chondrogenic potential.

OBJECTIVE: Articular cartilage contains mesenchymally derived chondroprogenitor cells that have the potential to be used for stem cell therapy. The aim of this study was to characterise the growth kinetics and properties of in vitro expanded cloned chondroprogenitors and determine if critical determinants of the progenitor phenotype were maintained or lost in culture. METHODS: Chondroprogenitors were isolated from immature bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cloned colonies were expanded in vitro up to 50 population doublings (PD). Growth characteristics were assessed by cell counts, analysis of telomere length, telomerase activity, expression of senescence-associated beta-galactosidase activity and real-time quantitative polymerase chain reaction to analyse the gene expression patterns of sox9 and Notch-1 in chondroprogenitors. RESULTS: Cloned chondroprogenitors exhibited exponential growth for the first 20 PD, then slower linear growth with evidence of replicative senescence at later passages. Mean telomere lengths of exponentially growing chondroprogenitors were significantly longer than dedifferentiated chondrocytes that had undergone a similar number of PD (P<0.05). Chondroprogenitors also had 2.6-fold greater telomerase activity. Chondroprogenitors maintained similar sox9 and lower Notch-1 mRNA levels compared to non-clonal dedifferentiated chondrocytes. Chondroprogenitors were induced to differentiate into cartilage in 3D pellet cultures, immunological investigation of sox9, Notch-1, aggrecan and proliferating cell nuclear antigen (PCNA) expression showed evidence of co-ordinated growth and differentiation within the cartilage pellet. CONCLUSION: Clonal chondroprogenitors from immature articular cartilage provide a useful tool to understand progenitor cell biology from the perspective of cartilage repair. Comparisons with more mature progenitor populations may lead to greater understanding in optimising repair strategies.

Effect of intermittent cyclical disodium etidronate therapy on bone mineral density in men with vertebral fractures.

OBJECTIVES: to investigate the effects of oral intermittent cyclical etidronate therapy on bone mineral density (BMD) in men with idiopathic vertebral osteoporosis. DESIGN: consecutive case series. SETTING: regional specialist clinic for metabolic bone disease. SUBJECTS: 42 men aged 35-81 (median 60.5) with established vertebral crush fractures and back pain, in whom secondary causes of osteoporosis had been excluded. INTERVENTION: repeated cycles of treatment with oral disodium etidronate 400 mg daily for 14 days followed by oral calcium 500 mg as citrate daily for 76 days. OUTCOME MEASURES: BMD measurement of the lumbar spine and femoral neck by dual energy x-ray absorptiometry at 6-12-month intervals; bone biochemistry (serum calcium, phosphate, alkaline phosphatase and urine calcium/creatinine and hydroxyproline/creatinine ratios) at 6-month intervals. RESULTS: all 42 men have been treated for more than 18 months, and 35 of them for more than 24 months. Median follow-up for the group as a whole is 31 months (range 18-45). The treatment was well tolerated. BMD at the lumbar spine increased by a mean of 0.024 g/cm2 per year of follow-up (95% confidence interval 0.017-0.032 g/cm2). This is equivalent to an average annual rate of change of 3.2% of baseline values. There was a small, non-significant rise in mean BMD at the hip equivalent to 0.7% of baseline values per year. Serum alkaline phosphatase tended to fall in the first 6 months of treatment, returning to baseline values at 2 years. Serum calcium and phosphate were unchanged and no decrease in urinary calcium/creatinine ratio or hydroxyproline/creatinine ratio was seen. CONCLUSIONS: intermittent cyclical etidronate therapy increased lumbar spine BMD over a 2-year period in an unselected group of men with osteoporotic vertebral fractures. This treatment warrants further evaluation in a randomized controlled trial.

Prebiotic transamination.

Biological amino acids and alpha keto acids directly condense with decarboxylation and transamination to yield product amino acids. This process is closely related to unusual amino acid decarboxylase enzymes in certain microorganisms and may represent a primordial mode of amino acid metabolism.

The effects of gestation dating on the calculation of patient-specific risks in Down's syndrome screening: multivariate case.

When screening for Down's syndrome using biochemical markers, the measurements are adjusted for the gestational age of the fetus because the concentrations of the markers are known to change with gestational age. This adjustment is performed by referring each marker measurement to the population median for that marker for the appropriate estimated gestational age group. The measurement of gestational age is subject to error, whichever method is used, and so the population median used is usually the median of a mixture of distributions for different true gestational ages. Most screening programmes aim for a specific number of weeks and this produces a concentrated distribution of true gestational ages. This fact, combined with dating errors, leads to an asymmetric mixture for each gestational age group and hence to bias in the estimates of the medians. In a previous communication we have shown how the proportions in this mixture distribution can be estimated and how the true medians corresponding to a true gestational age can be estimated. The calculations presented were performed using a single marker, and the details of our method were restricted to this situation. This paper extends the method to the multimarker situation and, as expected, leads to a gain in the detection rate for a specified false positive rate. The true patient-specific risk estimates are again markedly different from the quoted nominal value obtained by ignoring the dating errors. The data set on which the method is illustrated uses two markers, although the technique generalises in an obvious way to more than two.

All MoMs are not equal: some statistical properties associated with reporting results in the form of multiples of the median.

The statistical procedure for discriminating between a Down syndrome or neural tube defect (NTD) fetus and a normal fetus relies, to a great extent, on the reporting of maternal serum alpha-fetoprotein (MSAFP), hCG, and uE3 results in the form of multiples of the median (MoMs). Further, threshold MoMs values for MSAFP, such as 2.5 MoMs, are often used to define a reference range to identify an NTD fetus. We show that a constant threshold-MoMs cutoff for MSAFP values actually refers to different percentiles of MSAFP levels at different gestational ages and that the combining of MoMs values between centers and gestational ages, such as suggested by Wald et al. for deriving a patient-specific risk index, is highly questionable. The results presented in this paper are quite general and will apply to all situations where MoMs are used.