Biomedical Engineering Reference
In-Depth Information
of the nature of ECM development must be enhanced in order to develop
biomaterials with the appropriate host response. Little is known about how
the ECM is developed
in vivo
in the presence of biomaterials. Therefore
advances in cellular and molecular biology characterization techniques
offer the opportunity to investigate the function of biomaterials to assist
with specifi c cellular differentiation.
The application of assays in the fi eld of biomaterial evaluation has devel-
oped to provide relevant data from
in vitro
and
in vivo
models. The isolation
and purifi cation of nucleic acid and protein is used to determine differenti-
ation. The analysis of ribonucleic acid (RNA) and protein products is a key
technology in cell biology, molecular biology and proteomics.
4.2 Deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA) assays
Cellular differentiation involves gene expression changes epigenetically regu-
lated in a lineage or differentiation stage-dependent manner, and such changes
are examined to evaluate the progression of differentiation. In this section,
commonly used nucleic acid assays, such as quantitative real-time polymerase
chain reaction (PCR), multiplex PCR and PCR array, are discussed to pro-
vide insights into the principles and use of the assays (Table 4.1).
4.2.1 Extraction of total RNA and DNA
Isolation of total RNA and genomic DNA is the fi rst and critical step for
successful subsequent applications. Nucleic acids, RNA and DNA, can be
isolated from primary cells, tissues, and even from cultured cells residing
in three-dimensional biomaterial scaffolds in fresh, frozen or fi xed states.
Regardless of sample types or culture systems, isolated RNA and DNA are
utilized in subsequent applications in the same manner. In general, the isola-
tion procedure consists of three steps: (1) disruption and homogenization of
a starting material, (2) removal of cell debris and proteins, and (3) recovery
of nucleic acids. In this section, several RNA and DNA isolation methods
are introduced. The proper method is determined by the quantity and qual-
ity of nucleic acids required for subsequent applications, preparation time
and cost, and availability of equipment. For many scaffolding materials and
dense tissues, additional homogenization steps are required before nucleic
acid extraction ( Table 4.2 ).
Techniques for genomic DNA isolation
Organic extraction, salting-out, cesium chloride (CsCl) density gradient,
silica-based and anion-exchange methods are commonly used for DNA
