Method and membrane for regenerating mucosa

FIELD: medicine.

SUBSTANCE: the present innovation deals with the method to accelerate mucosal healing due to the following technique: one should apply a membrane consisted of purified collagenic material obtained out of natural collagen-containing tissue onto the part of affected mucosa to provide the chance for mucosal reconstruction in this part and, also, it deals with mucosa-regenerating preparation and application of purified collagenic material obtained out of collagen-containing natural tissue for preparing mucosa-regenerating preparation. The innovation provides more modified method that accelerates mucosal regeneration, as a whole, and, particularly, after surgical operations associated with the plasty of oral fornix.

EFFECT: higher efficiency.

12 cl, 3 dwg, 5 ex

 

Background of invention

The technical field of the invention

The present invention generally relates to a method of regeneration of the mucous membrane.

Discussion current level of technology

In recent years, there has been normal use of transplants of type "split flap" of skin grafts of the type "free flap" of the oral mucosa in the vestibular cavities and other types of vestibuloplasty in the oral cavity (plastic arch of the oral cavity). In addition, the grafts of the type "free flap" mucosa taken from the mucosa of the palate and the buccal region, used for the reconstruction of surfaces attached mucosa in areas deprived of teeth, alveolar process, in particular, in connection with the use of implants root type.

Despite the widespread use of such methods of transplantation, the development of effective alloplastic material or xenogenic material substitute for the transplant, which can be used for recovery and regeneration of normal mucous membranes, would significantly expand the use of surgical operations associated with the plastic arch of the mouth.

Thus, in this area there is a need to improve methods of accelerating the regeneration of the mucosa is blocki, in General, and in particular after surgical operations associated with the plastic arch of the oral cavity

Summary of the invention

According to the present invention mucosa regeneration speed by using a membrane consisting of purified collagen material derived from natural tissues containing collagen.

Brief description of drawings

Figure 1 presents a view in plan of the flap constituting a collagen membrane that covers the area of the mucous membrane, which is subject to treatment.

Figure 2 presents a side view of a membrane used according to the present invention.

Figure 3 presents a side view of another type of membrane used according to the present invention.

A detailed description of the preferred embodiments

The present invention relates to a method of accelerating the regeneration of the mucous membrane. Although the method is described in relation to the regeneration of the mucous membrane in the oral cavity after certain types of surgery in the oral cavity, it should be borne in mind that the method can be applied to accelerate the regeneration of the mucous membrane in any part of the body where there is damage to the mucosal tissue.

In preferred embodiments implement a method to accelerate the regeneration of letisti shell stage provides an overlay on the plot, subject to treatment, the flap of material representing a sheet of collagen membrane, where material, which is a collagen membrane comprises at least one layer having on one side a smooth surface, and the side opposite to the smooth surface has a fibrous surface, where the fibrous surface enables the cells to grow on it, and collagen membrane preferably contains a second layer, consisting mainly of collagen type II (collagen II) or a mixture of collagen type I (collagen I and collagen type III (collagen III), having outside spongy structure. A flap of material secured on the damaged area of the mucous membrane preferably using suture material and mucous membranes provide an opportunity to regenerate.

According to the present invention, as shown in Fig. 1, damage to the mucosa M restore, covering the damaged area of the flap 10 and securing the flap on the edges of the mucous membrane around the damage. Then give the possibility to regenerate the mucous membrane at the closed flap of the plot. As shown in figure 1, the flap 10 is fixed on the surface of the mucosa M using suture material 12. Alternatively, the flap can be fastened over the damaged area, attaches a compass is with an adhesive flap to the mucous membrane of the host or other structures, surrounding the surface to be treated, for example, using known in the field of organic glue, or using any other suitable method.

The flap 10 is made from the drug, which is a collagen membrane with the need for flexibility for the tight junction to the shaped surface area of the mucous membrane, on which it is placed. In a preferred embodiment, the collagen membrane consists of at least one collagen layer having a sufficient strength so that you can use suture material for its attachment to the mucosa and to protect the surface of the mucosa from injury in the healing process.

The first option collagen membrane, which can be used as a flap according to the present invention, is presented in figure 2, number 14. The membrane 14 has one collagen layer 16, having on one side a smooth surface 18 and textured or fibrous surface 20 on the other hand, the opposite smooth surface. Smooth surface 18 preferably should not be porous to provide mechanical protection, for example, in the process of eating. The configuration of the fibrous surface 20 is adapted to enable grew the cells and on it. In the practical use of the smooth surface is preferably oriented in the direction opposite to the surface to be processed, and the fibrous surface is preferably oriented in the direction of the area to be processed.

In preferred embodiments, the implementation of the collagen layer 16 is composed primarily of collagen I, collagen III or a mixture thereof One of the materials that can be used for this purpose is Biogide®manufactured by Ed. Geistlich Söhne AG für Chemische Industrie, which is the assignee of the present invention. Biogide®described in U.S. patent 5837278 included in the present description by reference.

Figure 3 presents the multilayered collagen membrane 14, which can be used as a flap according to the present invention. As is shown in Fig. 2, this membrane contains a first collagen layer 16 and the second collagen layer 22, having outside spongy structure. The second collagen layer 22 is preferably attached to the fibrous surface 20 of the first collagen layer 16 in order to place it on the surface of the mucous membrane to accelerate the regeneration of the mucous membrane. The second layer 22 may consist of collagen I, II, III, IV or VII or any combination of these types of collagens, but preference is sustained fashion it consists mainly of collagen I, collagen II or a combination of collagen I and collagen III (for example, 95% of collagen I and 5% collagen type III). When using the combination of the first (16) and second (22) of the layer increases the thickness of the membrane 14', which allows to simplify the application procedures and improve the healing process. The thickness of the membrane may vary depending on the purpose, but usually it is from about 0.5 to about 5 mm, preferably from about 2 to about 5 mm, the most preferred thickness is approximately 3 mm

For embodiments represented in figure 2 and 3, the first layer 16 can be obtained in various ways, including (but not limited to) the stage of the process described in the patent US 5837278; by deaeration and drying air suspension (resembling transparent film membrane); by deaeration and vacuum drying of the suspension (resembling transparent film membrane), or to use for him subjected to pressing the spongy material. The first layer 16 can be manufactured from collagen I, II, III or IV or a combination of these collagens types.

The second sponge layer 22 in the embodiment shown in Fig. 3, preferably obtained by freeze-drying the collagen suspension. The second layer 22 can be manufactured from collagen I, II, III, IV or VII sludge is a combination of these types of collagens. Preferably the second layer is made from collagen I, collagen II, or of a mixture of collagen I and collagen III

The second collagen layer 22 can be made from a substance derived from an organism of cows or pigs, preferably from the body of pigs.

In the variant presented in figure 3, the first (16) and second (22) layers of the membrane can be connected with each other or merge any suitable method. Three examples of methods that can be used for this purpose may include: attaching a first layer of the membrane to the second layer of the membrane using fibrin glue; attaching the first layer of the membrane to the second layer of membrane with collagen suspension; or applying the first layer of the covering membrane of the collagen suspension containing collagen type you want, with subsequent freeze-drying the obtained combination.

If necessary, the composition of the membrane can be entered growth factors such as EGF (epidermal growth factor), IGF-1 (insulin-like growth factor), β-FGF (fibroblast growth factor), PDGF (platelet-derived growth factor), TGF-β (transforming growth factor), which accelerate the regeneration of the mucous membrane, and/or they can be applied to the membrane, which is placed on the mucous membrane

Example 1

Receiving flap

(A) a First membrane layer receive according to the method is written in WO 93/11723 (drug containing purified collagen type I, obtained from pig skin), which is incorporated into this description by reference. The resulting washed fiber is not subjected to drying by freezing, but the product is subjected to vacuum deaeration when applied in strips and dried in a laminar air stream at room temperature.

(B) a Second membrane layer receive according to the method described in WO 93/11723 (preparation containing purified collagen type I was obtained from porcine tendons). The resulting washed fiber is not subjected to drying by freezing. Them again acidified with hydrochloric acid to obtain a pH of 2.9 to 3.2, while the dry matter content of 1.2 to 1.8%. The resulting suspension is dried by freezing, getting the product in the form of a sponge.

(C) First and second layers of the membrane unite by applying to one side of the first layer of the membrane above the suspension having a pH value of 2.9 to 3.2. The suspension turned into a product in the form of a sponge and dried by freezing.

Example 2

Receiving flap

(A) A First membrane layer receive according to the method described in patent US 5837278.

(B) a Second membrane layer receive according to the method described in WO 99/19005, which is included in the present description by reference, receiving a suspension of collagen type II. It should be noted that the process of obtaining Zam is Rosenau cartilage from the moment of taking her from freshly slaughtered pigs to the stage of washing with cold water takes a lot of time, during which the pH value is reduced to 3-3 .5. After this product 5 times subjected to homogenization and diluted with water, obtaining a homogeneous mass with a dry matter content of 0.5-2.0%.

(B) First and second layers of the membrane unite by applying obtained in the previous phase of the slurry on the first layer. After 30 min the combined product is dried by freezing.

Example 3

Receiving flap

(A) a First membrane layer receive according to the method described in patent US 5837278.

(B) a Second layer of the membrane is obtained by processing low-fat pork skin for approximately 4 hours at approximately 20°1 N. a solution of sodium hydroxide, then processing for approximately 2 hours at approximately 20°With 1 N. hydrochloric acid and homogenization according to the process described in example 2.

(C) First and second layers of the membrane unite by applying obtained in the previous phase of the slurry on the first layer. After 30 min the combined product is dried by freezing.

Example 4

Receiving flap

(A) A First membrane layer obtained by drying the suspension obtained according to the method described in example 3, removing the water with acetone, after which containing acetone wet product is clamped between two glass plates and the acetone removed under vacuum for approximately 48 h the ri pressure less than about 1 mbar and at a temperature of about 35° C.

(B) a Second membrane layer is obtained by mixing equal amounts of suspensions obtained in the second stage of examples 2 and 3, and freeze-drying the mixture to form a product in the form of a sponge.

(B) First and second layers of the membrane unite by gluing using fibrin glue.

Example 5

Testing

Intended for studying membrane material consisting of two layers was obtained from porcine collagen type I and type III. Deep or internal (second) layer of a collagen membrane had a rough surface, which was placed on the treated surgically site owner to facilitate the formation of a clot of blood and stimulation of neoangiogenesis. The outer (first) layer of the bilayer collagen membrane was smooth structure and had elastic characteristics, sufficient to ensure that the flap could be attached with suture material to the edges of the mucous membrane of the host and to protect the material of the graft (graft) from injury when placed in the mouth during the process of biodegradation and healing. In all cases, the plots for testing served four squares of the oral mucosa of adult baboons Papio anubis. In the vestibular areas of the canine and premolar cut out a section of an elliptic shape with a size of 3 cm×2 cm to a depth of layer, located n the d mucosa. This model allowed to obtain twelve samples for research in the areas of vestibular deepening.

In addition, to evaluate the effectiveness of the material when used as the basis for engraftment attached mucous membrane in deprived teeth areas of the alveolar process, used devoid of teeth sections attached to the mucosa of the upper jaw. Three animals carved on the surface area of the sky 1.5 cm×3 cm, located bilaterally along the crest of the ridge at a distance of 1 cm from the buccal surface and the crest of the ridge.

The membrane material was placed and fixed on the edges of the mucosal surface of the host using interrupted sutures using nylon nanovolokno suture material with the number of 4-0. (The total number of stitches for securing each collagen membranes ranged from 10 to 16). After surgery for four days, the animals were injected intramuscularly antibiotic drug containing 150000 units of penicillin, procaine and 150,000 units of bicillin. Animals were kept on normal diet and were given the opportunity to move freely circles in large cages.

After a three-week and six-week periods was carried out by biopsy of the vestibular areas subjected to chirurgeons the WMD intervention. Three weeks later performed a biopsy of each animal with one hand (2 square area) in the region extending from the top of the remaining native surface of the mucous membrane of the host through the surface material of the membrane to the opposite bottom edge of the portion of the mucous membrane graft owner. Surface samples were labeled accordingly with the help of suture material in order to identify their orientation when conducting histological studies. Subjected to biopsy the area was closed using interrupted sutures and left to heal. Two square plot for each animal kept for six weeks, then cut out the entire area and the surface of the mucosa was separated and brought together to implement primary closure.

Similarly performed the biopsy areas of attached mucosa: 3 weeks - on the one hand, and after 6 weeks on the other side of each animal, selecting each time for three samples.

In all cases, the suture was removed on the 14th day after surgery. Clinical postoperative period for animals took place without complications. Was not observed clinical signs of inflammation or infection, as well as separation of the membrane material. The membrane was left in place and the edges were evident post the military epithelium from peripheral areas of the surfaces of the mucous membrane of the host. Plots of the residual material of the membrane was detected in a clinical setting within 2-3 weeks after surgery. After 6 weeks all subjected to surgical intervention areas the epithelium was restored with the formation of the mucosa with a normal appearance.

After 3 weeks of biopsy testified to the epithelium at the edges of the plot with the formation of normal retinal structure. Were visible region decomposing collagen, including the epithelium, propagating on the membrane surface. In the Central part of the membrane approximately 0,5×0.3 cm (initial size 3×2 cm) recovery of the epithelium was not observed.

In samples taken after 6 weeks, there was a very good development of the mucosal surface in the entire area, closed by flap, according to the biopsy completely cut out of the closed flap of the site was observed neoangiogenesis in the layer located under the mucous membrane, and the formation of normal stratified squamous epithelium. There was complete settling down of the membrane material. You can see only small areas of residual collagen under the surface of the epithelium. As the dyes used trichromacy dye of cap Mason and hematoxylin and eosin.

To ensure a complete study of all closed the x flap areas have received a large number of sections from samples taken after six weeks, according to histological analysis found no residues of membrane material.

When you put the flap on the back part of the alveolar ridge and the surface of the palate of the upper jaw was observed substitution membrane, similar to that which occurred in the studied area type vestibular deepening. Three weeks was observed the presence of residues of membrane material in the Central part of the area covered by flap. You can find fibroblasts and epithelium extending from a peripheral edge region, closed the flap. All samples taken at six weeks, was discovered by a new attached mucosa on the crest of the alveolar ridge on the buccal side of the alveolar protrusion. It shows the complete engraftment of material and very good recovery of attached mucosa.

These histological analysis suggests that this material can be used for restoration of attached mucosa in the posterior maxilla using implants root type, although the efficiency for this area was not specifically studied in this experiment.

Plots for vestibular transplantation animals were selected in the vestibular mucosa of Obolo the key from the fangs on the upper and lower jaw and premolar. This area is most susceptible to injury in the baboon Papio anubis in the process of eating and grinding food, and it can serve as the most stressful test area. Thus the material was placed in conditions in which there is a high probability of injury with subsequent infection and separation of the flap. No such cases were observed in any of the investigated areas, the materials remained in place and were subjected to a very weak process of biodegradation in the cell activity.

Based on the results of these experiments it was concluded that the collagen membrane is used as a flap according to the present invention, is a very good Deputy autogenous soft tissue grafts. Some types alloplastic or allogeneic surface bandages tend to cause a wound contracture and scarring. For surface dressings of artificial leather tends either prematurely to biodegradation and flake off, or remain the same over an extended period of time, which is accompanied by a prolonged inflammatory reaction. When using the present invention did not reveal any scarring or a prolonged inflammatory response. Did not reveal any signs of fibrosis layer, nah is masegosa under the mucous membrane, sometimes when using surgical dressings.

Thus, apparently, the proposed material can be applied as Deputy transplants type of free flap of mucous membrane or graft-type "split flap" of skin while maintaining the height of the vestibular cavity and the restoration of attached mucosa in the area of insertion of the implant root type. One of the obvious advantages of this method is that the membrane (a) is subjected to biodegradation, without causing harmful inflammatory reaction, (b) enhances regeneration in areas with normal mucosa, and (b) technically suitable for use in surgical intervention and can withstand suturing.

As presented in this description of embodiments, the implementation can be done numerous modifications, variations and modifications of separate parts, it is assumed that all the options presented in the above description and illustrated in the accompanying drawings should be considered only as illustrations, which do not limit the scope of the invention.

1. The way to accelerate the regeneration of the mucous membrane, characterized in that the area of the damaged mucous membrane covering the rejuvenating kisitu the shell material, which includes a membrane consisting of purified collagen material derived from natural tissue containing collagen, fix the membrane in the area and provide an opportunity to recover the mucous membrane on the specified site, where this membrane is a barrier layer having a smooth external barrier surface and also having on the side opposite the specified smooth barrier surface, fibrous surface.

2. The method according to claim 1, where the barrier layer mainly consists of collagen type I, collagen type III, or a mixture thereof.

3. The method according to claim 2, where the membrane is a multilayer sheet of collagen membrane material that includes a matrix layer of collagen material, attached to the fibrous surface.

4. The method according to claim 3, where the matrix layer consists of collagen type I, collagen type II, collagen type III, collagen type IV, collagen type VII or any combination thereof.

5. The method according to claim 1, where the membrane has a thickness of approximately 0.5-5 mm

6. The method according to claim 4, where the matrix layer is composed primarily of collagen I.

7. The method according to claim 4, where the matrix layer consists predominantly of collagen II.

8. The method according to claim 1, where the membrane comprises at least one regenerating mucosa growth factor.

9. JV is the sob of claim 8, where at least one growth factor selected from the group including epidermal growth factor (EGF), insulin-like growth factor (IGF-1), fibroblast growth factor (β-FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF-P), or their mixture.

10. The method according to claim 1, where the mucosal damage is a damage to the mucous membranes of the mouth.

11. Regenerating mucosa, the drug designed to enhance regeneration of the mucous membranes, including membrane consisting of purified collagen material derived from natural tissue containing collagen, where this membrane is a barrier layer having a smooth external barrier surface and also having on the side opposite the specified smooth barrier surface, fibrous surface, for use according to claim 1.

12. The use of purified collagen material derived from natural tissue containing collagen, for the manufacture of regenerating mucosa of the drug, intended to accelerate the regeneration of the mucous membrane of claim 1.



 

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