FR2604905A1 - Method for obtaining a gingival epithelium substitute from human cells cultured in vitro, as well as the applications of this epithelium in odontology/stomatology - Google Patents

Abstract

According to this method, a superficial gingival micro-sample a few square millimetres in area and a few tenths of millimetres in thickness, essentially limited to the epithelial layer 4, is taken, this sample then being cultured and treated in a conventional manner in order to remove microorganisms and bacteria, and trypsinised in a conventional manner, and the cell suspension obtained being deposited on an irradiated support culture of the 3T3 type until confluent primary cultures are obtained, which cultures are again broken up by trypsinisation, inoculated and cultured until stratified confluent secondary cultures are obtained with formation of multiply stratified layers. The gingival epithelium obtained finds application in odontology/stomatology, and in particular in the production of gingival grafts.

Description

PROCESS FOR OBTAINING A GINGIVAL EPITHELIUM
EQUIVALENT FROM HUMAN CELLS CULTIVATED IN VITRO
AS WELL AS THE APPLICATIONS OF THIS EPITHELIUM
IN ODONTOLOGY-STOMATOLOGY
The present invention relates to a process for obtaining an equivalent gingival epithelium from human cells cultivated in vitro, as well as the applications of this epithelium in odonto-stomatology.

 Mucogingival surgery is defined as the set of plastic surgery techniques intended to correct or modify defects in the morphology, position and quality of the gingival tissue surrounding the tooth.

 Paradontologists have paid particular attention in recent years to the quantity and importance of the height of the gum tissue to maintain paradontal health and above all to avoid loss of the attachment system or epithelium joining it. tooth.

 The volume and shape of the alveolar bone and gum are closely related to the pathology and thereby modify the treatment data. The odonto-stomatologist clinician usually distinguishes the thick and flat periodontium from the thin and scalloped periodontium.

A classification established in 1980 by Maynard and Wilson identifies four types
1- Thick alveolar bone and thick and extended keratinized gum over a height of 3 to 5 mm (ideal)
2- Thick alveolar bone and thin and thin keratinous gum,
3- Alveolar bone of reduced thickness and "ideal" gum,
4- Alveolar bone of reduced thickness and little extended and thin gum.

 The different tissues of the periodontium are, in fact, concerned with mucco-gingival surgery, but it is the attached gum and its extent that this type of surgery usually refers to.

 The attached gum is a part of the keratinized gum itself delimited on the coronary side by the free gingival border and apically by the line of junction mucco-gingival. Beyond, the alveolar mucosa appears darker and easily mobilized with a thin non-keratinized epithelium. On the other hand, the gingival epithelium is thick and is underpinned by a dense connective tissue.

The clinical evolution of the attached gum is superimposed on the evolution of the depth of the gingival-dental groove in the absence of inflammation. The attached gingiva thus delimited presents a uniform appearance, specific to the keratinized epithelial coating. The clinician, however, observes only one and the same structure, whether it is attached to the underlying bone or to the tooth. However, in depth, the fixation of the gum comes from two distinct networks of connective fibers
- the gingivo-osseous fibers of the attached gingiva proper,
- the gingivo-cemental fibers of the connective attachment coronary to the bone crest.

 The single figure of the attached schematic drawing will allow a better understanding of the mechanism of the epithelio-conjunctive attachment.

 In the figure, the epithelial attachment is generally designated by 1 and the tooth by 2.

 We see at 3 the epithelial junction bordered by the internal basement membrane 6 in contact with the enamel 9 and the external basement membrane 7 on the side of the connective tissue 5; the gingival epithelium is designated by 4 and 8 designates the granulocytic and lymphocytic cells infiltrating the epithelial junction 3 and the connective tissue 5. 12 designates the cement covering the dentin 10 at the level of the root and 11 the amelo-cement junction.

This epithelio-conjunctive attachment is the true guarantor of the paradontal integrity and constitutes the essential concern of the therapist who can be in the presence of three situations
- sufficient or even large extent of the attached gum and the attachment system with the presence of gingivo-osseous fibers and gingivo-cemental fibers;
- brevity of the gum attached with exiguity of the attachment system reduced to some gingivo-osseous and gingivo-cementary fibers. In the extreme, there is only one bone attachment line with some gingivo-cemental fibers. Such a complex can be stable or, on the contrary, qualified as insufficient in the case of persistent pathology or progressive recession or aggressive therapy
- rupture of the attachment system associated with deep periodontal pathology.

Mucogingival surgery is an essentially reconstructive surgery, it is an additive surgery or the connective tissue supply
predictably changes an unfavorable environment for maintaining
periodontal health and the protective and aesthetic function of the tooth.

Rigorous operating protocols require handling
attentive to these fragile tissues and the stability of these tissues using
sutures anchored to the tooth or periosteum. Observing principles
biological is essential, not only to ensure the success of the intervention, but above all to avoid loss of the attachment system and alveolysis at the chosen donor site.

 It should also be noted that in muco-gingival surgery, the connective tissue plays an important role in the repair process.

Gingival features are not the result of functional adaptation. The gum is genetically determined and the differentiation of the gingival epithelium into keratinized tissue is done through the extracellular matrix largely produced by the cells of the underlying connective tissue. Thus, the success or failure of periodontal surgery interventions also depends on the quality of this connective matrix.

This surgery is performed, for example, in the following cases
- rupture of the attachment system (deep periodontal pathology),
- gingival and attachment system insufficiency,
- pre-prosthetic surgery,
- tissue development in children in connection with orthodontic treatment,
- gingival recessions,
Mucogingival surgery usually uses the following techniques
- frenotomies and frenectomies,
- vestibular deepening
- free gum grafts,
- coronally positioned flaps,
- laterally positioned flaps,
- flaps positioned apically.

 These techniques have many drawbacks, among which may be mentioned the pain caused and the duration of the interventions, the healing and repair times, the difficulty in obtaining acceptable donor sites qualitatively and especially quantitatively, and finally the need to protect donor sites, recovery of recipient sites and hemostasis.

 The object of the present invention is to remedy these drawbacks by providing paradontologists with a new means of avoiding the loss of the gum-tooth attachment system by providing an equivalent gingival epithelium obtained from human cells cultivated in vitro.

 The inventors have, in fact, discovered, surprisingly, that it was possible to apply to the production of an equivalent gingival epithelium certain methods of in vitro culture of human cells used for the preparation of skin grafts, in particular in the treatment of burns.

 The important problems which arise when applying these methods to the oral field were, in fact, such that it was not obvious, for the person skilled in the art, to envisage a simple extrapolation of conventional methods . It should, in fact, be remembered that the mouth constitutes an eminently septic environment and that the samples taken there come up against contamination problems that are not encountered when samples are taken from the skin.

 It is known, on the other hand, that the samples taken from the skin are of relatively large dimensions which can reach several cm 2; However, it is unthinkable to consider samples of this size on the gum, for obvious reasons of protection of the site and hemorrhage.

Furthermore, the only known methods of culturing gingival epithelium are limited to explant cultures, therefore comprising both connective tissue and epithelium, which therefore do not make it possible to manufacture epithelium alone (BUCHNER, A. And MLINEK , A. (1975) J. Periodontal Res. 10: 346-356).

 The inventors therefore applied themselves to providing a solution to these various problems, which led them to adapt both the conditions for graft removal and the very culture conditions, so as to obtain stratified cultures of original oral epithelial cells. human.

The process for obtaining equivalent gingival epithelium according to the invention is characterized in that a superficial gingival micro-sampling, a few tenths of a millimeter thick, is carried out, essentially limited to the epithelial layer, said sampling then being cultured and conventionally treated, in order to eliminate microorganisms and bacteria, conventionally trypsinized and the cell suspension obtained deposited on a support culture of type 3T3 irradiated until obtaining
confluent primary cultures which are again dissociated by trypsinisa
tion, seeded and cultivated until secondary crops are obtained
stratified confluences with the formation of multi-layered layers, and in this
that the secondary culture epithelium once confluent is then detached
of the culture support by enzymatic way then deposited on a support before
use.

According to the invention, the superficial sampling of buccal epithelium
is carried out using a razor-type device, which makes it possible to limit
essentially the removal from the epithelial layer; we have this
made, of a maximum of usable cells, with few losses due to
connective. The iso method is then applied to this buccal epithelium
and culture of epidermal cells described by O'CONNOR, NE,
CALLICO, G., COMPTON, C., KEHINDE, O. and GREEN, H., in "HUNT, TK,
HEPPENSTALL, KB, PINES, E. and ROVER, D., eds. "Soft and hard tissue
repair: Biological and Clinical aspects ". (New York: Praeger Scientific
Vol. 2: 283-29, 1984).

It is this process, which will be described in more detail below, which
provides layers of multi-layered epithelial cells.

The present invention will be better understood and its advantages
will emerge clearly from the following description of the process for obtaining
the equivalent gingival epithelium according to the invention, as well as its applica
tions in odonto-stomatology.

After anesthesia of the site and cleaning with merfen, we perform, on
a palate 4 to 5 / 10th of a mm thick, a sample of about 0.4 x
0.4 cm of oral epithelium using a scalpel with interchangeable blades,
a Klewanski epitome or a mucotome. This essential biopsy
made up of epithelial tissue, is immediately placed in
DMEM (Dulbecco's Modified Eagle Medium) supplemented with 100U / ml of
penicillin and 100 pg / ml of penicillin streptomycin (P / S).

The sample is rinsed successively in ten buffer baths
phosphate at pH 7.2 (PBS) with antibiotics (P / S). The biopsy is cut
as finely as possible and incubated with a 0.1% trypsin (VV) mixture
(in Hanks' without calcium or magnesium) -EDTA (Ethylenediamino Acid
tetracetic) 0.02 g / 100 ml (in PBS without Ca or Mg + P / S) in a
trypsinization bottle (CELSTIR 25 ml) with stirring at 370C for
thirty minutes. The supernatant is removed, added with an equal volume of
culture medium devoid of epidermal growth factor (EGF) and containing fetal calf serum (SVF) which stops the action of the enzyme.

 This supernatant is replaced in CELSTIR by an equal volume of trypsin-EDTA mixture, the flask is again stirred at 37 "C for thirty minutes. The supernatant is treated as for the first trypsinization. These two cell suspensions are centrifuged at 1200 rpm for five minutes; the pellet is taken up in culture medium without EGF. The dissociated epithelial cells (CE) can then be seeded in flasks (FALCON F25) at the rate of 5.105 cells / 25 cm2 on 5.10 3T3 fibroblasts ( strain 32) irradiated at 6000 Rads.

The cultures are maintained at 37 ° C. in a humid atmosphere at 10 Sb of carbon dioxide (CO2). After 48 hours, the medium without EGF is replaced by complete medium of composition given by O'CONNOR et Al.
DMEM. 3 volumes (Gibco),
HAM's F12 1 volume (Gibco),
+ 10, ó Fetal calf serum (Boerhinger)
(decomplemented: 30 minutes at 56 "C),
+ 100 U / ml Penicillin (Gibco)
+ 100 pg / ml Streptomycin (Gibco),
4 0.4 pg / ml Hydrocortisone (France Biochem),
+ 10 10M Cholera toxin (Sigma),
+ 5 llg / ml Insulin (Sigma),
+ 8.10 4M Adenine (Boerhinger),
+ 5 pg / ml Transferrin (France Biochem),
+ 2.10-9M Triiodothyronine (Rhône-Alpes Chimie),
+ 10ng / ml EGF = Epidermal Growth Factor (Sigma)
(add after 48 hours of primary culture).

 After fourteen to twenty days, the confluent primary cultures are dissociated with the trypsin-E.D.T.A mixture. (five minutes at 379C). As before, the trypsin is inhibited by adding complete medium, the suspension is centrifuged and the pellet taken up in complete medium. About 3.105 to 106 cells are isolated. Dissociated epithelial cells are cultured in the presence of irradiated 3T3 fibroblasts.

 This time, the keratinocytes are seeded at densities of between 0.7 and 5.105 cells per 25 cm2. The cultures are maintained under the same conditions as the primary cultures.

 As the epithelial colonies develop, the murine fibroblasts are repelled then detach and are eliminated from the vials during changes of environment. The epithelial cells after adhesion to the support have a polygonal appearance and are very contiguous.

After 14 to 16 days, the primary cultures reach confluence. Secondary cultures (subcultures) reach confluence more quickly,
6 5 to 6 days for a seeding density of 5.106 EC per 25 cm2.

The epithelium in secondary culture, once confluent, is detached from the support by an enzymatic route (dispase 11) and deposited on a vaseline gauze which serves as support for the culture-graft before surgical use.

The miniaturization of the method making it impossible to remove the connective tissue from the biopsy, one might have feared the proliferation of human fibroblasts. But the samples being very thin, this problem is not frequent. In addition, it is possible to detach fibroblasts adhering to the culture support by treatment with EDTA (0.02% in
PBS) for 3 minutes.

 The study in light microscopy makes it possible to determine that, at confluence, the culture has a single cell base and then stratifies. After treatment with the stratified cultures, it is possible to distinguish a layer of basal cells and a series of suprabasal layers, made up of more flattened cells.

 The study in electron microscopy makes it possible to determine that the buccal epithelium of pluristratified culture does not have a horny layer but only a basal layer surmounted by six to eight layers of suprabasal cells.

 Cultured cells have a polyhedral appearance; in section the different layers appear cuboid or flattened, that is to say analogous to what is observed in vivo at the level of the first layers of the buccal epithelium.

The epithelium was also detached from its culture support and placed in contact with a section of human dental enamel according to the following process
Healthy teeth extracted for orthodontic convenience are carefully cleaned (by scraping), passed with alcohol to 90 "then cut in section including exclusively the crown (Isomet BUEHLER). Before use, these sections are passed to alcohol and dried at the oven.

 The confluent secondary cultures are treated with the dispase, rinsed with culture medium and then delicately deposited on the side of the section constituting the surface of the tooth. The cell film is left with little medium for two hours at 37 "C to allow it to adhere to the enamel, then the" reconstituted "epithelium is covered with medium. The cultures in contact with the enamel are kept in the usual conditions for five days.

 After five days of enamel culture, the cells take on a more elongated appearance resembling the suprabasal cells of the junctional epithelium in vivo.

 In addition, these cells deposit on the enamel a structure comparable to the internal basement membrane of the junction epithelium composed of three sheets: lamina lucida, lamina densa, lamina (pars) fibroreticularis.

Dense plaques were also observed. These adhesion structures have strong analogies with hemidesmosomes (intracellular filaments, thickening of the cytoplasmic membrane). In vitro, on the morphological level, the adhesion of the epithelial cells to the enamel is quite comparable to what is observed in vivo in the junction epithelium.

 Dental enamel therefore induces on epithelial cells in vitro the differentiation of adhesion structures which are not observed during cultures on plastic, the properties of the substrate appearing to determine the nature and structure of the contact sites.

 The present invention also relates to the applications of the equivalent gingival epithelium obtained in odonto-stomatology and in particular the production of gingival grafts.

The transplant intervention is done under the following conditions:
- preparation of the recipient site by making a conjunctivo-periostate bed, or even cemento-conjunctivo-periosteum in cases of root covering requiring a favorable ratio: vascular surfaces / avascular surfaces;
- establishment of the graft culture (mounted on gauze) on the recipient bed previously covered with Tissucol to ensure "bonding" and the most intimate fixation possible between the graft and the recipient bed. If necessary, we realize a protective anchor by making sutures passing at a distance and guaranteeing a plating without displacement of the graft
- protection using a non-sticky screen of the gauze supporting the graft and fitting of a COE PACK type surgical dressing fixed on the teeth
- usual post-indications including mouthwashes, antibiotic therapy and if necessary anti-inflammatory analgesics
- removal of the surgical dressing eight days later, cleaning of the wound and its surroundings, placement of lighter protection for another week
- fifteen days after the operation, the dressings and sutures are removed
- post-operative control at one month, three months and six months.

 It is known, moreover, that the replacement of the dental organ by a prosthesis or implant (endo or justa-osseous) encounters difficulties in attaching the junction epithelium at the level of the collar of the material.

Under these conditions, we can understand the advantage of growing epithelial cells in multi-layered layers around the material to be implanted before carrying out the in situ placement in the patient. So,
Can the implant / epithelial cell interface be prepared in vitro, which eliminates the problems, very common in implantology, of bacterial spread between the implant, the conjunctiva and the underlying bone, the main cause of current failures.

 The invention has numerous advantages over the methods of the prior art and in particular over free gingival grafts.

- Sampling is always possible, because it is minimal q elques mm2 instead of 2 to 3 cm2 for free gingival grafts
- The sample is very superficial: 4 to 5 / 10ths of a mm thick against 1.5 to 2 mm for an epithelio-conjunctive sample. It leaves only a very minimal wound which is quickly repaired with little pain for the sick, while the usual samples (grafts) pose the problem of protecting the donor site still imperfect and giving uncomfortable consequences for eight to ten days
- There is no hemorrhagic problem due to the size of the sample and its thickness
- The possibilities offered by cultivation guarantee that there is always enough covering material whatever the extent of the intervention area to be treated and this in a single operating sequence.

In addition to the indications similar to those of free gingival grafts, cultures allow to consider immediate recovery:
- conjunctive and periosteal fenestrations in vestibular deepening (suppression of painful consequences and very significant acceleration of tissue repair)
- periosteal and bony areas exposed in the lateral or apical repositioning flaps
- compulsory sampling zones aimed at ensuring the gingival closure of bone graft fillers in modified Widman type flaps.

 Finally, the use of allograft and the establishment of available culture banks will avoid any waiting (three weeks) and any withdrawal before the intervention.

Claims (8)

 - CLAIMS  1- Method for obtaining equivalent gingival epithelium, characterized in that a superficial gingival micro-sampling of some mm 2 of surface and a few tenths of millimeters in thickness is carried out, essentially limited to the epithelial layer, said sampling then being cultured and treated in a conventional manner, in order to eliminate microorganisms and bacteria, conventionally trypsinized and the cell suspension obtained deposited on a support culture of type 3T3 irradiated until primary confluent cultures are obtained which are again dissociated by trypsinization, seeded and cultivated to obtain confluent secondary cultures stratified with formation of multi-layered layers.  2- A process for obtaining equivalent gingival epithelium according to claim 1, characterized in that the epithelium in secondary culture once confluent is detached from the culture support by enzymatic route, then deposited on a support before use.  3- A method for obtaining equivalent gingival epithelium according to claim I and claim 2, characterized in that the gingival sampling is carried out using a razor-type device.  4- A method for obtaining equivalent gingival epithelium according to one of claims I to 3, characterized in that the gingival sample is taken from the patient intended to subsequently receive the equivalent gingival epithelium.  5- A method for obtaining equivalent gingival epithelium according to any one of claims 1 to 4, characterized in that the gingival sampling is carried out on any patient and in that the equivalent gingival epithelium thus obtained is stored in a bank of culture to later allow the production of allografts.  6- A method for obtaining equivalent gingival epithelium according to any one of claims 1 to 5, characterized in that the confluent secondary cultures are treated with dispase, rinsed with culture medium, deposited on a section of enamel dental, then maintained for about five days in culture condition.  7- A method for obtaining equivalent gingival epithelium according to any one of claims 1 to 5, characterized in that the culture of epithelial cells in multi-layered layers is carried out around a material to be implanted or placed in the mouth, before placement of the implant or prosthesis in the patient.  8- Applications of the gingival epithelium obtained according to any one of claims I to 7 in dentistry stomatology.