Is macroporosity absolutely required for preliminary in vitro bone biomaterial study? A comparison between porous materials and flat materials

Abstract

Porous materials are highly preferred for bone tissue engineering due to space for blood vessel ingrowth, but this may introduce extra experimental variations because of the difficulty in precise control of porosity. In order to decide whether it is absolutely necessary to use porous materials in in vitro comparative osteogenesis study of materials with different chemistries, we carried out osteoinductivity study using C3H/10T1/2 cells, pluripotent mesenchymal stem cells (MSCs), on seven material types: hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and b-tricalcium phosphate (β-TCP) in both porous and dense forms and tissue culture plastic. For all materials under test, dense materials give higher alkaline phosphatase gene (Alp) expression compared with porous materials. In addition, the cell density effects on the 10T1/2 cells were assessed through alkaline phosphatase protein (ALP) enzymatic assay. The ALP expression was higher for higher initial cell plating density and this explains the greater osteoinductivity of dense materials compared with porous materials for in vitro study as porous materials would have higher surface area. On the other hand, the same trend of Alp mRNA level (HA > β-TCP > α-TCP) was observed for both porous and dense materials, validating the use of dense flat materials for comparative study of materials with different chemistries for more reliable comparison when well-defined porous materials are not available. The avoidance of porosity variation would probably facilitate more reproducible results. This study does not suggest porosity is not required for experiments related to bone regeneration application, but emphasizes that there is often a tradeoff between higher clinical relevance, and less variation in a less complex set up, which facilitates a statistically significant conclusion. Technically, we also show that the base of normalization for ALP activity may influence the conclusion and there may be ALP activity from serum, necessitating the inclusion of "no cell" control in ALP activity assay with materials. These explain the opposite conclusions drawn by different groups on the effect of porosity.

Figure 1

XRD spectra of porous and dense: (a) hydroxyapatite, HA; (b) α-TCP and (c) β-TCP discs; and (d) the corresponding matches from the database (the spots on the spectrum indicate the positions of the five most intense peaks from the powder X-ray data of the database).

Figure 2

Surface topography of porous materials and dense materials (HA, α-TCP and β-TCP) revealed by SEM at 30× and 5000×.

Figure 3

Relative quantification of gene expression of Alp normalized with β-Actin of 10T1/2 cells seeded on HA, α-TCP, β-TCP and plastic after 6 days of culture (n = 5, technical duplicate in batch 1 and triplicate in batch 2; mean ± SEM; ** p < 0.01, ⁺ p < 0.1).

Figure 4

Effect of cell plating density on ALP expression and total protein amount of 10T1/2 cells seeded on a plastic culture plate (a) ALP enzymatic activity estimated from p-NPP assay after 2 and 7 days of culture, with background subtraction using values obtained in the “no cell” control (n = 3 wells; mean ± SD; ⁺ p < 0.1, * p < 0.05, ** p < 0.005); (b) Protein amount after 2, 5 and 7 days of culture estimated from Bradford assay (n = 3 wells; mean ± SD; ⁺ p < 0.1, * p < 0.05, ** p < 0.005); (c) Phase contrast photos of 10T1/2 cells seeded on a 96-well plastic culture plate with different plating densities and photos captured the day before the assays (i.e., at day 1, 4 and 6) (scale bar =50 μm).

Figure 5

(a) 10T1/2 cells viewed under a fluorescent microscope with DAPI staining the nuclei of cells cultured for 3 days; (b) SEM photos of 10T1/2 cells cultured on porous HA for 1 week; SEM photos of (c) pore surface at 1,000×; (d) pore surface at 10,000×; (e) pore edges at 1,000× and (f) pore edges at 10,000×.

Figure 6

ALP activity of cells on materials detected using ALP enzymatic assay (9 × 10⁴ cells plated per well of a 48-well plate, assayed after 3 days of culture; n = 2; The experiment was repeated and the high background ALP activity detected for the “no cell” control of porous materials was consistently observed in the repeated experiments).

Figure 7

Schematic showing the relation between porosity, surface area and cell density.

Figure 8

Factors involved in deciding scaffold type.

Figure A1

ALP activity of different dilution of cell culture medium (2.5 μL, 5 μL and 10 μL) of different serum concentration (0%, 5% and 10% FBS) with PBS as the reference.

Figure A2

(a) Different causes of ALP activity elevation; (b) Events of a mammalian cell cycle; (c) Relations between cell density and the bases of normalization (area coverage, protein amount, cell number and DNA amount).