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A novel method was developed for calcium deficient hydroxyapatite (CDHA) scaffold 3D printing, through which a bilayer scaffold was fabricated by the integral molding of individual CDHA and poly(lactic-glycolic acid copolymer) (PLGA). The hydration reaction of α-tricalcium phosphate (TCP) was utilized to form CDHA, and a mixed solution of gelatin, glycerine and glutaraldehyde was applied as the dispersant and adhesive. The concentration of the glutaraldehyde (1‰(v/v)) and the mixing ratio of α-TCP (0.6, 0.8, 1.0 and 1.2 g ml−1) were studied with regard to the effect on the forming ability of the CDHA ink. The influence of α-TCP proportion (0.6, 0.8, 1.0 and 1.2 g ml−1) on the formation of CDHA was also researched in phase analysis, morphology and compressive strength measurements. The CDHA/PLGA bilayer scaffold was fabricated with a good combination of the two components by 3D printing. The in vitro degradation, cytotoxicity and cell proliferation behavior were studied. Meanwhile, the in-vivo performances in terms of surgical safety, biodegradation and osteogenic capacity were investigated with a cortical bone defect model in a rabbit femur. The results showed that the CDHA/PLGA bilayer scaffold had excellent biocompatibility and no cytotoxicity. The scaffolds were successfully implanted and presented remarkable osteogenic capacity within 6 months through analyses in radiography and histology. In conclusion, the method has a potential clinical application in diverse bone repair practices by varied 3D-printing fabrication.

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reversephase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS. (Hypertens Res 2000; 23 Suppl: S21-S27)