Biomaterials and their effect on wound healing: a practical example
As explained thoroughly in Section 4.1, wound healing is a very complex process that requires spatial and temporal fine-tuning of multiple genes in several different cell populations. Any researchers studying biomaterial development for applications in cutaneous wound-healing context need to factor this knowledge into their experimental design.
Figure 6.7 Experimental design of hypothetical experiment to investigate the effect of biomaterial “X” on gene expression in wound healing. (a) Animal model and downstream assays. (b) Cell types and time points of interest in the three phases of wound healing.
To probe how gene expression could be utilized in the assessment of a biomaterial on wound healing, we will use the dorsal full-thickness wound-healing model in C57BLKS-Leprdb (also named db/db) mice. db/db mice have a mutation in the leptin receptor (LEPR), which leads to the development of type II diabetes, and the mice harbor a well-described impairment in skin healing (Michaels et al., 2007). This model is commonly used in wound-healing experiments and is a useful tool to study wound healing in a manner representing impaired repair (Nambu et al., 2009; Tkalcevic et al., 2009; Van Slyke et al., 2009). The experimental design of our practical example illustrated in Fig. 6.7 will be the creation of four full-thickness 6-mm wounds on the back of each db/db mouse, with a soft biomaterial “X” implanted into half of the wounds (see Fig. 6.7(a)). In this impaired wound-healing model, the inflammatory phase is ongoing from day 0 to day 7, the proliferative phase begins around day 4 to day 15, and the remodeling phase starts at wound closure and is ongoing for several months (Tsuboi and Rifkin, 1990). Knowing how the impairment influences the timing of each stage of healing in the db/db mice is very important information, since the spatiotemporal expression of the target genes related to the phases of healing will vary, and how gene expression patterns are changed provides essential information on the efficacy of the biomaterial (Cooper et al., 2005; Deonarine et al., 2007; Lefebvre-Lavoie et al., 2005).
So what should be considered with respect to changes in gene expression? If scaffolds speed healing, the temporal gene expression pattern expected in db/db mice and the phases of healing would be accelerated. This would change which genes will be active based on cell recruitment and which genes should be analyzed at any selected time point. Therefore prior to performing gene expression studies, it is prudent to perform closure kinetic assays, as we have done, to assess where the effect of the scaffold starts to change the closure kinetics. This allows assessment of whether the repair speed is increasing and therefore the phase of healing has changed, i.e., from inflammatory to proliferative. Closure kinetics illustrates which phase of healing is being affected by the presence of the scaffold. For example, if the wounds are reduced in size in the presence of the material between days 1 and 3 compared to controls, it is very likely that the scaffold is altering a number of inflammatory processes. In contrast, if wound closure were faster in the presence of the biomaterial versus controls between days 4 and 7, the scaffold is very likely to influence processes part of the proliferative phase of healing. However, it should also be considered that measuring closure kinetics has limitations. For instance, it is possible that a biomaterial could alter inflammatory events that do not manifest in changes in closure rates until the proliferative phase.
Finally, although gene expression can give a lot of information on the effects of the scaffolds on the resident cell types of the wound, it is important to also include assessments at the protein level. This is particularly important since it is recognized that several posttranscriptional modifications affect the regulation of genes involved in wound healing. Besides, discoveries have also highlighted more than 21 microRNAs that can act as regulators of gene transcripts implicated in wound healing (Moura et al., 2014).