Characterization of mesoporous calcium phosphates from calcareous marine sediments containing Si, Sr and Zn for bone tissue engineering.

Calcium phosphates (CAPs) can be produced from either biologically sourced materials or mineral deposits. The raw materials impart unique properties to the CAPs due to innate trace amounts of elements that affect the crystal structure, morphology and stoichiometry. Using calcium carbonate (CaCO3) precursors derived from fossilized calcareous marine sediments (FCMSs), we have synthesized a novel class of CAP biomaterials, termed fm-CaPs, with defined Ca/P molar ratios of 1.4 and 1.7 using a wet synthesis method. Compared with commercially available CAP biomaterials, such as hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP), fm-CaP1.7 had a biphasic composition consisting of an HA phase (in a hexagonal system) and a ß-TCP phase (in a rhombohedral crystalline system), which is desirable for the current design of bone substitutes, whereas fm-CaP1.4 consisted of an HA phase and a beta-dicalcium pyrophosphate phase (in a tetragonal system). These bioceramics exhibited a fringe structure of regular crystallographic orientation with well-ordered mesoporous channels. The FCMS raw material imparted trace amounts of silicon (Si), strontium (Sr) and zinc (Zn) to fm-CaPs; these are elements that are important for bone formation. The cyto-compatibility of these biomaterials and their effects on cellular activity were evaluated using osteoblast cells. Cell proliferation assays revealed no signs of cytotoxicity, whereas cells growth was equal to or better than HA and ß-TCP controls. The SEM analysis of the cell and material interactions showed good cell spreading on the fm-CaP materials that was comparable to ß-TCP and in vitro assays suggested robust osteogenic differentiation, as seen by increased mineralization (alizarin red) and upregulation of osteogenic gene expression. Our results indicate that fm-CaP1.7, in particular, has chemical, physical and morphological properties that make this material suitable for applications that promote bone tissue regeneration.

Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences.

Mandibular osteoblasts originate from the neural crest and deposit bone intramembranously, mesoderm derived tibial osteoblasts by endochondral mechanisms. Bone synthesized by both cell types is identical in structure, yet functional differences between the two cell types may exist. Thus, both matched juvenile and adult mandibular and tibial osteoblasts were studied regarding their proliferative capacity, their osteogenic potential and the expression of osteogenic and origin related marker genes. Juvenile tibial cells proliferated at the highest rate while juvenile mandibular cells exhibited higher ALP activity depositing more mineralized matrix. Expression of Hoxa4 in tibial cells verified their mesodermal origin, whereas very low levels in mandibular cells confirmed their ectodermal descent. Distinct differences in the expression pattern of bone development related genes (collagen type I, osteonectin, osteocalcin, Runx2, MSX1/2, TGF-ß1, BAMBI, TWIST1, ß-catenin) were found between the different cell types. The distinct dissimilarities in proliferation, alkaline phosphatase activity, the expression of characteristic genes, and mineralization may aid to explain the differences in bone healing time observed in mandibular bone when compared to long bones of the extremities.

A novel xylene substitute for histotechnology and histochemistry.

Propylene glycol methyl ether (PGME) exhibits excellent solvent and coupling properties. A toxicity database provided evidence suggesting that PGME might be a useful substitute for xylene in histotechnology and histochemistry applications. Tissue specimens were fixed, cleared in either PGME or xylene, embedded in paraffin wax, then dewaxed in either PGME or xylene. Sections were treated with the following stains: hematoxylin & eosin (H & E), three special stains of the Gordon/Sweet silver staining method, PAS, and Masson's trichrome, and immunostains including actin, CD3, CD34, CK, CK7/CK9, Ki-67, and ER/PR. The sections were mounted in a resinous medium consisting of PGME and pinene copolymer, then examined under a microscope. Variables such as water tolerance, dimension change, organic solvency, and anti-fading efficacy also were assessed. Depending on the application, PGME performance was equal to or exceeded that of xylene. PGME provided better optical clarity and nuclear detail, did not harden the tissue samples, conserved tissue antigenicity, and was amenable to resinous mounting. Tissues not dehydrated with absolute ethanol also were processed properly. Tissues treated with PGME did not warp or contract compared to those treated with xylene (p < 0.0001). PGME, however, exhibited less organic solvency than xylene. There was no discernible change in the colors of stains in sections processed with PGME even after storage for two years. These results suggest that PGME is a novel xylene substitute for applications in histotechnology and histochemistry.