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A Role for Proteoglycans in Mineralized Tissue-Titanium Adhesion

Laboratory for Bone and Implant Sciences (LBIS), The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, 10833 Le Conte Avenue (B3-081 CHS), Box 951668, Los Angeles, California 90095-1668, USA

View larger version (42K): [in this window] [in a new window]   Figure 1. Expression of bone-related proteoglycan genes in the bone-marrow-derived osteoblastic cultures analyzed by reverse-transcriptase/polymerase chain-reaction (RT-PCR). The osteoblastic cells were cultured on either a polystyrene dish, a machined titanium surface, or an acid-etched titanium surface. (A) A representative PCR analysis, at multiple time-points of culture, visualized on a 1.5% agarose gel with ethidium bromide staining. (B) The expression level at multiple time-points normalized to GAPDH mRNA expression.

View larger version (88K): [in this window] [in a new window]   Figure 2. Immunochemical localization of various glycosaminoglycans (GAGs): chondroitin sulfate for A, B, C; keratan sulfate for D, E; heparan sulfate for F, G; dermatan sulfate for H, I. The bone-marrow-derived osteoblastic cells were cultured for 28 days on the titanium-coated polystyrene (A,B,D,F,H) or cell-culture-grade polystyrene (C,E,G,I). Ti, titanium; P, polystyrene. See text for the details of the histological preparation, immunostaining, and titanium coating. Bar is 20 µm.

View larger version (24K): [in this window] [in a new window]   Figure 3. Hardness and elastic modulus of the day 24 osteoblastic mineralized tissue obtained from nano-indentation. The mineralized tissue was formed after culture on either the polystyrene, the machined titanium, or the acid-etched titanium surface. From days 21 to 24, the mineralized tissues were treated with the following glycosaminoglycan (GAG) degrading enzyme: ChAC (chondroitinase AC); ChB (chondroitinase B); He (heparinase); Ke (keratanase). Control: untreated control culture. Data are shown as the mean ± SD (n = 9).

View larger version (67K): [in this window] [in a new window]   Figure 4. Results of nano-scratch tests performed on the day 24 mineralized tissue specimens. (A) An optical micrograph of the scratch performed on the machined titanium with no enzyme treatment. Bar is 50 µm. (B,C) Serial SEM images depicting a scratch path near a determined point of delamination (black line in panel C), which was performed on the non-enzyme-treated machined titanium. Tissue peeling away from the substrate is vividly observed (white arrowheads in panel C). Bar is 10 µm. EDS elemental analyses immediately before (D for spot d in panel B) and after (E for spot e in panel C) the point of delamination. (F,G,H) Serial SEM images depicting a scratch path near a point of delamination (black line in panel C), which was performed on the non-enzyme-treated acid-etched titanium. Tissue peeling away from the substrate can be seen (white arrowheads in panel H). Bar is 10 µm. EDS elemental analyses immediately before (I for spot i in panel H) and after (J for spot j in panel H) the determined point of delamination. (K) Critical load values of the tissue cultured on either polystyrene, machined titanium, or acid-etched titanium, with or without a glycosaminoglycan-degrading enzyme (either of ChAC [chondroitinase AC], ChB [chondroitinase B], He [heparinase], or Ke [keratanase]). Control tissue was not treated with enzyme. Data are shown as the mean ± SD (n = 9).