Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: Phenotypic and genotypic responses observed in vitro

Abstract

Time-dependent phenotypic response of a model osteoblast cell line (hFOB 1.19, ATCC, and CRL-11372) to substrata with varying surface chemistry and topography is reviewed within the context of extant cell-adhesion theory. Cell-attachment and proliferation kinetics are compared using morphology as a leading indicator of cell phenotype. Expression of (alpha2, alpha3, alpha4, alpha5, alphav, beta1, and beta3) integrins, vinculin, as well as secretion of osteopontin (OP) and type I collagen (Col I) supplement this visual assessment of hFOB growth. It is concluded that significant cell-adhesion events-contact, attachment, spreading, and proliferation-are similar on all surfaces, independent of substratum surface chemistry/energy. However, this sequence of events is significantly delayed and attenuated on hydrophobic (poorly water-wettable) surfaces exhibiting characteristically low-attachment efficiency and long induction periods before cells engage in an exponential-growth phase. Results suggest that a 'time-cell-substratum-compatibility-superposition principle' is at work wherein similar bioadhesive outcomes can be ultimately achieved on all surface types with varying hydrophilicity, but the time required to arrive at this outcome increases with decreasing cell-substratum-compatibility. Genomic and proteomic tools offer unprecedented opportunity to directly measure changes in the cellular machinery that lead to observed cell responses to different materials. But for the purpose of measuring structure-property relationships that can guide biomaterial development, genomic/proteomic tools should be applied early in the adhesion/spreading process before cells have an opportunity to significantly remodel the cell-substratum interface, effectively erasing cause and effect relationships between cell-substratum-compatibility and substratum properties. IMPACT STATEMENT: This review quantifies relationships among cell phenotype, substratum surface chemistry/energy, topography, and cell-substratum contact time for the model osteoblast cell line hFOB 1.19, revealing that genomic/proteomic tools are most useful in the pursuit of understanding cell adhesion if applied early in the adhesion/spreading process.

Figure 1

Different approaches to bioadhesion with some archetypical literature citations. Each of these approaches effectively probe different phases of bioadhesion and describe cell adhesion in different terms.

Figure 2

Schematic illustration of cell adhesion and proliferation kinetics identifying quantitative parameters that can be extracted from measurement of number of attached cells (expressed here as percentage of (viable) cell inoculum; %I) with time. %I_max is the maximum percentage of a cell inoculum that adheres to a surface from a sessile cell suspension and t_1/2 measures half-time to %I_max. The proliferation rate (k) and cell-number doubling time (t_d) measure viability of attached cells (adapted from ref. [60]).

Figure 3

Correlation of % I _max (see Fig. 2) with substrata surface energy for hFOB. Surface energy is here measured by water adhesion tension ${\tau }^o={\gamma }_{lv}^{o}cos\theta$, where ${\gamma }_{lv}^o=72.8\text{dyne}/\text{cm}$ at 20° C for pure water and θ is the angle subtended by a water droplet on the surface understudy (advancing θ = filled symbols, receding θ = open symbols; adapted from ref. [60]). Error bar represents standard deviation (N ≥3). Trend-line through advancing and receding data is guide to the eye; ▲= TCPS; ▼;= BGPS (bacteriological grade polystyrene); ● = glass; ■= quartz; ◆ = PTPS (plasma-treated polystyrene); ⬢ = biodegradable polymers of PLGA 5/5 (M_n = 80 k), PLGA 7/3 (M_n = 96 k), PLA (M_n = 160 k), PCL (M_n = 80 k), PLCL 7/3 (M_n = 82 k), PLGCL 2.5/2.5/5 (M_n = 60 k), PLGCL 3.5/3.5/3 (M_n = 54 k). M_n = number-average molecular weight by GPC. PLGA = poly(lactide-co-glycolide); PLCL = poly(lactide-co-caprolactone); PLGCL = poly(lactide-co-glycolide-co-caprolactone). See ref. [60] for details on materials preparation and characterization.

Figure 4

Correlation of cell proliferation rate constant k with substratum surface energy for hFOB. Surface energy is here measured by water adhesion tension ${\tau }^o={\gamma }_{lv}^{o}cos\theta$, where ${\gamma }_{lv}^o=72.8\text{dyne}/\text{cm}$ at 20° C for pure water and θ is the angle subtended by a water droplet on the surface understudy (advancing θ = filled symbols, receding θ = open symbols; adapted from ref. [60]). Error bars represent standard deviation of N ≥ 3. Trend-line through advancing and receding data is guide to the eye. Material identification is the same as in Fig. 3.

Figure 5

Variation in hFOB morphology on different surfaces after 4, 24, 48, and 96 hours of culture as assessed by SEM (hydrophilic surfaces: PTQ = plasma-treated quartz, PTG = plasma-treated glass, TCPS = tissue-culture grade polystyrene; hydrophobic surfaces: BGPS = bacteriological grade polystyrene, STQ = silane-treated quartz). Scale bar = 10 μm. Note that variation in cell morphology abates with time, especially for hydrophobic specimens.

Figure 6

Variation in hFOB shape on plasma-treated glass (PTG) after 4 hours culture as assessed by SEM showing widely-varying morphological response to apparently cytotoxic SiO_x glass. Scale bar = 10 μm.

Figure 7

Dimensional analysis by image analysis (Image J, NIH) of Coomassie-blue-stained hFOB cultured on hydrophilic surfaces (PTQ = plasma-treated quartz, TCPS = tissue-culture grade polystyrene) and hydrophobic surfaces (STQ = silane-treated quartz) for 24 hours. Statistical significance indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).

Figure 8

Actin, vinculin, and composite immunofluorescent images (400X) of hFOB cultured for 3 and 24 hours on hydrophilic substratum (PTQ = plasma-treated quartz, TCPS = tissue-culture grade polystyrene) and hydrophobic surfaces (STQ = silane-treated quartz).

Figure 9

Short- and long-term growth dynamics of hFOB on tissue-culture grade polystyrene (TCPS) spanning 30 days in continuous culture without subculture. Inset expands short-term attachment rates using a linear ordinate.

Figure 10

Variation in long-term hFOB morphology on tissue-culture grade polystyrene (TCPS) assessed by cross-sectional TEM (Panels A: Scale bar = 5 μm; B: Scale bar = 10 μm) showing formation of multiple cell layers. Note that apoptotic bodies were clearly evident after 30 days of culture. Apoptotic cells (green) among normal cells (red, Sytox Orange) visualized using confocal microscopy confirms an increase in apoptosis with culture age (Panels C, D: Scale bar = 50 μm). Percent apoptotic bodies noted in lower right of Panels C,D were estimated by image analysis (see ref. [112] for experimental details).