Large Mucosal Defects Research Articles

Effect of donor age on cultivation of human oral mucosal keratinocytes

Cultured mucosal grafts (CMG) are among recently developed biological grafting materials to cover large oral mucosal defects following resection of mucosal pathology. This study evaluates the effect of donor’s age on the cultivation process of oral mucosal keratinocytes for grafting. Human mucosal epithelial cells were utilized and classified into three donor age groups: 3–30 years (14 patients); 31–60 years (9 patients); and >60 years (6 patients) (11 males and 18 females). Isolation and cultivation of oral mucosal keratinocytes were according to Rheinwald and Green [Cell 6 (1975) 331], originally developed for epidermal keratinocytes. Isolated primary cell lines were seeded and cultivated. Propagation of cell lines (“passages”), time period required to reach confluence, yield of cells and plating efficiency were recorded. All cells propagated well up to the fourth passage. Thereafter, a decline was observed and was more distinct with age. Period to confluence was longer among the old age group. Yield of cells in fourth passage was high among the young age group and decreased with age. Plating efficiency in passages 4–6 decreased with age. These results suggest that age-related changes in cultivation of oral keratinocytes are not general phenomena, but rather limited to the donor age of 60 years and above. In this age group all the parameters studied were adversely affected. Oral mucosal keratinocytes may be a useful model for oral aging.

Autologous cultured mucosal graft to cover large intraoral mucosal defects: A clinical study

Purpose: The study evaluated the performance of cultured mucosal grafts (CMG) for large intraoral mucosal defects caused by surgical excision of mucosal pathology. Patients and Methods: Eleven patients (10 men and 1 woman; mean age, 52.4 ± 14.1 years) were treated using CMG following mucosal defects. A biopsy specimen (0.2 to 0.5 cm2) was taken from a clinically healthy oral mucosa a few weeks before the surgery. Mucosal epithelial cells were cultured in vitro over a feeder layer of fibroblasts. Usually, within 3 to 4 weeks, multilayered sheets (50 to 250 cm2) were generated. The cultured sheets were placed on the mucosal defects (48.4 ± 21.7 cm2; 8 to 70 cm2) and anchored to the adjacent tissue with sutures. Results: One week after surgery, the CMG survived and adhered to the wound bed. Three weeks postoperatively, the grafted site was smooth and keratinized, without infection or scar contraction. Three months postoperatively, the grafted area was covered with a healthy mucosa, indistinguishable from the adjacent mucosa. Conclusion: CMG is a useful grafting material for large intraoral mucosal defects. © 2003 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 61:169-173, 2003

Application of Banked Autologous Mucosal Cells to Graft Large Intraoral Mucosal Defects.

Purpose: This study was aimed to investigate the conditions that affect the generation of autologous cultured mucosal grafts (CMG) to promote their clinical application for large intraoral defects, following surgical excision of mucosal pathology. Specific parameters studied were the effect of patient's age and cell banking on the in vitro development of CMG, and on their clinical performance in vivo.Patients and methods: Twelve patients (10 M, 2F; mean age of 50.7+/-14.3 years) with intraoral mucosal pathologies were included in this study. Autologous mucosal cells derived from 0.2-0.5 cm(2) biopsies, taken from clinically normal oral mucosa, were cultured in vitro and banked, as cell suspensions, in liquid nitrogen. Two to three weeks before the scheduled surgery, multilayered CMG were developed according to the anticipated area of the excised tissue (8-100 cm(2)), from banked or proliferating autologous cells (for 7/12 and 5/12 patients, respectively). Following excision, CMG sheets were placed on the mucosal defects and anchored to the adjacent tissue with sutures, which were removed a week later.Results: Both the production of CMG, and their clinical performance were unaffected significantly by patient's age. Three weeks postoperatively, the grafted sites were smooth and keratinized, without infection or scar contraction, with complete and comparable healing among the two groups, treated with banked and non-banked cells.Conclusion: This study indicates that CMG is a proper dressing for large intraoral mucosal defects of various etiologies, for a large range of patients' age, using either banked or non-banked cells.

Corrective Transduction of Human Epidermal Stem Cells in Laminin-5-Dependent Junctional Epidermolysis Bullosa

Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.

A cure for xerostomia? Free jejunal patch graft in oral reconstruction

A case of oral carcinoma associated with extensive leukoplakia and severe xerostomia is presented. The advantage of using a free jejunal patch graft to restore large intra-oral mucosal defects and concurrently eradicate xerostomia is demonstrated. Further, it is recommended that a tracheostomy be used routinely in such cases to avoid postoperative aspiration of the graft secretions. Following healing the use of osseointegrated implants can complete oral rehabilitation without compromising the graft.

Reconstruction of the anterior commissure with a free mucosal flap

Introduction: To prevent stenosis and adhesion of the anterior commissure in the laryngeal reconstruction, we devised a technique of relining using a free labial mucosal graft and human fibrin glue. Materials and Methods: A single large piece of mucosal flap was prepared, folded at the anterior commissure, and sutured onto the raw wound of the larynx from both sides of the posterior glottis. Alternate suturing from both right and left sides was performed anteriorly, and the wound on the laryngeal lumen was fully covered by the mucosal graft. We applied this technique on a patient with laryngeal stenosis and two patients with laryngeal cancer. Results: Without the use of a stent, we successfully reconstructed the anterior commissure and obtained a good result in voice quality in each case. Conclusion: Large mucosal defects including the anterior commissure should be relined to acquire good voice quality. Relining using a free mucosal flap was a useful method since there were no limitations to size and placement. The adhesive property of human fibrin glue was indispensable in this procedure.

The combined sensate radial forearm and iliac crest free flaps for reconstruction of significant glossectomy‐mandibulectomy defects

The loss of motor and sensory function of the tongue following ablative surgery has a devastating effect on oral function. At the present time, there is no way to restore lost tongue musculature following partial glossectomy. The use of sensate cutaneous flaps has been shown to restore sensory feedback to reconstructed areas of the oral cavity. No single composite flap supplies a sensate soft-tissue component together with an osseous component of sufficient bone stock for functional mastication. In this article, the combination of the radial forearm free flap with the iliac crest osteocutaneous or osteomyocutaneous free flap is reported. The radial forearm free flap was used to resurface the resected portion of the tongue to provide maximum mobility and sensation. The lingual nerve was the recipient nerve for anastomosis to the antebrachial cutaneous nerves in all but one case. The iliac bone was used to reconstruct the mandible, with the iliac skin paddle or the internal oblique muscle used to reconstruct the neoridge. This combination of flaps was used in 10 patients. There was one flap failure due to vascular kinking from "piggybacking" the iliac crest to the distal end of the radial forearm flap. As a result, the use of two separate sets of recipient vessels is now advocated. Although a single composite free flap offers an excellent form of oromandibular reconstruction in most cases, it has been shown that oral function deteriorates when large areas of anesthesia are present in the oral cavity. We believe that this combination of two free flaps offers an opportunity for superior function in select patients with significant glossectomy and/or large mucosal defects.

In VitroReconstitution of Differentiated Human Epithelia

Human keratinocytes obtained from skin biopsies can be serially cultured in vitro. When plated on lethally irradiated 3T3 fibroblasts, keratinocyte colonies reconstitute a stratified squamous epithelium devoid of stratum corneum. The expression of a mature cornified epidermis, expressing all the morphological and biochemical markers of the in vivo epidermis, can be obtained by the “emerged dermal equivalent” culture system. Melanocytes grown under the same culture conditions, maintain a physiological melanocyte/keratinocyte ratio, are organised in the basal layer of the cultured epidermis, and maintain differentiated functions such as dendritic arborisation, melanin synthesis and melanosome transfer. This allows the reconstitution of an epidermis physiologically populated by functionally active melanocytes. Epithelial cells from different mucosal body sites, namely palate, urethra, conjunctiva, cornea and vagina, can also be cultured and maintain the characteristics of the original donor sites. The in vitro reconstituted human epithelia, permanently transplanted onto patients presenting large epidermal or mucosal defects, retain the characteristics of the original donor site, suggesting an intrinsic site-specific differentiation programme. These three-dimensional human epithelium models could prove useful in standard cytotoxicity assays and could be used as a tool to study the effects of a variety of compounds on normal human epithelia in vitro.

The free radial forearm flap for closure of large defects of upper lip and adjacent cheek tissue

Four cases with large defects of the upper lip and adjacent cheek, reconstructed with radial forearm free flaps, are presented. The defects were secondary to tumor excision, three recurrent and one primary. Because of the extensive infiltration of the tumor into the adjacent tissues, the excisions were extensive. There were also large mucosal defects. Free radial forearm flaps were chosen to repair these defects instead of local cheek or lip flaps. The free flap was easily folded to repair skin and mucosa simultaneously. Although the patients had partial loss of orbicularis oris muscle, they did not completely lose upper lip function. Drooling was controlled, and there was no microstomia. This is the free flap of choice to repair a large defect of the upper lip and adjacent cheek tissue in selected cases.