The progress of early phase bone healing using porous granules produced from calcium phosphate cement.

OBJECTIVE: Bone grafting is a vital component in many surgical procedures to facilitate the repair of bone defects or fusions. Autologous bone has been the gold standard to date in spite of associated donor-site morbidity and the limited amount of available donor bone. The aim of this study was to investigate the progress of bone regeneration and material degradation of calcium phosphate granules (CPG) produced from a calcium phosphate self-setting cement powder compared to the use of autologous bone grafting in the treatment of "critical size defects" on load-bearing long bones of minipigs. METHODS: A critical size defect in the tibial metaphysis of 16 mini-pigs was filled either with autologous cancellous graft or with micro- and macroporous carbonated, apatic calcium phosphate granules (CPG) produced from a calcium phosphate self-setting cement powder. After 6 weeks, the specimens were assessed by X-ray and histological evaluation. The amount of new bone formation was analysed histomorphometrically. RESULTS: The semi-quantitative analysis of the radiological results showed a complete osseous bridging of the defect in three cases for the autograft group. In the same group five animals showed a beginning, but still incomplete bridging of the defect, whereas in the CPG group just two animals developed this. All other animals of the CPG group showed only a still discontinuous new bone formation. Altogether, radiologically a better osseous bridging was observed in the autograft group compared to the CPG group. Histomorphometrical analysis after six weeks of healing revealed that the area of new bone was significantly greater in the autograft group concerning the central area of the defect zone (p<0.001) as well as the cortical defect zone (p<0.002). All defects showed new bone formation, but only in the autograft group defects regenerated entirely. CONCLUSIONS: Within the limits of the present study it could be demonstrated that autologous cancellous grafts lead to a significantly better bone regeneration compared to the application of calcium phosphate granules (CPG) produced from a calcium phosphate self-setting cement powder after 6 weeks. In the early phase of bone-healing, the sole application of CPG appears to be inferior to the autologous cancellous grafts in an in vivo critical size defect on load-bearing long bones of mini-pigs.

Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase.

BACKGROUND: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who possess a certain type of clinical analyzer. We describe a two-point manual method for CB that could be adapted as a rate assay to automated clinical analyzers. METHODS: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum. RESULTS: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized and delta-bilirubin is not oxidized at all. Results obtained with the bilirubin oxidase method agreed well with those obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from healthy adults, were 0.0-1.2 mg/L, with a mean value of 0.2 mg/L. CONCLUSIONS: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Effects of substrate concentration on results of determination of prostatic acid phosphatase with thymolphthalein monophosphate.

The relation between concentration of thymolphthalein monophosphate substrate and catalytic activity was investigated for the determination of prostatic acid phosphatase. This study, an extension of previously reported work (Clin. Chem. 27: 1372, 1981), shows that lot-to-lot variation in purity of thymolphthalein monophosphate preparations is reflected in substrate-velocity curves. Plateau regions in these curves at 1.5-2.5 g/L result from the combined effects of (a) substrate concentrations that are an order of magnitude below Km and (b) a further decrease in available substrate caused by formation of substrate aggregates in the presence of serum. To simplify the identification of superior lots of thymolphthalein monophosphate, we give a mixed-substrate protocol for testing different lots.

Response monitoring and action limits: use of ADC numbers in understanding the operational characteristics of the Beckman ASTRA chemistry system.

The Beckman ASTRA is a microprocessor-controlled multichannel chemistry analyzer. The output of each module is available to the operator as analog-to-digital conversion (ADC) numbers, which we record during each calibration (Response Monitoring). After so studying three instruments for a total of 33 months, we have established limits for Calibrator ADC numbers that indicate possible operator action (Action Limits). These Action Limits are tighter than the microprocessor's programmed limits, and alert the operator to short- and long-term drift. These tighter limits warn of (a) impending failure of the instrument to calibrate or (b) possible inaccuracies in results for patients. We have instituted changes in preventive maintenance based on our studies of each module's operational characteristics, and have replaced electrodes that failed to meet our Response Monitoring specifications. Response Monitoring and Action Limits based upon ADC numbers have significantly enhanced our understanding of the ASTRA system and thus improved its operational efficiency and analytical reliability. Estimates of precision and accuracy (true value) were satisfactory in comparison to our prior single-channel continuous-flow and flame photometry analytical measurement systems.

Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes.

Isolated myocytes were prepared from adult canine hearts using a combined technique of myocardial perfusion followed by incubation with collagenase. More than 60% of the cells routinely excluded trypan blue dye. Disruption of the myocytes was accomplished using high pressure nitrogen cavitation. After differential and sucrose gradient centrifugation, the peak sarcolemmal fraction averaged 100-fold enrichment in ouabain-inhibited K+-stimulated p-nitrophenyl phosphatase and 82-fold in ouabain-inhibited (Na+,K+)-ATPase. These sarcolemmal membranes are enriched in phospholipid phosphorus (1.98 mumol/mg of protein) and more than 4-fold in sphingomyelin and cholesterol. Polyacrylamide gels revealed three major protein peaks at 50,000, 91,000, and 140,000 apparent molecular weights. This work demonstrates the feasibility of preparing highly pure cardiac sarcolemma from isolated adult myocytes. The problem of cellular cross-contamination due to heterogeneity of cell types in whole myocardial tissue has been circumvented. The level of enrichment exceeds all reported preparations of cardiac sarcolemma from whole myocardium and cultured myocytes. This preparation should prove to be useful as an in vitro model for studies of physiological, pharmacological, and pathological perturbations of sarcolemmal structure and function.