Rat Gingivectomy Model

Fifteen adult female Wistar rats aged 8 to 20 weeks weighing between 160 and 370 g were used in experiments (a generous gift from the lab of Dr. Stephen Sims, Western University, London, ON, Canada). All animal procedures were approved by the University Council on Animal Care of Western University. Gingivectomy was performed on the maxiallary palatal gingiva or mandibular buccal gingiva by one operator (Figure 2.2). Animals were anesthetized by intraperitoneal injection of

Ketamine-Xylazine (75 mg/kg Ketamine and 10 mg/kg Xylazine). A small, round dental instrument was used to disrupt the junctional epithelium and a No. 15 scalpel blade was used to raise a full-thickness 900 µm wide gingival flap along the first, second and third molars. The No. 15 scalpel blade was then used to excise the soft connective tissue flap and a small dental blade was used to thoroughly remove any remaining soft connective tissue on the tooth or bone. To maintain hemostasis, cotton swabs were held over the wound with slight pressure. Wounds received either PCL alone, low bFGF or high bFGF scaffolds or no scaffold (empty defect) as a control (n = 6 wounds receiving low bFGF and high bFGF scaffolds and n = 9 wounds receiving PCL alone scaffold or an empty defect). Two sterilized scaffold strips 1 mm x 3 mm in size were placed into the

experimental surgical wounds on top of one another to create a double layer and held in place with a small amount of Vetbond tissue adhesive (Figure 2.2). A subset of animals (n =3) with empty defects and PCL alone scaffolds were euthanized by carbon dioxide inhalation immediately following surgery completion to serve as a day 0 baseline. Remaining animals received 0.5 mg/kg Buprenorphine by subcutaneous injection twice daily for 48 hours post-surgery as an analgesic. Animals were maintained on a standard

lab chow powdered food diet and were allowed food and water ad libitum for the

Figure 2.2 - Gingivectomy in a rat model and scaffold placement.

Gingivectomy was performed in adult female Wistar rats. Soft gingival tissue was

removed and two layers of sterilized scaffolds were placed in the defect. A) Macroscopic view of scaffold placement in the gingival wound. White arrow – Molars, Black arrow – Scaffold placement. B) Uninjured gingival tissue. B – Alveolar bone, G – Gingiva, D – Dentin, E – Enamel space. Dashed red line demarcates approximate location of wound edge to be created following gingivectomy. C) Immediately following gingivectomy in an empty defect. Dashed red line indicates wound edge. D) Immediately following gingivectomy in a PCL alone treated defect. Dashed red line indicates wound edge. Black arrow indicates the scaffold.

Animals were euthanized 3 (n = 6) or 7 (n = 6) days post-gingivectomy by carbon dioxide inhalation. Heads were fixed in 10% neutral buffered formalin (Sigma)

overnight at 4°C. Jaws were excised and tissues were subsequently decalcified at 4°C for 48 hours in two changes of Cal-Ex Decalcifier (Fisher Chemical, Waltham, MA, USA).

Molars were processed en bloc for frozen sections or by routine paraffin embedding.

Serial 5 µm sections were taken in the mesio-distal plane encompassing the second molar.

Sections were rehydrated and stained with hematoxylin (Sigma) and eosin (alcoholic; Leica) (H&E), Van Gieson, and Masson’s Trichrome. Images were taken with a Leica DM100 light microscope (Leica). Histomorphometric analysis of H&E stained sections of the wounds was completed using ImageJ 1.48v software (National Institutes of Health, USA) to measure the epithelial migration distance, the area of regenerated gingival connective tissue, and biologic width; as indicated by connective tissue attachment and junctional epithelium length (Khuller and Sharma 2009) (Figure 2.3). Connective tissue attachment was measured from the crest of the alveolar bone to the apical most epithelial attachment. Junctional epithelium length was measured from the most apical epithelial attachment to the bottom of the gingival sulcus. Biologic width was the combined measurement of connective tissue attachment and junctional epithelium length (Figure 2.3).

Figure 2.3 - Biologic width.

Measurements of biologic width were made on H&E stained sections of rat gingival wounds. Measurement parameters are indicated on uninjured tissue. Horizontal green lines demarcate boundaries between which measurements are made, vertical lines indicate distances measured. Biologic width (BW) is the combined measurement of connective tissue attachment (CTA) and junctional epithelium length (JE). Sulcular depth (SD) is indicated, however does not contribute to the biologic width measurement. Red dashed line demarcates the approximate location of the wound edge which would be created following gingivectomy.

Immunohistochemical staining for arginase-1 (V-20:sc-18354, Santa Cruz), alpha smooth muscle actin (α-SMA) (clone 1A4, A5228, Sigma-Aldrich) and proliferating cell nuclear antigen (PCNA) (ab2426, Abcam) was performed to visualize M2 phenotype

macrophages, myofibroblasts (α-SMA positive cells not associated with blood vessels) and proliferating cells in the wound area, respectively. Sections were rehydrated, permeabilized using 0.1% Triton X-100 in PBS for 5 minutes at RT, rinsed in PBS and blocked using 10% horse serum. Sections were incubated with primary antibodies against arginase-1, α-SMA and PCNA diluted 1:100 in 10% horse serum for 1 hour at room temperature. Sections were rinsed in PBS and incubated, protected from light, for 1 hour at RT with an appropriate Cy5 conjugated secondary antibody. Sections were rinsed with PBS to remove unbound antibody and mounted using Vectashield Mounting

Medium with DAPI. Sections were imaged using an AxioImager.M2m (Zeiss) with Zen software (Zeiss). Negative control sections were created in which the primary antibody was omitted and used to set the threshold level for fluorescence detection (Appendix L). ImageJ 1.48v software (National Institutes of Health, USA) was used to quantify the number of myofibroblasts and proliferating cells in the wound area.