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

Juliana T Y Lee,Wai-Hung Tsang,Kefeng Wang,King L Chow

doi:10.3390/jfb2040308

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 β-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.

Highlights

It has been widely accepted that the porosity or pore size of materials influence the cell behavior and the osteogenesis process [1]
The results in this study show that the cells on dense flat materials have higher alkaline phosphatase gene (Alp) expression compared with porous materials for HA, α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) (Figure 3)
Porous materials are highly preferred in bone substitute application and cellular behavior on these materials were intensively studied [4,7,10,12,14], our results suggest that high porosity may not be absolutely required for in vitro osteogenesis assay

Summary

Introduction

It has been widely accepted that the porosity or pore size of materials influence the cell behavior and the osteogenesis process [1]. Porous materials with interconnected pores provide the space for the ingrowth of blood vessels and allow better formation of bone in the implants. This is one of the major reasons for the necessity of porosity for bone formation in animal models (in vivo). Continuing intensive efforts have been made to prepare and characterize highly porous scaffolds for bone substitute applications [2,3,4,5,6,7,8,9,10,11,12,13] and assess cellular behavior on these materials [4,7,10,12,14]

Results

Discussion

Conclusion