## 4 Discussion  
### Time-Evolution of Cell Populations in Surgical Vocal Fold Injury and Repair  
Multi-parametric flow cytometry was used to analyze dynamic changes to cell composition of vocal folds up to four weeks following injury. Results are generally consistent with the temporal dynamics of immune cells (neutrophils and macrophages) known in general wound healing literature. In dermal wound healing, neutrophils arrive at the wound site in the first few hours, peak in abundance one day after the injury, and reduce rapidly by the third day after injury [101; 102; 103; 104; 105; 106; 107]. Macrophages peak slightly later, by the second day after the injury, but also demonstrate rapid decreases on the third day after surgery [101; 102; 103; 104]. Given the similarities in the temporal dynamics of neutrophil and macrophage changes following injury in vocal folds and other types of tissue, comparable cellular functions of immune cells underlying the inflammation and repair processes are speculated. In addition to immune cells, the temporal pattern of endothelial cells following vocal fold damage also resembles the general wound healing literature [105; 106]. In this study, endothelial cells contributed about 9% of the total cell population in uninjured controls that were likely originated from the endothelium layer of undisturbed blood capillaries in native vocal fold mucosae. A rise in the endothelial cell population was observed in Week 2 after surgery, representing about 14% of total cell populations. An active angiogenesis might have started between Week 1 and Week 2 post- surgery. To our best knowledge, this study is the first to evaluate the population of endothelial cells in vocal fold injury and healing. Further investigation on the precise role of endothelial cells, their interaction with other cell types as well as their implications in vocal fold scarring is warranted before we could reliably formulate agent-rules of endothelial cells in VF-ABM. Fibroblasts normally arrive at the wound site around Day 3 and start the proliferative phase [37; 103; 105; 106]. These cells usually reach their maximum concentrations between Day 5 \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Time-Point** & **Cell Types** & \
\begin{tabular}{c} **Empirical Data of** \\ **Cell Quantities** \\ \end{tabular} &
\begin{tabular}{c} **95\% Confidence Interval (and Mean)** \\ **of ABM-Simulated Cell Quantities** \\ \end{tabular} \\ \hline Day 7 & Neutroph & 214 & 198.5–228 * (212.29) \\  & Macrophages & 359 & 310–406 * (359.15) \\  & Fibroblasts & 5751 & 4920.5–7541.5 * (6083.45) \\  & **Empirical Data within 95\% CI** & **100\%** \\ \hline Week 2 & Neutroph & 180 & 188.5–222.5 (204.87) \\  & Macrophages & 186 & 103.5–155.5 (126.8) \\  & Fibroblasts & 3854 & 1759–2640 (2142) \\  & **Empirical Data within 95\% CI** & **0\%** \\ \hline Week 4 & Neutroph & 214 & 192.5–227 * (210.16) \\  & Macrophages & 252 & 10–27 (17.07) \\  & Fibroblasts & 4347 & 254–378 (304.45) \\  & **Empirical Data within 95\% CI** & **33\%** \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Cell quantities from empirial and simulation experiments from Day 7 to Week 4. * denotes the empirical data within the 95% confidence interval (CI) of VF-ABM outputs. and Day 7, and start to decrease gradually by the end of Week 1 [101; 102; 103; 104]. Interestingly, fibroblasts were predominantly present in injured rat vocal folds spanning from the early acute inflammatory to later remodeling phases of wound healing as shown in this study. Previous studies reported the abundant presence of fibroblasts up to one week after surgical vocal fold injury [36; 66; 108]. Our data further indicated a notable accumulation of fibroblasts at the wound site up to four weeks after surgery, likely associated with their important roles in ECM remodeling. Further, fibroblasts remain the dominant cell type at Day 1 (53.81%) and Day 2 (29.37%) despite the infiltration of neutrophils and macrophages into the acute wound environment.