Claims:We claim

1. A guided tissue regeneration multilayer collagen membrane exhibiting the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like, the claimed multilayer collagen membrane comprising a significantly purified collagen multilayer membrane with at least about 90% by weight native collagen, having two distinct surface textures on either side of the said multilayer membrane comprising a dense smooth surface and embossed porous surface wherein the dense smooth surface forms the top layer thereby contacts with soft tissue to prevent epithelial down growth and wherein the embossed porous surface forms the bottom layer thereby contact with bones and enhances bone growth, wherein the said multi-layer membrane is formed by adhering atleast two purified collagen membranes through collagen gel and compressing with special equipment thereby forming embossed porous surface on one face and dense smooth surface on opposite face.

2. The membrane as prepared in claim 1 wherein the said purified collagen membrane prepared by the process comprising of;

a. harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet
b. subjecting the sheets of step (a) to dehydration treatment to form sheet having soft dense surface
c. cutting the obtained soft sheets of step (b) into required size and dipping in 1% solution of PCL and allowing to dry at normal room temperature to form dry purified collagen membrane.

3. The membrane as claimed in claim 1 wherein the said collagen gel is prepared by the process comprising of

a. harvesting collagen from bovine tendon followed by cutting and mincing the said collagen to formed minced collagen
b. subjecting the minced collagen of step (a) to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue
c. subjecting the tissue of step (b) in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material collagen gel.

4. A method of preparing guided tissue regeneration multilayer collagen membrane having two distinct surface textures on either side of the said multilayer membrane comprising a dense smooth surface and embossed porous surface, which membrane exhibiting the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like and comprising at least about 90% by weight native collagen, the claimed preparation method comprises of following steps,
a. harvesting collagen from bovine Tendon followed by cutting and mincing the said collagen to form minced collagen
b. subjecting the minced collagen of step (a) to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue
c. subjecting the tissue of step (b) in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material in the form of gel
d. harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet
e. subjecting the sheets of step (d) to dehydration treatment to form sheet having soft dense surface
f. cutting the obtained soft sheets of step (e) into required size and dipping in 1% solution of PCL and allowing to dry at normal room temperature to form dry sheets
g. pasting atleast two sheets obtained in step (f) with the gel obtained in step (c) and allowing to dry at normal room temperature to obtain multilayer membrane and
h. finally compressing the multilayer membrane obtained in step (g) using special equipment to form the guided tissue regeneration multilayer collagen membrane having embossed porous surface on one face and dense smooth surface on opposite face.

5. A guided tissue regeneration multilayer collagen membrane prepared by the process as claimed in claim 4.
6. The guided tissue regeneration multilayer collagen membrane of any of the preceding claims has a thickness of 0.15mm to 0.17mm when dry.
7. The guided tissue regeneration multilayer collagen membrane of any of the preceding claims has longest in vivo stability time of 20-24 weeks, which accounts for the sustained function during the bone repair process.

Dated this 27th day of August 2015
For COLOGENESIS HEALTHCARE PRIVATE LIMITED
By its Patent Agent
Dr.B.Deepa
, Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“MULTILAYER COLLAGEN GTR MEMBRANE AND METHOD OF PREPARATION THEREOF”

in the name COLOGENESIS HEALTHCARE PRIVATE LIMITED, an Indian national having address at Plot No 51,The Salem Co-Operative Industrial Estate Udayapatti,Salem-636140 Tamil Nadu, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed
FIELD OF THE INVENTION:
The present invention generally relates to wound healing. More particularly the present invention relates to a collagen membrane implant for use in guided tissue regeneration.
BACKGROUND OF THE INVENTION AND PRIOR ART:
Experimental and clinical application of the guided tissue regeneration concept led to the use of different materials in periodontal regenerative therapy. The first material clinically used in periodontal surgery allowing regeneration of cementum, periodontal ligament and alveolar bone, was cellulose acetate laboratory filter. Thus for the first time periodontal regeneration as a response to GTR was histologically verified. Since that time membranes from different materials for achieving periodontal regeneration have been developed and modified.
Biomaterial is a non-viable material used in medicine and dentistry intended for interaction with biological systems. Any material introduced into the human organisms such as GTR membranes has to fulfil two important requirements: safety and efficacy. Safety is assessed through wide selection of in vitro and in vivo assays for testing specific aspects of biocompatibility. Cell culture cytotoxicity, subcutaneous implantation, blood compatibility, haemolysis, carcinogenesis, mutagenecity, pyrogenecity, short and long term histological tissue reaction are some of the assays used to evaluate biocompatibility.
There are many forms of GTR membrane available in the Market such as resorbable Membrane and Non-resorbable membrane.
Non resorbable membrane retains their build and form in the tissues requiring a second surgical procedure for removal, thus adding to the trauma of the periodontal tissues and to patient Discomfort as well as raising the costs and duration therapy. This led to development of resorbable membranes. whereas in the case of resorbable membrane it does not require a second surgery, reduce patient discomfort and costs and eliminates potential surgical complications.
Resorbable membrane sometimes pose problem such as form and size of the defect probably due to compromised space maintenance. Some membranes even show significant inflammatory reaction.
US 5622707 discloses Biomaterials comprised of biodegradable, biocompatible, and bioabsorbable composite membranes for use in surgery for the guided regeneration of tissues. The composite membranes are comprised of threads embedded in a matrix, wherein both the matrix and the threads can be comprised of esters of hyaluronic acid, used singly or in combination, or esters of hyaluronic acid in combination with esters of alginic acid or other polymers.
US 6155831 discloses a dental appliance consisting of a biocompatible, non-absorbable retrievable membrane material may be used as a separation and isolation barrier following periodontal surgery to promote tissue regeneration. The appliance is configured with a cord woven into the inferior aspect of the membrane and extends up from the apical border of the membrane through a loop in the superior border. The free end of the cord extends through the gingival sulcus and remains exposed while the membrane is in place. At the appropriate time for membrane removal, the sutures holding the membrane in place are released and the membrane removal cord is use to lift the membrane through the sulcus by gently pulling on the cord.
US 5,297,563 discloses a device for facilitation of tissue and bone guided regeneration of a bony deficit comprising a non-resorbable barrier placed over a tissue deficit. The barrier must be surgically removed.
US 6752834 discloses a multi-layer membrane comprising a matrix layer predominantly of collagen II and having an open sponge-like texture, and at least one barrier layer having a close, relatively impermeable texture.
US 8268361 methods and compositions useful in the regeneration of damaged, lost and/or degenerated tissue in humans and animals. In certain embodiments, the invention provides an acellular bioabsorbable tissue regeneration matrix, methods of making such a matrix, and methods of using such a matrix for the regeneration of damaged, lost and/or degenerated tissue.
From the detailed prior art review it is inferred that the various available guided tissue regeneration membrane suffers various draw back which includes the following.
o Histological evaluation of Resorbable membranes shows significant inflammation reaction.
o The size of the membrane poses a defect.
o Some membrane derived from bovine corium is no more effective in humans.
o Non resorbable membrane causes complications such as pain, purulence and swelling.
Thus there exists a need in the state of art to develop a novel tissue regeneration membrane which is devoid of above said draw backs.
OBJECT OF THE INVENTION:
The main object of the present invention is to develop a safe, efficient and highly stable guided tissue regeneration collagen multilayer membrane.
Another object of the present invention is to develop the multilayer membrane possessing the property of biocompatibility, cell exclusion, space maintenance and tissue integration.
Yet another object of the present invention is to develop a method of preparing guided tissue regeneration multilayer collagen membrane.
Yet another object of the present invention is to develop a guided tissue regeneration multilayer collagen membrane having two distinct surface textures on either side comprising a dense smooth surface for contacting the soft tissue to prevent epithelial down growth and embossed porous surface for contacting with bone and favouring bone in growth.
Yet another object of the present invention is to develop a guided tissue regeneration multilayer collagen membrane which has property adoptable to surface and which should have controlled resorption time.
Yet another object of the present invention is to develop a guided tissue regeneration multilayer collagen membrane which can withstand masticatory forces, flap tissues tension or reduction of wound spaces and also active in the future.
Yet another object of the present invention is to develop a guided tissue regeneration multilayer collagen membrane which is resorbable and does not demand second surgical procedure for its removal.
Further object of the present invention is to utilize the developed guided tissue regeneration multilayer collagen membrane for periodontal regenerative therapy and the like.
SUMMARY OF THE INVENTION:
The present invention discloses a guided tissue regeneration multilayer collagen membrane exhibiting the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like. The present invention further discloses method of preparation of guided tissue regeneration multilayer collagen membrane. The multilayer collagen membrane comprises of a significantly purified collagen membrane with minimum of 2 layers and with at least about 90% by weight of native collagen. The membrane has two distinct surface textures on either side comprising a dense smooth surface for contacting the soft tissue to prevent epithelial down growth and an embossed porous surface for contacting with bone and favoring bone in growth. The multi-layer membrane is formed by adhering atleast two purified collagen membranes through collagen gel and compressing with special equipment to form embossed porous surface on one face and dense smooth surface on opposite face.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses a guided tissue regeneration collagen multilayer membrane and method of preparation thereof.
The GTR membrane invented was comprised of both collagen in native form and of the reconstituted form which helps in the healing of the wound and also acts as barrier for the microorganisms, moreover the GTR membrane enhances the growth of the cell by inducing epithelial proliferation.
The multilayer membrane of the present invention is prepared as follows. The raw material tendon was cut into small bits and minced and then subjected to Non-ionic Detergent treatment followed by alkali and salt treatment and enzymatic treatment and final product in the form of tissue is obtained.
A known quantity of tissue is taken and swelled in the acid water at the PH not exceeding 3.5 to 4.5 the swelled material in the form of Gel is taken for pasting of the sheets.
For the preparation of native collagen the raw material Serosa is taken and subjected to Non-ionic Detergent washing, alkali and enzymatic treatment and pure collagen in the form of sheets is obtained. The obtained sheet is then dehydrated by by some known methods. Now pure collagen sheets with soft nature is obtained. Then these sheets are cut into required sizes and dipped in 1% solution of PCL and allowed to dry under normal room temperature. Later these sheets are pasted with the help of Gel prepared by using Tendon and allowed to dry under Normal room temperature.
Characteristics of GTR membrane was next evaluated for its biocompatibility, cell exclusion, space maintenance, tissue integration ease of use and biological activity .
The product of the present invention possesses excellent cell exclusion property needed for the membrane to separate gingival flap from coagulum in the wound space.
The next property is space maintenance for new alveolar bone, periodontal ligament and tooth cementum. Space maintenance plays a vital role in grafting process. If the product occupies a larger space it hinders the placing of the material in appropriate place which would favour the growth of new bone. Since the material of the present invention is only 0.5mm to 0.7mm in thickness it easily gets attached to space without obstructing the growth of the new bone material.
Since this multilayer membrane consists of native collagen from serosa in layer which is more flexible. The membrane can withstands masticatory forces, flap tissue tension and prevent collapse of tissues (or) reduction of the wound space.
Tissue integration property of the product of the present invention ensures wound stabilization and inhibition of epithelial migration, resulting in gain of attachment level.
This multilayer GTR membrane being flexible, stronger and adaptable the clinician conduct the surgical procedure without undue difficulties.
The non resorbable membranes retain their build and form in tissues, requiring a second surgical procedure for removal, thus adding to the trauma of the periodontal tissues and to patient discomfort as well as raising the costs and duration therapy. To overcome the above issues the multilayer collagen GTR membrane was developed that would get resorbed on 20-24 weeks.
Thus the product of the present invention is resorbable membrane and the material gets resorbed easily within 20-24 weeks and there is no need of second surgery.
Thus the multilayer membrane of the present invention posses all the important requirements that should be fulfilled for a material to be a safe scaffold for human organism. The inventiveness of the present invention lies in the following
• The multilayer membrane is produced to provide an optimized degree of flexibility and rigidity combined with the most predictable resorptive process.
• The multilayer membrane of the present invention comprises two distinct surface textures on either side of the membrane –a dense smooth surface and embossed porous surface. The dense top layer is in contact soft tissue to prevent epithelial down growth and the porous embossed surface is in contact with bone and favours bone in growth.
• The membrane is flexible in nature that it can be shaped or modified into the required sizes where it should be placed and withstand suturing due to its strength.
• The membrane is easily adaptable in nature that once wet with blood tends to adapt extremely well to the bone wall.
• The multilayer membrane is stiff so that it does not collapse over the defect it spans
• The membrane is stiff enough to withstand the pressure exerted by the overlying flaps and designed to withstand mastication until the blood clot building underneath the membrane has matured enough to support it.
• Since the membrane is easily adaptable in nature it facilitates the safe and precise trimming of the membrane easy insertion and the formation of a good seal between the root of the affected tooth and the membrane hindering the intrusion of soft tissue cell into space.
• Since the sheets are pasted with the gel when dried these gel forms a sheet which helps the materials to possess enough tear strength to be immobilized with sutures.
• It is easy to form a seal between the root of the affected tooth and the membrane by using a sling around the tooth.
• The pliability of the material helps to strictly appose membrane to the tooth when pulled tight with the suture.
• It has definite geometry in vivo stability and mechanical strength for use as a barrier material.
• It has longest in vivo stability time 20-24 weeks, these accounts for the sustained function during the bone repair process.

In one of the preferred embodiment the present invention shall disclose a guided tissue regeneration multilayer collagen membrane. The membrane exhibits the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like. The multilayer collagen membrane comprises of a significantly purified multilayer collagen membrane with at least about 90% by weight native collagen. The membrane has two distinct surface textures on either side comprising a dense smooth surface and embossed porous surface. The dense smooth surface forms the top layer and contacts with soft tissue to prevent epithelial down growth. The embossed porous surface forms the bottom layer and contacts with bones and enhances bone growth. The multi-layer membrane is formed by adhering atleast two purified collagen membranes through collagen gel and compressing with special equipment to form embossed porous surface on one face and dense smooth surface on opposite face.
As per the invention the purified collagen membrane prepared by the process comprising of harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet . The formed sheet is subjected to dehydration treatment to form sheet having soft dense surface. Finally the obtained soft sheets is cut into required size, then dipped in 1% solution of PCL and allowed to dry at normal room temperature to form dry purified collagen membrane.
In accordance with the invention the collagen gel is prepared by the process comprising of harvesting collagen from –bovine tendon followed by cutting and mincing the collagen to formed minced collagen. The minced collagen is subjected to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue. The formed tissue is subjected in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material collagen gel.
In another preferred embodiment the present invention shall disclose a method of preparing guided tissue regeneration multilayer collagen membrane having two distinct surface textures on either side of the multilayer membrane comprising a dense smooth surface and embossed porous surface. The membrane exhibits the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like. The membrane comprises of at least about 90% by weight native collagen. The preparation method comprises of harvesting collagen from bovine Tendon followed by cutting and mincing the said collagen to form minced collagen. The minced collagen is subjected to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue. The formed tissue is subjected in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material collagen gel. Subsequently harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet. The formed sheet is subjected to dehydration treatment to form sheet having soft dense smooth surface. The obtained soft sheets is cut into required size, then dipped in 1% solution of PCL and allowed to dry at normal room temperature to form dry purified collagen membrane sheets. At least two dried sheets are pasted together with the obtained collagen gel and allowed to dry at normal room temperature to obtain multilayer membrane. The obtained multilayer membrane is finally compressed using special equipment to form the guided tissue regeneration collagen multilayer membrane having embossed porous surface on one face and dense smooth surface on opposite face.
As per the invention the guided tissue regeneration multilayer collagen membrane can be prepared by the process as described above.
In accordance with the invention the guided tissue regeneration multilayer collagen membrane has a thickness of 0.15mm to 0.17mm when dry.
According to the invention the guided tissue regeneration multilayer collagen membrane has longest in vivo stability time of 20-24 weeks, which accounts for the sustained function during the bone repair process.

EXAMPLE 1:
Preparation of collagen gel
100gms of tissue swelled in 2000ml of acid water for overnight at PH 2.5-3.5 and homogenize it thoroughly on next day.The gel formed is suitable for processing.
Ingredients required: Tissue-100gms
Acid: 5ml
EXAMPLE 2:
Preparation of multilayer membrane:
The treated serosa was subjected to 2-propranol treatment during which the serosa turns into soft latter these membrane is cut into required size and dipped in 1% polycaprolactone and allowed to dry under normal room temperature.
The PCL treated membranes are then pasted with the help of gel totally 5 layers are pasted one above the other and allowed to dry.
The dried membranes are then compressed with the special equipment to get embossed surface on one side and the soft surface on the other side.
Ingredient used: 1. Treated serosa
2. Polycaprolactone: 1 gm
3. Dichloromethane: 100 ml
4. 2-propranol: 500ml
5. Gel for binding: 10 Gms
Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. The above-mentioned details are provided to serve the purpose of clarifying aspects of the invention and it will be apparent to one skilled in the art that they do not serve to limit the scope of the invention. All modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the present invention. It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention.

We claim

1. A guided tissue regeneration multilayer collagen membrane exhibiting the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like, the claimed multilayer collagen membrane comprising a significantly purified collagen multilayer membrane with at least about 90% by weight native collagen, having two distinct surface textures on either side of the said multilayer membrane comprising a dense smooth surface and embossed porous surface wherein the dense smooth surface forms the top layer thereby contacts with soft tissue to prevent epithelial down growth and wherein the embossed porous surface forms the bottom layer thereby contact with bones and enhances bone growth, wherein the said multi-layer membrane is formed by adhering atleast two purified collagen membranes through collagen gel and compressing with special equipment thereby forming embossed porous surface on one face and dense smooth surface on opposite face.

2. The membrane as prepared in claim 1 wherein the said purified collagen membrane prepared by the process comprising of;

a. harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet
b. subjecting the sheets of step (a) to dehydration treatment to form sheet having soft dense surface
c. cutting the obtained soft sheets of step (b) into required size and dipping in 1% solution of PCL and allowing to dry at normal room temperature to form dry purified collagen membrane.

3. The membrane as claimed in claim 1 wherein the said collagen gel is prepared by the process comprising of

a. harvesting collagen from bovine tendon followed by cutting and mincing the said collagen to formed minced collagen
b. subjecting the minced collagen of step (a) to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue
c. subjecting the tissue of step (b) in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material collagen gel.

4. A method of preparing guided tissue regeneration multilayer collagen membrane having two distinct surface textures on either side of the said multilayer membrane comprising a dense smooth surface and embossed porous surface, which membrane exhibiting the property of biocompatibility, cell exclusion, space maintenance, tissue integration, resorbable and ease of use in periodontal regenerative therapy and the like and comprising at least about 90% by weight native collagen, the claimed preparation method comprises of following steps,
a. harvesting collagen from bovine Tendon followed by cutting and mincing the said collagen to form minced collagen
b. subjecting the minced collagen of step (a) to Non-ionic Detergent treatment followed by alkali treatment, salt treatment and enzymatic treatment to obtain a product in the form of tissue
c. subjecting the tissue of step (b) in the acid water at the pH not exceeding 3.5 to 4.5 to obtain a swelled material in the form of gel
d. harvesting serosa from a subject and subjecting to Non-ionic Detergent treatment followed by alkali treatment, and enzymatic treatment to form a pure collagen in the form of sheet
e. subjecting the sheets of step (d) to dehydration treatment to form sheet having soft dense surface
f. cutting the obtained soft sheets of step (e) into required size and dipping in 1% solution of PCL and allowing to dry at normal room temperature to form dry sheets
g. pasting atleast two sheets obtained in step (f) with the gel obtained in step (c) and allowing to dry at normal room temperature to obtain multilayer membrane and
h. finally compressing the multilayer membrane obtained in step (g) using special equipment to form the guided tissue regeneration multilayer collagen membrane having embossed porous surface on one face and dense smooth surface on opposite face.

5. A guided tissue regeneration multilayer collagen membrane prepared by the process as claimed in claim 4.
6. The guided tissue regeneration multilayer collagen membrane of any of the preceding claims has a thickness of 0.15mm to 0.17mm when dry.
7. The guided tissue regeneration multilayer collagen membrane of any of the preceding claims has longest in vivo stability time of 20-24 weeks, which accounts for the sustained function during the bone repair process.

Dated this 27th day of August 2015
For COLOGENESIS HEALTHCARE PRIVATE LIMITED
By its Patent Agent
Dr.B.Deepa