Rodan and Noda (1991) reviewed the models that are used to study osteoblastic gene expression in vitro. These include primary cultures, osteosarcoma derived cell lines, non-transformed cell lines, and experimentally immortalised cell lines. In implantology research, the use of primary cells, especially from the site where implants are to be inserted is ideal. Primary cultures allow better transferability of results obtained in vitro to osteoblasts in vivo, as they are likely to be more representative of the cells in the tissue from which they were derived, and if the cell culture conditions are appropriate, should express patterns of gene expression consistent with those found in vivo (Aufmkolk et a l, 1985).
Primary cultures from bone tissues may be marrow, calvarial, or explant derived, isolated from mature or foetal bone donor sources, including humans (Davies, 1988; Doglioli and Scortecci, 1991; Puleo et al, 1991; Bowers et al., 1992; Chehroudi et al,
1992; Groessner-Schreiber and Tuan, 1992; Swart et al, 1992; Massas et al, 1993; Howlett et al, 1994; Keller et al, 1994; Riccio et al., 1994; Yliheikkilâ et al, 1995; De Santis et al., 1996; Komu et al, 1996; Wilke et al., 1998).
Bone tissues, primarily calvariae, from rat and rabbit are frequently used as a convenient source of bone cells. However, their use may have limitations in their relevance to adult human biology. Tomas et al. (1997) compared the differences in the responses of rat, rabbit and human bone marrow cells to the corrosion products of a cobalt-chromium orthopaedic alloy and metal salts containing cobalt and chromium ions. Growth of human cells was inhibited when exposed to chromium ions, while there was no effect on the growth of rat and rabbit cells. Alkaline phosphatase (AP) activity of rat cultures was also much stronger than the activity of rabbit and human cultures. Although all cultures were able to form mineralised nodules, the nodules in rat cultures were bigger in size than the nodules in human and rabbit cultures.
Besides species variation, primary cultures are also not always available, difficult to establish, and are usually heterogeneous. Because of their ready availability, researchers have used human osteosarcoma derived cell lines, like the MG-63 and SaOS-2 cells (Martin et al, 1995; Gronowicz and McCarthy, 1996; Price et al., 1997). However, there are concerns about the predictability and differentiation state of these cells, and their phenotype may not reflect that of normal osteoblasts. This is a result of the deregulation of growth control which occurs in transformed cell lines, so that they may not follow the pattern of cell contact, proliferation and differentiation operative in normal cells (Lian and Stein, 1992). Malignant and transformed cells are also known to be less adhesive than normal cells, and the lost in anchorage-dependent growth control is one of the major characteristics of tumour cells (Plantefaber and Hynes, 1989).
MG-63 cells have some of the properties of normal human OB cells, as 1,25(0H)2D3 also stimulates their AP and osteocalcin synthesis (Mahonen et al., 1990;
Lajeunesse et al., 1990; Clover and Gowen, 1994). Unlike MG-63 cells, the AP levels in HOS cells were not increased by l,25(OH)2D3, and no osteocalcin was detected in supernatants from HOS cells, even following treatment with l,25(OH)2D3 (Clover and Gowen, 1994). Like HOS cells, the U-2 OS cells also do not produce osteocalcin, with or without the addition of l,25(OH)2D3, and their basal AP level is also not significantly raised by treatment with l,25(OH)2D3 (Mahonen et al, 1990).
The conditions for MG-63, HOS and U-2 OS cells to produce matrix which would mineralise in culture are not known (Hughes and Aubin, 1998b; Lincks et al.,
1998c), although other human osteosarcoma cell lines, like the HOS and SaOS-2 cells, have been reported to do so under appropriate culture conditions (Carvalho et al., 1995; Ahmad et al., 1998). Ahmad et al. (1998) observed mineralisation in SaOS-2 cells grown on metallic discs in culture supplemented with ascorbate, with or without 3 mM P-glycerophosphate. Mineralisation increased with time over a six week period and was based on the expression of the non-collagenous proteins, bone sialoprotein and osteocalcin, in addition to the presence of mineralised nodules. However, it was unclear if true calcification had occurred. Although calcification on the Ti alloy used was confined to the nodules formed, calcification on the cobalt-chromium alloy was diffuse across the surface of the discs. This may indicate dystrophic, rather than physiological calcification, as no microanalysis of the nodules formed was carried out to determine their precise mineral composition.
As an alternative to the use of human osteosarcoma cell lines, experimentally immortalised cell lines of human origin could be used. An example is the SV40 conditionally transformed adult osteoblastic cell line, HOB-02-C1 (Bodine et al., 1996), which exhibits a transformed phenotype under specific culture conditions, but will revert to a normal phenotype when these conditions are modified. This would allow homogeneous cell populations to be maintained, and terminal differentiation of the cells could also be assessed on biomaterials, as the extracellular matrix (ECM) would mineralise in culture. A search of the literature did not reveal that such a cell line of human origin has been used in cell-biomaterial interaction studies.
The purpose of this part of the study was to compare the adhesion and differentiation of OB cells cultured on commercially pure titanium (cpTi) and zirconium (cpZr) surfaces with different roughnesses in vitro. Although there have been several studies reporting in vitro responses of different cells to various implant and orthopaedic materials, there have been no published reports of studies comparing the effects of material surface properties on the three human OB cell lines used in this study (MG-63, HOS and U-2 OS). Even though transformed cell lines may not be representative of normal cells, these cells are suitable as osteoblastic models for this study as two of the cell lines (MG-63 and HOS), exhibit some of the normal OB cell behaviour in culture, particularly their adhesion properties and integrin expression (Clover and Gowen, 1994). MG-63 cells have been used in previous investigations of cell-biomaterial reactions (Martin et al., 1995; Price et al., 1997), while HOS and U-2 OS cells have not been used in any published reports of this kind.
3.2
Materials and Methods
3.2.1 Cell culture