ARTÍCULO CIENTÍFICO

 

In vitro culture with collagen and human fibroblasts of a full-thickness oral mucosa equivalent*

Cultivo in vitro con colágeno y fibroblastos humanos de un equivalente de mucosa oral de espesor total*

 

 

 

S. González Mendez¹, L.M. Junquera Gutiérrez², I. Peña González³, V. García Díaz⁴, L. Gallego López³, E. García Pérez⁴, A. Meana Infiesta⁵

1 Especialista en Cirugía Oral y Maxilofacial. Práctica Privada.
2 Profesor Titular Vinculado de Cirugía Oral y Maxilofacial. Universidad de Oviedo. Hospital Universitario Central de Asturias. España
3 Médico Residente. Servicio de Cirugía Oral y Maxilofacial. Hospital Universitario Central de Asturias. España
4 Bióloga. Banco de Tejidos.
5 Coordinador del Banco de Tejidos.
Centro Comunitario de Sangre y Tejidos del Principado de Asturias. Oviedo. España

* Premio "Beca de Investigación Básica, Dr. Gómez Iglesias"

Correspondence

 

 

 

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ABSTRACT

Objectives. The objective of this study was to obtain, by in vitro culture, sheets of oral tissue in which complete oral mucosa structures can be identified. Clinical application of the findings of this study will allow the replacement of free skin grafts or autologous oral mucosa grafts by this technique in certain cases.
Material and Method. Primary keratinocyte cultures were prepared from small biopsy samples of oral mucosa. Secondary cultures were prepared from these primary cultures on an artificial submucosa constituted by collagen and human fibroblasts. The cell cultures were analyzed histologically in vitro and then used for graft implants in athymic mice to study their behavior in vivo.
Results. The primary cultures were confluent within a minimum period of 10 days and maximum of 12 days, which is similar to the period that the secondary cultures required to reach confluence. The time from sampling to achieving a complete artificial mucosa ranged from 20 to 22 days. The artificial mucosa showed histologic characteristics of a normal mucosa. After 17 days of graft implantation in immunoincompetent mice without any clinical contingency, histologic and immunohistochemical characterization (cytokeratins 19 and 13, collagen IV, and laminin) confirmed the similarity of the mucosa in vitro to healthy oral mucosa.
Conclusion. A complete oral mucosa equivalent can be prepared with collagen and fibroblasts using in vitro culture techniques. Although this mucosa shows considerable retraction, its clinical handling is very favorable.

Key words: In vitro cultive; Collagen; Fibroblasts; Oral mucose.

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RESUMEN

Objetivos. El presente trabajo tiene por objetivo obtener, mediante cultivo in vitro, láminas de tejido oral en las que se pueda identificar las estructuras de una mucosa oral completa. La aplicación clínica del presente estudio permitiría, en determinados casos, la sustitución del empleo de injertos libres de piel o autólogos de mucosa oral por esta técnica.
Material y Método. A partir de pequeñas biopsias de mucosa oral se hicieron cultivos primarios de queratinocitos. A partir de estos cultivos primarios se realizaron cultivos secundarios sobre una submucosa artificial constituida por colágeno y fibroblastos humanos. Se analizaron histológicamente sus características in vitro, y ulteriormente se procedió a la realización de injertos en ratones atímicos para conocer su comportamiento in vivo.
Resultados. Los cultivos primarios fueron confluentes en un plazo mínimo de 10 días y máximo de 12 días, periodo similar al observado para la confluencia de los cultivos secundarios. El tiempo transcurrido desde la toma de la muestra hasta la obtención de una mucosa artificial completa osciló entre los 20 y los 22 días, mostrando las características histológicas de una mucosa normal. Tras 17 días de injerto en ratones inmunoincompetentes, sin ningún tipo de contingencia clínica, la caracterización histológica e inmunohistoquímica (citoqueratinas 13 y 19, colágeno IV y laminina) confirmó la similitud de la mucosa in vitro con la mucosa oral sana
Conclusión. Es posible mediante técnicas de cultivo in vitro la obtención de un equivalente de mucosa oral completa con colágeno y fibroblastos. Si bien esta mucosa muestra un importante grado de retracción, su manejo clínico es muy favorable.

Palabras clave: Cultivo in vitro; Colágeno; Fibroblastos; Mucosa Oral.

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Introduction

In 1975, Rheinwald and Green described a method for culturing sheets of epithelial tissue in vitro using a layer of lethally irradiated mouse fibroblasts (3T3 cells) as primer cells in a medium containing fetal bovine serum and cellular growth factors. The first line of keratinocytes was obtained from a murine teratoma.¹ Two years later these authors reported the technique for obtaining human keratinocytes by in vitro culture.² Autologous human epithelium obtained by culture has been used successfully since 1980 as a permanent covering for large skin defects.^3–6 It is interesting to note that the transplanted epithelium conserves the characteristics of the donor site. This technique is currently a recognized form of treatment for major burns.⁵

In 1990, De Luca et al.4 used oral mucosa obtained by culturing keratinocytes from the palate in clinical practice for the treatment of gingival defects of periodontal origin. They demonstrated that large amounts of cultured epithelium capable of maintaining the properties of the donor zone could be prepared from a biopsy sample of 1-3 mm². Ragoebar et al.⁷ used the in vitro culture technique for oral mucosa to cover defects that occur during vestibuloplasty. In their study, half of the denuded area was covered with mucosa obtained conventionally from the palate as a free graft, and other half with palatal mucosa obtained by culture. At three months of surgery, the mucosa covering both halves was similar to the palatal mucosa; no reaction against the graft obtained by culture in vitro was evident. Light microscopy and electron microscopy demonstrated that both grafts formed a fully differentiated mucosa that was ultrastructurally consistent with the palate.

However, difficulties of culture transport, fixation to the receptor bed, and permanence in the receptor bed in response to trauma in the oral medium limit the clinical applicability of this technique due simply to the intrinsic fragility of exclusively epithelial tissue. Various authors have developed full thickness cutaneous models in culture using several types of skin substrate^8-12 that exhibit better clinical behavior than isolated epithelial cultures. Fewer investigations of this type have been made in relation to oral mucosa.^13,14 The objectives of the present study were: 1. Prepare a complete oral mucosa using a gel of collagen I and human fibroblasts as the epithelial substrate. 2. Evaluate the clinical behavior of the mucosa by graft implantation in immunoincompetent mice. 3. Characterize the behavior of the mucosa immunohistochemically 17 days after graft implantation.

 

Material and method

Primary culture of human keratinocytes

Three samples of oral mucosa were obtained in patients intervened in the outpatient surgery of the Oral and Maxillofacial Surgery Department of our center during an intervention for a benign oral pathology with local anesthesia. The surface area of the samples in every case was less than 0.25 cm². All the patients gave written consent to allow the sample to be collected after receiving documented information about the nature of the study. The samples were processed within 4 hours of collection. They were divided mechanically until fragments of the smallest possible size were obtained. The resulting material was submitted to enzymatic digestion in 4 ml of Trypsin/EDTA (T/E) during 30 minutes at 37ºC and with gentle agitation. The supernatant was collected and centrifuged for 10 minutes at 1400 rpm, so that the cells suspended in fluid were deposited at the bottom of the test tube. The resulting pellet was diluted in 0.5 ml of culture medium to count the cells obtained. The cells obtained were cultured at a density of 5,000 to 12,000 cells/cm2 on cell culture plates in the presence of lethally irradiated mouse fibroblasts (3T3; European Collection of Animal Cell culture 85022108). The medium was changed every 3 days. After the first change, EGF (Epidermal Growth Factor, Austral Biologicals) was added to the medium. The cultures were kept in an oven with a humid atmosphere and 5% CO₂ at 37ºC.

Preparation of an artificial submucosa consisting of collagen and human fibroblasts

The collagen source used was obtained from dissected tendons of Wistar rat tail. The tails were kept in 70º alcohol for 2 hours and then dissected using sterile technique. Once the skin was removed, the tendinous bundles of the tail were separated carefully and the blood vessels were eliminated to ensure that pure collagen was obtained. Once the tendons were individualized, they were divided into small pieces and washed twice in distilled water. Later the tendons were dried between sterile gauze pads. The material obtained was weighed and dissolved in sterile acetic acid in a proportion of 0.01% (100 ml of solution per gram of tissue). The solution containing the previously prepared tendinous bundles was incubated at 4ºC with gentle agitation for at least 48 hours. Once the digestion process was complete, the solution was centrifuged for 30 minutes at 30,000 G and the resulting pellet was lyophilized and stored in sterile tubes. The purity of the collagen obtained was analyzed by electrophoresis on polyacrylamide gel.

The fibroblasts used were obtained from human foreskins removed from pediatric patients treated for phimosis at our center, after previously obtaining permission from their legal guardians. These samples were fragmented mechanically and then digested enzymatically with T/E while being mechanically agitated. Every 30 minutes the T/E medium was replaced with fresh medium. Finally the solution was centrifuged during 10 minutes at 1,400 rpm to recover the cells from the sample. The cells were seeded at a density of 100,000 cells/cm² in a fibroblast culture medium. When the cells of this culture were confluent, the culture bottle was washed twice with T/E and then incubated at 37ºC with T/E until the cells separated from the substrate. The cells were recovered and seeded in other culture bottles at a density of 5,000 to 10,000 cells/cm² (secondary culture). The fibroblasts were maintained by successive cultures or were frozen in fibroblast freezing medium for later use.

The collagen and fibroblast gel was prepared using 0.8 ml of collagen solution with 0.1 ml of NaOH and 0.1 ml of Ham-F12 10X. Fibroblasts were added to this solution at an approximate concentration of 30,000 to 50,000 cells/ml. For a 25-cm² plate, 5 ml of gel was used. The material obtained by culture according to the technique described above was studied histologically by fixation in 10% formalin and staining with hematoxylin-eosin.

Animal experimentation The cultured cells were grafted in three athymic mice (BALD/ c nu/un) following the technique described by Barrandon et al.¹⁵ (Fig. 1). At 17 days of graft implantation, the animal was killed and a biopsy was made of the graft tissue for histologic analysis. Immunohistochemical analysis was made with kits for cytokeratins 13 and 19, collagen IV, and laminin. A diagram of the main stages of work is shown in figure 2.

 

Results

Primary cultures

At three or four days of culture, incipient formation of keratinocyte colonies surrounded by 3T3 fibroblasts was observed. Starting in the first week of culture, fibroblasts were displaced to the periphery of the colonies and their number diminished considerably. When the epithelial colonies were confluent, the 3T3 fibroblasts were not visible (Fig. 3). The primary cultures were confluent within a minimum period of 10 days and a maximum period of 12 days. At this moment the secondary culture was made on the collagen and fibroblast gel prepared. The yield was approximately 1000 cells per cell cultured in this period.

Secondary cultures

The secondary cultures also were confluent at 10 days, when they were fixed for histologic study. At this time and from the first moments of culture, a rapid process of the keratinocyte retraction was observed on the collagen gel. In any case, the time from sampling until a complete artificial mucosa was obtained from collagen and human fibroblasts ranged from 20 to 22 days.

Histologic analysis of the material obtained found a continuous epithelium of cuboid cells similar to what we find in the basement layer of normal oral epithelium. In some zones, the epithelium had two or three rows of cells, but no sign of keratinization (Fig. 4). Stellate fibroblasts adhered to each other were recognized subepithelially. The presence of anarchically distributed bundles exhibiting collagen-like staining properties and a rudimentary vascular network also was evident.

Animal experimentation

The animals were kept alive for 17 days. During this time no mouse presented complications, whether directly related to surgery or other. Microscopic study at low magnification of the interface between the graft tissue and native mouse tissues detected continuity. The epithelial portion exhibited several layers of cells. At higher magnification, cellular layers organized similarly to normal unkeratinized oral mucosa were apparent. In some areas, epithelial disorganization was evident (Fig. 5). None of the grafts exhibited keratinization; nuclei were evident even in distal keratinocyte layers. In the subepithelial tissue abundant proliferating fibroblasts were appreciable in the gel matrix. Papillae were not recognized, although slight invaginations of the epithelial tissue were seen in some areas in the submucosa.

Cytokeratin 13 staining was positive in all the layers of the epithelium, whereas cytokeratin 19 staining showed only limited areas of immune labeling. Immune labeling of collagen IV produced marked staining of the submucosa and most basal layers of the epithelial portion, but laminin staining was concentrated in the basal portion of epithelium (Fig. 6).

 

Discussion

Early attempts at in vitro culture of keratinocytes were plagued by major drawbacks: 1. 1. Marked retraction was observed when the cultured tissue was separated from the culture bottle. 2. Grafts are extremely thin to their exclusively epithelial constitution. 3. The grafted tissue is fragile and early graft loss is possible. These drawbacks are directly related to the absence of an adequate substrate for the constitution of a normal basement membrane and the complete and adequate differentiation of epithelial tissue.¹⁶

In order to resolve these problems, investigations have focused on developing epithelial cell cultures with connective support.^17,18 Different authors have proposed a variety of systems that can be grouped as: 1. Use of cryopreserved cadaver dermis transplanted onto the graft receptor bed.¹⁹ 2. Use of three-dimensional synthetic structures of absorbable materials as an autologous fibroblast substrate or as acellular dermis.²⁰ 3. Synthesis in vitro of a substrate of dermal proteins for the growth and development of human fibroblasts.²¹ At present, in vitro synthesis is enjoying the most intensive investigative efforts of the scientific community. A number of gels have been synthesized from human proteins, such as collagen type I, glycosaminoglycans, and fibrin.²² Another line of investigation has the objective of obtaining a protein base containing live fibroblasts. It is known that in the normal development of epithelial tissues the synthesis of basement membrane proteins (laminin and collagen IV) is a keratinocyte function, but the other component of the basement membrane, anchorage fibrils, is not synthesized in the absence of fibroblasts of the dermis. It has been demonstrated that human fibroblasts maintained in fibrin gel are capable of synthesizing the same substances as in vivo, making it a possible substrate for keratinocyte growth.²³ The preparation of submucosa from collagen in our study was technically simple and quick, but it was accompanied by intense culture retraction that began in the first 48 hours of the secondary culture and ultimately resulted in a large reduction in the size of the usable graft. A similar observation has been highlighted by other authors who have produced complete oral mucosa using collagen as the protein substrate.²² Nevertheless, it was extraordinarily easy to separate the cultures made on a gel of collagen and human fibroblasts and, in some cases, the epithelial layer separated spontaneously. The resistance of the submucosal substrate allowed perfect handling at any time, without requiring any special care.

In the microscopic study of the oral mucosa obtained after grafting, we identified small destructured areas with hydropic degeneration. Although some authors 24 claim that the graft evolution of destructured areas is unfavorable, immunohistochemical characterization of these areas in our study did not show any differences with respect to the cultured mucosa that did not have structural alterations. Microscopic examination of the submucosa revealed the existence of rudimentary subepithelial vascularization consisting of small caliber vessels, which did not condition or limit the development of the epithelial layer. It is possible that collagen, unlike fibrin, does not favor the incorporation of new vessels in the injured area.

Cytokeratin 13 (K13) is typically expressed by the suprabasal cells of the oral epithelium, keratinized or unkeratinized, and also was strongly positive in the cells of the fetal oral mucosa. Cytokeratin 17 also showed strong positivity in our samples, perhaps attributable to the differentiation process that the tissue was experiencing 17 days after graft implantation. Cytokeratin 19 (K19) is a typical marker of unkeratinized mucosa that also was expressed in our preparations, including in keratinocytes located in the suprabasal layers of the areas of more disorganized epithelium.

According to several authors,^22,23 the structure of the basement membrane is completely normalized at three weeks of culture. This consists of a regular deposition of collagen IV and laminin and stable contacts between the epithelium and connective tissue, as a consequence of the mediation of alpha-6-beta-4 integrin. This regularization is fundamental in obtaining an artificial mucosa with a normal phenotype. In our study, immunohistochemical staining was positive for collagen IV and laminin 17 days after graft implantation.

The present study shows that collagen gel was a good substrate for the growth of fibroblasts and oral keratinocytes. Synthesis of the gel was simple and predictable. Its management was extraordinarily easy and no exceptional precautions were necessary. However, marked retraction occurred and it was difficult to obtain a large amount of tissue, which was one of the objectives of the technique. Nonetheless, we also observed that the retraction process ceased when the graft is implanted and the grafted tissue conserved its diameter when the animals were killed.

 

Acknowledgements

We would like to thank the Spanish Society of Oral and Maxillofacial Surgery and Industrias Tarma, S.A. for supporting our work (Dr. Gómez Iglesias Basic Research Grant),

as well as Ficyt (Eva García Perez) and MBA (Verónica García Díaz) for their technical support.

 

 

Correspondence:
L.M. Junquera
Universidad de Oviedo. Escuela de Estomatología
Catedrático José Serrano s/n
33009 Oviedo. España
E-mail: Junquera@uniovi.es

Recibido: 15.11.07
Aceptado: 28.01.09

 

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