All PhD Theses

R. Ophof

Construction and application of substitutes for oral mucosa in cleft palate repair


A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 2nd of June 2010


The aim of the present study was to develop a substitute for oral mucosa, which can prevent the iatrogenic effects of surgical cleft palate closure.

Chapter 1 describes the background of the study and reviews the literature on orofacial clefts, cleft palate surgery, and tissue engineering of oral mucosa. After cleft palate surgery in young patients, maxillary growth and dento-alveolar development are severely impaired. Two events during the wound healing process, contraction and scar formation, are responsible for these adverse effects of surgery. Scar tissue formation is still one of the main causes of maxillary growth inhibition. A possible solution is to supplement the tissue shortage after palatal repair with a cellular or acellular mucosal substitute using tissue engineering techniques. To investigate this, several approaches have been taken. Firstly, a cellular substitute was constructed and evaluated in vitro. Secondly, the biocompatibility of this substitute and several dermal substrates was tested in an animal model. Finally, the effects of an acellular implanted substitute on the development of the dento-maxillary complex were evaluated in a dog model.

Chapter 2 describes an in vitro study on the culture and characterization of substitutes containing dog keratinocytes. Epidermal and oral keratinocytes were cultured on several skin-derived and collagen-based substrates. The results show that oral keratinocytes cultured on the skin-derived substrates closely resemble normal oral epithelium. Epidermal keratinocytes cultured on skin-derived substrates form an epithelium that is similar to dog epidermis. In contrast, keratinocytes cultured on the collagen-based substrates invade the substrate without the formation of a multi-layered epithelium. It is concluded that dog oral keratinocytes cultured on skin-derived substrates exhibit a tissue organization that resembles normal dog oral mucosa.

Chapter 3 describes a study on the in vivo behavior of five different dermal substrates. This study was performed in order to obtain a suitable material for implantation on the palate of the beagle dog. Three collagen based substrates and two skin-derived substrates were implanted in open wounds on the palate. Histology was performed at 3, 10, and 20 days post-surgery. It was concluded that all collagen-based substrates were well-tolerated. However, it was unclear whether AlloDerm was rapidly degraded or that it was sequestrated and lost.

In chapter 4 it was investigated whether cultured mucosal substitutes composed of skin-derived substrates and dog oral keratinocytes can be used as a graft material. Two different types of cultured mucosal substitutes composed of skin-derived substrates (DED and AlloDerm) and autologous oral keratinocytes were implanted in palatal wounds in six beagle dogs. The cultured substitutes were compared with a sham and a control group. The animals were sacrificed in pairs up to 12 weeks after surgery. It was shown that before implantation all cultured substitutes possessed a multilayered epithelium, closely resembling normal oral epithelium. After implantation however, the epithelium was rapidly lost and an inflammatory response was observed in the first week. After 3 and 12 weeks, the implanted substitutes had completely disappeared and epithelial migration had occurred from the wound margins. This in vivo study further demonstrated that the substitutes did not improve the healing of palatal wounds. It was suggested that the revascularization of the substitutes is too slow to allow survival and integration of the substitutes.

In chapter 5 the influence of the silicon top layer of Integra on palatal wound healing was investigated. Wounds were made in the palate of four beagle dogs. Integra with and without the silicon layer was implanted into the full-thickness wounds. In some of the wounds, the silicone layer was removed after 14 days. No implants were placed in the control wounds. At two and four weeks post-surgery, the wounds were assessed for epithelialisation, inflammation, number of myofibroblasts, and general histological characteristics. It was concluded that wounds implanted with Integra without the silicon layer showed less myofibroblasts and inflammatory cells than the control wounds. In addition, collagen fibres were more randomly oriented than in the sham group. Wound closure was found to be retarded and many inflammatory cells were present when Integra with silicone was implanted. Moreover, the silicon layer was lost within 4 weeks from the wounds. It was concluded that in the moist oral environment the silicon layer of Integra is not required.

Chapter 6 compared the dento-alveolar development in beagle dogs after palatal repair with and without the implantation of Integra without the silicon layer. Nineteen young beagle dogs were assigned to two experimental groups and a control group. Palatal surgery was performed by the Von Langenbeck technique. Thereafter, a substitute was implanted in one experimental group, while the other served as sham group. Dental casts were made prior to surgery, and at several time-points in all groups. Dento-alveolar development was measured on the dental casts. These were used to determine the transversal distances, arch depth, tipping, and rotation. Histology was performed at 3, 7, and 15 weeks after surgery. It was found that the scar tissue in both experimental groups was firmly attached to the palatal bone by means of Sharpey’s fibers. Moreover, it was shown that bone was deposited inside the Integra substrate after implantation. Transversal dento-alveolar development was similar in both experimental groups but significantly smaller than in the control group at some time-points. Therefore, it was concluded that the implantation of the dermal substrate does not improve the dento-alveolar development after the Von Langenbeck procedure for palatal repair.

Finally, in chapter 7 the results of the previous chapters are discussed and suggestions are made for further research. In spite of the disappointing results of the implanted substrates found in this thesis, these materials still might be useful in cleft palate surgery. Loading of a substrate with growth factors may promote angiogenesis and further reduce contraction and scar formation. Therefore, future research both in vitro and in vivo is needed to better understand material characteristics, and cell-tissue interactions to the materials before using these materials in human cleft palate surgery. In summary, the following conclusions are drawn from the studies: 1. Seeding oral keratinocytes onto a skin-derived substrate, a fullthickness oral mucosal substitute can be established. However, these substitutes containing autologous keratinocytes were lost after implantation. It was suggested that the revascularization of the substitutes is too slow. 2. The implantation of Integra in palatal wounds yields a wound with less parallel collagen fibers and less myofibroblasts. However, this positive effect on the structure of the scar tissue was counteracted by its attachment to the palatal bone. Therefore, it was not surprising that palatal surgery combined with implantation of the dermal substitute had no positive effect on dento-alveolar development.