Abstract
The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin–eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.
Since the classical experiments of Green’s group demonstrating the possibility of human keratinocyte cultivation in vitro , cultured autologous keratinocytes have been widely used as grafts in the treatment of severe burns and chronic wounds. Such multilayered cultured epithelial autografts have some disadvantages, such as the relatively long time required to obtain the graft, its fragility, sensitivity to infection and the unpredictability of engraftment. The use of preconfluent keratinocytes (cultured to the stage before they form a compact sheet) has shown considerable advantages in wound treatment procedures. Such cells have better proliferative capabilities, an optimal integrin profile for attachment to the wound bed and require less time to be cultivated in vitro . Among different methods of subconfluent keratinocyte delivery to the wound, suspension of keratinocytes in fibrin glue has been shown to be an effective method of treatment in cases of burns, chronic wounds and leg ulcers in animal models and patients.
In contrast to the extensive research on skin wound healing, studies on the effect of cultured oral keratinocytes on healing of oral mucosal wounds are scarce and the procedure using preconfluent keratinocytes in fibrin glue has not been tested. The aim of the present study is to assess the influence of an oral keratinocyte suspension in fibrin glue on the healing rate of oral mucosal wounds in the rabbit model.
Materials and methods
The study was carried out on New Zealand white rabbits, in which a fragment of right buccal mucosa approximately 1 cm in diameter was excised under general (0.2 ml Combelen, Biowet, Poland and 1.5 ml 2% Rometar, Spofa, Czech Republic) and local anaesthesia (1% lignocaine, Polfa, Poland). The animals were separated into three groups with 12 animals in each. In group 1 the wound was covered by suspension of cultured autologous keratinocytes in fibrin glue (Tissucol Kit, Baxter, Austria) and secured by biological dressing (Codogard, Tricomed, Poland). In group 2 the wound was covered by fibrin glue and secured with a biological dressing. In group 3 (control) the wound was secured with a biological dressing.
On postoperative days 1, 3, 7 and 14, the buccal mucosa containing the wound area was excised under general anaesthesia with a 0.5 cm margin and the animals were killed afterwards. Progress in wound healing was examined in paraffin-embedded sections of the mucosa using light microscopy and digital image analysis software. The experiment was approved by the local ethical commission.
Collection and culture of autologous keratinocytes
Under general and local anaesthesia, a small (0.5 cm 2 ) fragment of the left buccal mucosa was excised and the wound was sutured. The mucosal fragment was placed in buffered deionized saline (Sigma, Germany) containing antibiotics (200 U/ml penicillin, 200 μg/ml streptomycin, Sigma, Germany) and incubated for 16 h at 4 °C in the same solution containing 6 U/ml dispase (Gibco, USA). The epithelial layer was gently separated from the mucosa and trypsinized (0.05% trypsin with EDTA, Sigma, Germany, 5 min, room temperature). The keratinocyte suspension obtained was centrifuged at 1000 rpm for 5 min, the cell pellet was suspended in 4 ml Dulbecco modified Eagle medium (DMEM) containing 10% foetal calf serum and the suspension was placed in culture dishes. After 24 h DMEM was replaced by KGM-2 medium supplemented with KGM-2 Single Quots (Clonetics, Cambrex Bio Sciences, USA). The cells were cultured up to preconfluence stage for 8–10 days at 37 °C, with the medium changed every 72 h.
For preparing suspension of keratinocytes in fibrin glue, the cultured cells were trypsinized (2 min, 37 °C) to detach them from the culture dish, suspended in 5 ml DMEM and centrifuged. The cell pellet was added to 0.5 ml of fibrin solution, a component of the Tissucol Kit. Cell viability was assessed using the trypan blue exclusion test.
Histological techniques and morphometry
Mucosal samples were fixed in 4% buffered formalin for 24 h at room temperature. The samples were embedded in paraffin and sectioned at 6 μm in the plane perpendicular to the wound surface. Sections were stained with haematoxylin–eosin and examined under an Olympus BX50 light microscope with an Olympus DP71 camera coupled to a PC equipped with the Olympus AnalySIS FIVE image analysis system.
The following parameters of wound epithelization were measured: marginal epithelization (i.e. the length of new epithelium measured from the original wound margin); and the percentage of wound area covered by new epithelium. For each wound, 3 sections from different wound areas were examined and values of the measured parameters were calculated.
Statistical analysis
The measured values were expressed as medians or means and analyzed using ANOVA Kruskal–Wallis, Mann–Whitney and Fisher exact tests, with p < 0.05 as the level of significance.
Results
The wounds in all groups showed progressive healing manifested by epithelization of the wound surface and reconstruction of the stromal tissue. The epithelization mostly proceeded by proliferation and migration of epithelial cells from the wound margin, although some focal (insular) epithelization not associated with the marginal epithelium was also observed.
There were no significant differences in the epithelization parameters on postoperative day 1. On days 3 and 7, the epithelization degree was significantly higher in keratinocytes plus fibrin glue treated wounds (Group 1) compared to fibrin glue alone treated wounds and untreated wounds ( p = 0.006 and 0.009 for marginal epithelization, p = 0.04 and 0.01 for epithelization area, respectively) ( Figs. 1 and 2 ). On day 14, in the majority of rabbits the epithelization was complete or nearly complete ( Figs. 3 and 4 ) and differences between the groups were insignificant. No significant differences were found between wounds treated with fibrin glue and the untreated ones, with the exception of marginal epithelization on day 7 ( Tables 1 and 2 ).
| Postoperative day | Keratinocytes + fibrin glue (I) | Fibrin glue (II) | Control (III) | Significance |
|---|---|---|---|---|
| 1 | 291 [61–598] | 131 [55–139] | 172 [99–321] | NS |
| 3 | 819 [720–1220] | 397 [224–551] | 385 [264–530] | p = 0.006 I/II, I/III: S II/III: NS |
| 7 | 1305 [596–2635] | 918 [440–1564] | 409 [342–839] | p = 0.009 I/II, I/III, II/III: S |
| Postoperative day | Keratinocytes + fibrin glue (I) | Fibrin glue (II) | Control (III) | Significance |
|---|---|---|---|---|
| 1 | 8.0 [0.9–19.8] | 2.6 [2.1–3.4] | 3.3 [2.3–7.2] | NS |
| 3 | 20.8 [17.4–25.8] | 9.9 [5.5–24.8] | 6.4 [4.9–12.8] | p = 0.04 I/II, I/III: S II/III: NS |
| 7 | 59.4 [24.2–67.7] | 30.7 [22.3–38.1] | 18.8 [13.4–27.2] | p = 0.01 I/II, I/III: S II/III: NS |
| 14 | 100 [100] | 100 [94.5–100] | 78.7 [66.1–100] | NS |
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