A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions:importance of fixative osmolarity for maintaining morphology

The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a ‘physiological’ level of ~280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids (~280mOsm). These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues

Synthesis, characterization, and cytocompatibility of potential cockle shell aragonite nanocrystals for osteoporosis therapy and hormonal delivery

Alhaji Zubair Jaji,1,2 Md Zuki Bin Abu Bakar,1,3 Rozi Mahmud,4 Mohamad Yusof Loqman,5 Mohamad Noor Mohamad Hezmee,1 Tijani Isa,3 Fu Wenliang,3 Nahidah Ibrahim Hammadi1 1Department of Veterinary Pre-Clinical Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 2Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Ilorin, Ilorin, Kwara, Nigeria; 3Molecular Biomedicine Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 4Department of Imaging, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 5Department of Companion Animal Medicine and Surgery, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Abstract: Calcium carbonate is a porous inorganic nanomaterial with huge potential in biomedical applications and controlled drug delivery. This study aimed at evaluating the physicochemical properties and in vitro efficacy and safety of cockle shell aragonite calcium carbonate nanocrystals (ANC) as a potential therapeutic and hormonal delivery vehicle for osteoporosis management. Free and human recombinant parathyroid hormone 1-34 (PTH 1-34)-loaded cockle shell aragonite calcium carbonate nanocrystals (PTH-ANC) were synthesized and evaluated using standard procedures. Transmission electron microscopy and field emission scanning electron microscopy results demonstrated highly homogenized spherical-shaped aragonite nanocrystals of 30±5 nm diameter. PTH-ANC had a zeta potential of −27.6 ± 8.9 mV. The encapsulation efficiency of the formulation was found to be directly proportional to the concentrations of the drug fed. The X-ray diffraction patterns revealed strong crystallizations with no positional change of peaks before and after PTH-ANC synthesis. Fourier transform infrared spectroscopy demonstrated no detectable interactions between micron-sized aragonite and surfactant at molecular level. PTH-ANC formulation was stabilized at pH 7.5, enabling sustained slow release of PTH 1-34 for 168 h (1 week). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytocompatibility assay in Human Foetal Osteoblast Cell Line hFOB 1.19 showed that ANC can safely support osteoblast proliferation up to 48 h whereas PTH-ANC can safely support the proliferation at 72 h and beyond due to the sustained slow release of PTH 1-34. It was concluded that due to its biogenic nature, ANC is a cytocompatible antiosteoporotic agent. It doubles as a nanocarrier for the enhancement of efficacy and safety of the bone anabolic PTH 1-34. ANC is expected to reduce the cost, dosage, and dose frequency associated with the use of PTH 1-34 management of primary and secondary forms of osteoporosis. Keywords: bone, FESEM, FT-IR, MTT viability, PTH 1-34, sustained release, TEM, XRD, zeta potentia