Harvesting abdominal donor grafts

The 15 experimental rabbits (Group B) had dermis–fat grafts procured from their lower abdomen ( Fig. 1 ). The autogenous grafts were harvested via a 2 cm × 0.5 cm elliptical incision to a depth of 0.5 cm in the lower abdomen of each rabbit. Only the epidermal layer was carefully removed by sharp dissection from the dermis–fat grafts. The donor site wound was primarily closed with 4/0 vicryl sutures. Each graft (2 cm × 0.5 cm × 0.5 cm = 0.5 cm ³ ) was passively inserted into the left TMJ and the joint capsule securely closed. The greatest cross-sectional area of each graft was 50 mm ² .

Dermis–fat graft being harvested from the lower abdomen. The skin remains attached to the fat tissue bed until the epidermal layer is removed to leave the underlying dermis before the graft is raised.

Surgical technique for TMJ

A horizontal skin incision was made from just posterior to the lateral canthus of the eye to just anterior to the external acoustic meatus. The zygomatico-squamosal suture line was exposed and a section of the zygomatic process overlying the TMJ capsule was carefully removed. In the 6 control rabbits (Group A) a left side discectomy using sharp dissection and 5 mm condylectomy using fine bone ronguers was performed and the wound was immediately repaired without any graft. In the 15 Group B rabbits, a left side discectomy using sharp dissection and 5 mm condylectomy using fine bone ronguers was performed and an autogenous piece of dermis–fat graft (0.5 cm ³ ) was passively placed in the resultant surgical cavity without any suture anchorage. The joint capsule was closed and the surgical wounds were repaired in layers with 4/0 vicryl sutures.

The animals were killed using IV sodium pentobarbitone (2 mg/kg) 4, 12 and 20 weeks following surgery ( Table 1 ). The animals were decapitated and conveyed to the histopathology laboratory where the left TMJs were dissected out and placed in formalin. The specimens were decalcified prior to histological sectioning. Coronal sections of each TMJ specimen were prepared for histological evaluation under light microscopy. At least 3 sections, 3 mm apart, from each joint specimen were prepared and stained with haematoxylin–eosin for histological examination under light microscopy. Pertinent findings were recorded using digital photography and histomorphometric analysis was performed with virtual microscopy.

Virtual microscopy (quantitative) analysis

The haematoxylin–eosin stained histological slides were digitally scanned using the ScanScope T3 virtual microscopy slide scanner (Aperio, Vista, CA, USA) and ScanScope Console software v7.00.08.1020 provided the user interface. After all the slides were scanned, the digital images were analysed using the ImageScope(r) software package. The fat was selected using the ‘pen tool’ and the ‘positive pixel count’ algorithm was run on the selected tissue. The colour saturation threshold was calibrated for each group, based on the intensity of the stain of the positive control slide, containing adipose tissue alone, to achieve uniformity in measuring the stain for all sections of that group. The same procedure was repeated for the (non-fat) fibrous and epithelial elements to determine the background stain. The number of positive pixels was divided over the surface area to obtain the number of positive pixels per mm ² for each slide. This value was subtracted from that of the negative control slides, containing non-fat, fibrous and epithelial elements, to exclude background stain and provide an absolute value of the area of fat present for each slide.