WO2014021797A2

WO2014021797A2 - A cement material for renewal of damaged dental tissues

Info

Publication number: WO2014021797A2
Application number: PCT/TR2013/000228
Authority: WO
Inventors: Akman MELEK; Celik ILHAMI; Durmus ERCAN
Original assignee: Melek Akman; Ilhami Celik; Ercan Durmus
Priority date: 2012-07-30
Filing date: 2013-07-24
Publication date: 2014-02-06
Also published as: WO2014021797A3

Abstract

The invent is related to preparation of a regenerative dental material to use as a root canal sealer in the pulp capping, apexification, resection, amputation, root and bifurcation perforations, root fractures, surgical and non-surgical approaches to fill the root tip, in the treatment of primary teeth pulpatomy, Cvek pulpotomy, prophylactical partial pulpatomy of dense invaginatus, root canal treatment of permanent teeth, in root canal treatment of persistent primary teeth, restoration of resorptive defects; as a barrier material in-channel whitening procedures in endodontically treated teeth; as a filling material in the closure of commissure region and endodontically treated teeth.

Description

DESCRIPTION

A CEMENT MATERIAL FOR RENEWAL OF DAMAGED DENTAL TISSUES

TECHNICAL AREA

The invent is related to preparation of a dental material to use as a root canal sealer in the pulp capping apexification, resection, amputation, root and bifurcation perforations, root fractures, surgical and non-surgical approaches to fill the root tip, treatment of primary teeth pulpatomy, Cvek pulpatomy, prophylactic partial pulpatomy of dense invaginatus, root canal treatment of permanent teeth, root canal treatment of persistent primary teeth, restoration of resorptive defects; as a barrier material in-channel whitening procedures in endodontically treated teeth; as a filling material in the closure of commissural region and endodontically treated teeth. PRIOR ART

Dental pulp is in a close connection with lateral periodontal tissues through apical foramen and lateral channels. Dental pulp is separated from oral environment via enamel, dentin and gingival attachment. In order to maintain oral and dental health, the pulp and periodontium must maintain their vitality and health. When the pulpal and periodontal tissues are directly connected with oral cavity, due to dental caries, trauma, resorption and perforation caused by a physician, these tissues are inevitably affected by micro organisms and toxins. Dental pulp openings, furca and root canal perforations caused by a physician should be occluded with a restorative material in order to prevent bacterial leakage. However, the restorative materials must be biocompatible, not dissolve in oral fluids, must have sufficient physical properties to prevent leakage, trigger the desired tissue regeneration and prevent invasion of microorganisms.

For these purposes, zinc oxide eugenol-based cements such as Mineral Trioxide Aggregate (MTA), dental amalgam, Super-EBA, zinc oxide-eugenol based cements such as IRM, glass ionomer cements and composite resins are used. An ideal cement material to be used in these treatments should; 1 Be biocompatible,

2 Stimulate dentin bridge construction,

3 Have antiseptic activity,

4 Have alkaline reaction,

5 Have neutralization ability of acids in the caries,

6 Not either shrink nor expand,

7 Low thermal conduction coefficient,

8 Prevent bacterial leakage,

9 Have sedative effect

10. Easily be placed.

Some negative features of these materials such as micro-leakage, toxicity at different levels, moisture sensitivity, lack of both the antimicrobial efficiency and stimulating activity of tissue regeneration led the investigators to seek new cement materials.

BRIEF DESCRIPTION OF THE INVENTION

Eggshell consists of a thin proteinous film layer called as the cuticle, which is the outermost layer of the eggshell, a mineralized calcium carbonate layer in the calcite form of crystals and a pair of shell membranes.

An external spongy layer consisting of the vertically oriented crystals, and the inner mamillary layer form mineralized layer of the eggshell. When membranes included, 91.87% percent of the eggshell consists of inorganic salts. Inorganic salts contain 98.4% calcium carbonate, 0.8% magnesium carbonate and 0.8% of tricalcium phosphate. The eggshell contains 64% protein, 1.7% water 0.03% lipids. The eggshell is a composite of various minerals, especially calcium carbonate and water-soluble and insoluble matrix proteins. Mineral composition is constituted of calcium carbonate (CaC03), which is in the calcite form. Calcified eggshell is formed in the avian (uterus shell gland) of the avian species. More than 97% of eggshell is mineral matrix, and construction of the shell matrix is the fastest and the highest amounts of calcium accumulating bio-mineralization process in the nature, in which calcium carbonate (CaCO3) deposition rate is approximately 0.33 g/h. The remaining part of the inorganic portion contains small amounts of phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), zinc (Zn), manganese (Mn), iron (Fe) and copper (Cu). The eggshell contains relatively high amounts of strontium (Sr). Lead (Pb), aluminum (Al), cadmium (Cd), mercury (Hg), vanadium (V), boron (B), iron (Fe), zinc (Zn), phosphorus (P), magnesium (Mg), nitrogen (N), fluorine (F), selenium (Se), copper (Cu) and chromium (Cr) levels are below the measurement limits. Fluor (F), selenium (Se), copper (Cu), chromium (Cr) and strontium (Sr) levels vary depending on the dietary levels of the animal and it is an advantage to obtain the desired eggshell, which is rich in the elements mentioned above. About 24.1 1 % of dry matter of the fraction obtained by eggshell decalcification is calcium, 0.04% is phosphate and 0.23% is magnesium. In elemental analysis of the eggshell, 32% calcium, 3% magnesium, 20% carbon, 43% oxygen have been detected. However, trace amounts of chlorine (CI), nitrogen (N), phosphorus (P), potassium (K), sodium (Na) and sulfur (S) were found. Strontium (Sr) exerts an anabolic effect on the bone in humans and strengthens enamel layer of the teeth. Although effective daily dose is not certain, when taken 170 mg a day, it increases the bone density. When taken at the appropriate amounts, the Sr adhered onto hydroxyl apatite crystal increases bending resistance of the bone. Eggshell has a relatively high (30,000 ppm feed level corresponds to 1 1 .0% eggshell strontium) Sr content. Other elements of the eggshell, such as fluorine (F), copper (Cu) and selenium (Se) also has a positive effect on the bone metabolism.

Biochemical analysis of chicken calcified eggshell extracts showed the presence of ovotransferrin, ovocleidin-17, ovocleidin-1 16, osteopontin, ovalbumin, ovocalyxin-21 , ovocalyxin-25, ovocalyxin-32, ovocalyxin-37 clusterin and lysozyme proteins, which play significant roles in bone formation and bone remodeling. These proteins are the bioactive molecules that regulate precipitation speed and morphology of the calcite crystal, during the calcified shell formation. Ovocleidin-17 facilitates aggregation of calcite crystal and modifies its morphology at 500pg/ml concentration. Ovotransferrin reduces crystal size and leads to elongation of the crystals at 500pg/ml concentration. Another protein, ovocalyxin-32 plays a significant role at the terminal phase of crystal formation. Therefore, ovocalyxin-32 is located in the outer regions of the calcified eggshell, in both palisade and cuticle layers. It has been suggested that ovocalyxin-32 related to termination of mineralization. Ovocalyxin-36 is also effective in the control of the shell formation. Ovocleidin-116 settles in the palisade layer, and its primary role is the control of the mineralization. Another shell matrix protein, osteopontin (OPN) plays significant roles in the bone modeling and remodeling, via increasing adhesion of osteoblasts to organic matrix and binds to hydroxyapatite. OPN locates in the core of protein fibers of the calcified eggshell. OPN prevents calcite crystal growth in vitro. Clusterin is found in both mamillary and palisade layers. This protein possibly provides protein stability via preventing their precipitation under the stress conditions, because that it serves as a chaperon.

Large amount of glucose amino glycans (GAGs), which are the most important components of amorphous fundamental substance of the connective tissue, are also found in the calcified eggshell. It is well known that they have many physiological functions including water retention in addition to the control of electrolytes. GAGs are anionic polysaccharides consisting of repeating units of uronic acid- hexosamine dimers. Most of the GAGs attach to the core proteins to form proteoglycans. GAGs in the chicken calcified eggshell contain equal proportions of galactose amino glycans (GAAGs), and hyaluronic acid. Dermatan sulphate and chondroitin sulphate are dominant GAGs found in the GAAGs. Keratan sulphate plays a significant role at the nucleation stage of crystal formation. Ovoglycan, which is consisted of a dermatan sulphate chain, is an acidic and poly anionic GAG. It has high affinity for calcium ions and regulates the crystal growth. GAGs have widely been used in pharmaceutical, cosmetic and food industries. For example, hyaluronic acid is used in cosmetics as a moisturizer, and in the treatment of inflammatory joint disorders, i.e. arthritis. Cartilage protective and anti-atherogenic effects of chondroitin sulphate have been revealed in experimental animals. Small amounts of transforming growth factor-1 (TGF-1 ), which controls the cell differentiation and proliferation, calcitonin and progesterone have also been identified in the calcified eggshell. In recent years, a number of antimicrobial proteins, such as histones and beta-defensins have been identified in acid-soluble fraction of the calcified eggshell matrix proteins. There are some anti-bacterial agents preventing the proliferation of Gram-negative bacteria species including Pseudomonas aeruginosa and E. coli D31 , in protein fraction of the calcified eggshell matrix. Antibacterial calcified eggshell proteins also prevent proliferation of some of the Gram-positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. This activity depends on lysosomal activity in the protein extract of outer parts of the calcified eggshell, especially in the cuticle layer.

Lysozyme is an bacterial cell wall destroying enzyme, which has 14.4 kDa molecular weight and 29 amino acid length. Its antibacterial activity depends on hydrolysing capacity the 1 ,4-beta-ligands between both N- acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglicans, and the ligands between N-acetyl-D-glucosamine residues in chitodextrins.

In addition to its antibacterial activity, lysozyme also plays significant roles in the calcified eggshell formation, as it defines calcite crystal morphology. Lysozyme is also abundant in both organic matrix of the eggshell and albumen. Because of its anti-bacterial properties, this enzyme might be widely used in oral care products, such as toothpaste, tooth gel and mouthwash in the prevention oral mucosa infections and periodontitis. Oral and topical lysozyme application has been found to be effective in the prevention of herpes simplex virus and also against HIV. Oral and topical application of lysozyme has been shown to be effective against both herpes simplex virus and HIV. The matrix proteins of the calcified eggshell also have a defensin mechanism. Pseudomonas aureginosa, Bacillus cereus and Staphylococcus aureus are inhibited in the presence of 100 mg/ml of soluble calcified eggshell matrix protein. On the other hand, Escherichia coli and Salmonella enteritidis are inhibited weakly, when exposed to the calcified eggshell matrix protein up to 4 hours. The proteins exert their effect by destroying bacterial cell wall.

Transferrins are also found in the calcified eggshell. Ovotransferrin locates near to pores of the shell and shows the antibacterial activity by binding iron at higher concentrations than 1 mg/ml. Ovocalyxin-32 (OCX-32) is another antibacterial substance located in the cuticle and outer layers of the calcified eggshell. OCX-32 inhibits proliferation of Bacillus Subtilis with its carboxypeptidase inhibitory activity. Ovocalyxin-36 (OCX-36) is also isolated in the calcified eggshell matrix. This protein has close similarities with lipopolysaccharide-binding proteins and member of PLUNC protein family, which act as defense factors of the eggshell by increasing bacterial membrane permeability. Potentially anti-microbial proteins of the acid-soluble fraction of egg shell matrix proteins, such as histon and avian beta-defensins have been found to be effective against P. aeruginosa, Bacillus cereus and S. Aureus.

When consider the eggshell membranes, there are two eggshell membranes, which one of them is outer layer and attaches onto the eggshell, and the second one is the inner membrane, which completely surrounds the egg white. Organic component constitutes 70% of the membranes, 10% is made of inorganic elements and 20% is water. The membranes contain 16% nitrogen, 2% saccharides and 1.35-1.40% is lipid. The ratio of neutral lipids to complex lipids is 86:14. 63% of complex lipids is phosphatidylcholine and 12% is sphingomyelin. Water-insoluble membrane proteins form a fibrous network. The membranes acquire a semi-permeable feature depending on their fibrous proteins. The shell membranes have a significant amount of cystine. 5- hydroxylysine content shows that the shell membranes contain significant amounts of collagen. The inner and outer shell membranes are composed of Type-I, Type-V and Type-X collagens. Desmosin, isodesmosin and a non- elastin protein (a cross-linking agent) are also found in the eggshell membranes as cross-linking proteins. Hyaluronic acid ratio of the membranes was determined between 0.1-2%, with a colorimetric method. The ratio was found between 5-10% (1-5% in practice) by ELISA on the basis of wet weight. The membranes also contain 2-5% hexosamines (w/w), 0.3-3% chondroitin (w/w) and 5-30% collagen (w/w). Collagen ratio was calculated as 4.5% on wet weight basis and hydroxy-proline amino acid content. Type-I collagen is the most abundant collagen. The amount of Type-V and Type-X collagens is lesser. Type-X collagen is known that to control tissue mineralization. Glucosamine and chondroitin ratios were determined as 10%, and 9% respectively, on the basis of wet weight.

The shell membranes include high amounts of arginine, glutamic acid, methionine, histidine, cystine and proline amino acids. At the same time, hydroxy proline, hydroxy lysine and desmosin amino acids are also abundant. Cystine is a sulphure-containing amino acid, which is necessary for healthy skin, hair, bone and connective tissue. Alpha-keratin protein, which forms nail, hair skin is abundant in the oral mucosa contains high amount of cystine. Cysteine improves skin elasticity, because that it takes part in the formation of tissues containing collagen. Thus, cysteine supplementation accelerates healing of burn wounds and other wound types, and aslo increases the flexibility of the inflamed joints.

The shell membranes contain high amounts of N-acetyl glucosaminidase, which is a bacteriolytic enzyme. Some membrane components decrease resistance of Gram-positive and Gram-negative disease causing bacteria (Salmonella enteritis, Escherichia coli 0157: H7, Listeria monocytogenes and Staphylococcus aureus) to high temperatures. The eggshell membranes contain acid glucose amino glycans (GAGs) and sulphated glycoproteins. Dermatan sulphate and chondroitin sulphate are the most important sulphated glycoproteins. Glycoproteins contain hexosamine, hexose, fucose and hyaluronic acid. Among the other components of the eggshell membrane, ovotransferrin, desmosin, isodesmosin and lysil oxidase are significant. Oral and injectable preparations containing some of substances, such as GAGs, chondroitin sulfate, hydrolyzed or natural form of collagen, sodium hyaluronate, ascorbate chelate of manganese, and L-malic acid, which are present in the calcified eggshell, have largely been used in the prevention and treatment of the connective tissue and skin diseases. The composition of these substances is effective in maintaining tissue health, accelerates synthesis activity of chondrocytes and wound healing. Suspension preparations containing collagen and GAGs, which used for acceleration of wound healing are on the market. The use of the shell membrane products also can provide significant benefits in the treatment of gingival lesions. Eggshell membranes contain lysil oxidase enzyme (EC 1.4.3.13), which is a copper-containing amine oxidase with an active site having lysine quino-cofactor tyrosyl quinone (LTQ). This enzyme plays an important role in the development and repair of the connective tissues. In case of copper deficiency with nutritional origin, connective tissue damage occurs in gingival tissue as in the other regions. For this reason, oral care products that have egg shell membrane might contribute to maintaining the gingival epithelium and connective tissue health.

Although collagen, glucosamine, chondroitin sulfate and hyaluronic acid can also be obtained from other sources, the rations of these substances are higher in the eggshell membrane, and additional processing methods should be applied to achieve the desired purity from other sources. Hyaluronic acid, glucosamine, chondroitin and collagen constituents can be obtained in a relatively easier manner from unprocessed and mechanically processed eggshell membrane.

Chicken eggshell membrane has been used in the cell culture, bone defects, dressing of burn wounds as a biomaterial. A protein, which is derived from the eggshell membranes stimulates production of Type III collagen fibers in addition stimulation growth of human skin fibroblasts by binding to their cell membrane receptor molecules.

The products of animal origin have the high risk of zoonotic disease transmission, such as mad cow disease (bovine spongious encephalopathy, BSE) and other prion diseases. Therefore, there is a need of new resources including higher amounts of compounds with therapeutic, cosmetic and nutraceuticals to prepare oral care products.

Calcified eggshell and the membranes are the only animal product, which does not contain DNA. Therefore, their antigenitciy is very low. Moreover, treatment of the eggshell with 2.5% glutaraldehyde completely eliminates the risk of viral agent. Eggshell powder including the eggshell membranes has a powerful potential to use in oral care products, such as tooth gels, because that it contains the substances controlling the shape and growth of calcium crystals (CaC03), the substances preventing the bacterial growth, and also high amounts of Sr and Mg. Low level of toxic heavy metal of the calcified eggshell powder also makes it a more advantageous source than other calcium and strontium sources.

Tyndalization; A sterilization method of fluids and tissue culture media containing heat-sensitive materials such as protein, carbohydrates and blood serum to remove germs, by applying intermittent heating. Water proof container; It has the vital importance to know storage conditions of protein cantaining material to maintain the freshness. Airtight containers can be used to maintain the freshness of the protein-containing materials. Different products should not be stored in the same container. Inert substance; In chemistry, the phrase "inert" is used for qualifying chemically inactive substances. "Inert containers" describes a container, which it does not react with the stored material and not change its structure.

DETAILED DESCRIPTION OF THE INVENTION

The invent is related to processing separately ostrich eggshell and shell membranes, preparation of a water-soluble eggshell membrane protein solution and coating the eggshell particles with the water-soluble eggshell membrane protein, preparation of a gel from the coated particles, preparation of a regenerative cement by addition of different amounts of tricalcium aluminate, sodium aluminate, activated carbon, water, protein polymers, 1 .5 mM calcium chloride (CaCI2) and 0.9 mM kalium dihydrogen phosphate (KH₂P0₄) containing 20 mM HEPES (pH 7.0) buffer solution, calcium oxide (CaO).

1. Preparing ostrich eggshell and eggshell membranes for processing;

The contents of fresh ostrich eggs are emptied, the eggshell and shell membranes are mechanically separated and divided into bulky pieces. Each material is thoroughly are washed 3 times with pressurized distilled water and transferred into 70% ethyl alcohol. These materials are fixed overnight in 2,5% glutaraldehyde, washed thoroughly 3 times by shaking in distilled water, every one hour, and the fragments are dried overnight under 300 mbar vacuum, at 50°C.

2. Powdering eggshell particles;

The eggshell particles prepared in the item 1 are grinded into a fine powder with a suitable mill by preventing from over heating. 3. Processing the prepared membrane material;

3.1. Removing calcium remnants and lipids in the membrane pieces;

The pieces of the eggshell membrane prepared as in the item 1 are first treated for 1 hour with 1 ,5 M hyrochloric, and then overnight with ether-aceton (v/v) by mixing every 2 hours. Following filtration, the membrane pieces are dried overnight at 50°C in the oven. The dried membrane material is divided into 2 parts. The first part is treated with sodium trimetaphosphate (STMP), and the second part is grinded into a powder in order to prepare water-soluble membrane proteins. 3.2. Treating the membrane material with sodium trimetaphosphate (STMP);

The membrane pieces prepared as in the item 3.1 are treated with 3200 U/mg pepsine prepared with 1 % (w/v) 0.5M acetic acid solution, for 48 hours by agitating every 2 hours at 37°C in order to remove the proteins except collagen. At the end of the treatment, enzymatic activity is blocked by adding 0,2 mg /100 ml pepstatine. Following washing with distilled water, the membrane pieces are treated with 0,2 M sodium trimetaphosphate (STMP, pH 1 1 ,5) for 24 hours at room temperature (22 °C), in order to bind phosphate groups to the collagen fibers. The filtered membrane pieces are dried overnight at 50°C, and then powdered. 3.3. Preparing water-soluble membrane proteins from the powdered membrane material;

The powdered membrane material prepared as in the item 3 is used. For this purpose, 3 different method are followed;

3.3.1. Isolation of water-soluble eggshell membrane protein fraction with 3-mercaptopropionic acid treatment;

600 mg of the shell membrane powder prepared as in the item 3.1 , is added into 20 ml of 1 ,25 N mercaptopropionic acid prepared in aqueous 10% acetic acid (v/v), at room temperature (22°C). The temperature of the mixture is brought to 90°C and continuously stirred for 12 hours, in order to dissolve completely the membrane fragments. Then, the mixture is cooled to room temperature. Insoluble particulates are precipitated by centrifugation. Supernatant is transferred into another tube, and its pH is adjusted to 5 with 5M NaOH. The precipitate is filtered, washed with absolute methanol, and then dried.

3.3.2. Isolation of water-soluble eggshell membrane protein fraction with NaOH and absolute methanol treatment;

The membrane powder prepared as in the item 3.1 is treated with a mixture of 3 volumes of 1 ,5 N NaOH and 1 volume of absolute ethanol (v/v) for 3 hours, at 50°C. When the dissolution of the membrane fragments is completed, liquid phase is evaporated. The remaining part at he bottom is the water-soluble membrane fraction. This fraction is washed with absolute methanol and dried overnight, at 50°C.

3.3.3. Isolation of water-soluble eggshell membrane protein fraction with performic acid treatment;

The membrane pieces prepared as in the item 3.1 are treated overnight with performic acid prepared by agitating in 10 of ml hydrogen peroxide and 90 of ml formic acid at 25°C. The material is filtered through the glass filter and washed with distilled water, and then treated with 3200 U/mg pepsine prepared in 0.5M 1 % acetic acid (w/v) by agitating every 2 hours at 25°C for 48 hours. Enzymatic activity is blocked by addition of 0,2 mg /100 ml pepstatine. Following centrifugation, membrane protein rich supernatant is removed, diluted with distilled water and protein fraction is separated by lyophilization. 3.3.4. Isolation of water-soluble eggshell membrane protein fraction with salicylic acid treatment;

The membrane pieces prepared as in the item 3.1 are treated overnight with performic acid prepared by agitating 10 of ml hydrogen peroxide and 90 of ml formic acid and 1 g/ml (w/v) at 25°C. The material is filtered through the glass filter and washed with distilled water, and then treated with 3200 U/mg pepsine prepared in 0.5M 1 % acetic acid (w/v) by agitating every 2 hours at 25°C for 48 hours. Enzymatic activity is blocked by addition of 0,2 mg /100 ml pepstatine. Following centrifugation, membrane protein rich supernatant is removed, diluted with distilled water and protein fraction is separated by lyophilization.

4. Preparing water-soluble protein solution:

50 g of the water-soluble membrane protein fraction, which is prepared as in the item 3.3.1 , is dissolved in 10% aqueous acetic acid solution (w/v). 50 g of the water-soluble membrane protein fraction, which is prepared as in the item 3.3.2, is dissolved in 10% aqueous acetic acid solution (w/v).

50 g of the water-soluble membrane protein fraction, which was prepared as in the item 3.3.3, is dissolved in 10% aqueous acetic acid solution (w/v).

50 g of the water-soluble membrane protein fraction, which is prepared as in the item 3.3.4, is dissolved in 10% aqueous acetic acid solution (w/v). 5. Coating the eggshell particles with water-soluble eggshell membrane proteins;

The eggshell powder prepared as in the item 2 is added into sufficient volume of the protein solution, which was prepared as in the item 4, mixed well and particles are soaked well. The mixture is dried overnight under vacuum (300 mbar), at 60°C. The coated powder is treated trice with 70% ethyl alcohol and dired overnight under vacuum (300 mbar), at 60°C. 6. Gel preparation;

6.1. Gel preparation by using eggshell powder coated with membrane protein, which was isolated with 3-mercaptopropionic acid treatment;

a) 10 g of carboxy methyl cellulose (CMC)

b) 150 g of eggshell powder coated as in the item 5

c) 10 gr of membrane powder treated with STMP as in the item 3.2.

d) 100 ml of 0.1 M phosphate buffer

Carboxy methyl cellulose (CMC) and the eggshell powder prepared as in the item 5 are mixed. Phosphate buffer is heated to 50°C and CMC is gradually added and pH of the mixture is brought to 7 with sodium bicarbonate.

6.2. Gel preparation by using eggshell powder coated with membrane protein, which was isolated with NaOH and absolute methanol treatment;

a) 10 g of carboxy methyl cellulose (CMC)

b) 50 g of eggshell powder coated as in the item 5

c) 10 gr of membrane powder treated with STMP as in the item 3.2.

d) 100 ml of 0.1 M phosphate buffer

6.3. Gel preparation by using eggshell powder coated with membrane protein, which was isolated with performic acid treatment;

a) 10 g of carboxy methyl cellulose (CMC)

b) 150 g of eggshell powder coated as in the item 5

c) 10 gr of membrane powder treated with STMP as in the item 3.2.

d) 100 ml of 0.1 M phosphate buffer

Carboxy methyl cellulose (CMC) and eggshell powder prepared as in the item 5 are mixed. Phosphate buffer is heated to 50°C and CMC is gradually added and pH of the mixture is brought to 7 with sodium bicarbonate. Some hardener and additional materials should be added into the gel to gain sufficient physical and biological properties for application as a cement material without loosing its biological characteristics.

6.4. Gel preparation by using eggshell powder coated with membrane protein, which was isolated with salicylic acid treatment;

a) 10 g of carboxy methyl cellulose (CMC)

b) 150 g of eggshell powder coated as in the item 5

c) 10 gr of membrane powder treated with STMP as in in the item 3.2.

d) 100 ml of 0.1 M phosphate buffer

Carboxy methyl cellulose (CMC) and eggshell powder prepared as item 5 are mixed. Phosphate buffer is heated to 50°C and CMC is gradually added and pH of the mixture is brought to 7 with sodium bicarbonate.

Some of the materials can be added into the gel, which is prepared from the eggshell and shell membrane in the preparation of cement can be as follows;

Carbon is the most common nonmetallic element in the nature. It is the most versatile, which can form different compounds with almost every element found in the nature. Carbon is the unique element in the nature having the ability to form a wide variety of complex substances, which meet basic functions of life. Certain carbon compounds comprise approximately 18% of living matter (the rest of it is is mostly water). These materials serve as the basic building blocks of the cell.

Among the industrial adsorbents used in the control of environmental pollution, activated carbon has a wide use with its high porosity. With its a large crystal form and a quite large internal porous structure, "activated carbon" is the general term used in defining family of carbon adsorbents. They are harmless to the public health and have a very high internal surface area and porosity.

Activated carbons are referred as "adsorbent" because that they can attract the molecules and ions in a given solution on their internal surfaces through the pores. Porous system of the activated carbon is very important in adsorption and desorption processes. Micro pores construct majority (~%95) of the internal surface. Although macro pores do not play significant roles in adsorption, they are important conductors to accelerate diffusion rate towards micro pores. Briefly, in the adsorption process, macro pores facilitate diffusion of a molecule into activated carbon, meso pores transfer the molecule more interior and micro pores adsorb the molecule.

Tricalcium aluminate plays an important role in the chemical stability and characteristics of cement.

Sodium aluminate is an inorganic chemical, which is suitable for industrial and technical applications and aluminum hydroxide origin. It can be used as sodium aluminate solution or as an additive.

Natural polymers are the polymers, those are biologically produced and have unique functional features. Proteins (collagen, gelatin, elastin, actin, etc.), polysaccharides (cellulose, starch, dextran, chitin, etc.) and polynucleotides (DNA, RNA) are the main natural polymers. Proteins are the most important biopolymers of life. Protein molecules are constituted of hundreds of amino acids bound with peptide bonds. Natural polymers, due to their different functional properties have a wide use and they can be used as biomaterials.

Some features of these natural polymers are as follows;

Collagen; It is a filamentous protein family, which is very important for the body. Their resistance to stress and tension is very important for biological functions of these proteins. The collagen is longest known protein. Collagen is synthesized by through a regulated stepped polymerization reaction in-vivo. Fibrous alpha-helical structure (alpha helix) structure provides a high tensile resistance and mechanical resistance. Gelatine: An industrial product derived from collagen. Polypeptide chains are broken, when dilapidated bone or skin remnants are boiled in an autoclave, under 5-10 atm. at 150°C. Gelatin is obtained after water content of the solution is evaporated and refined (color and odor are eliminated). Because that its water-dissolved product is a macromolecular solution, it has the features of colloids. Elastin; Because that elastin is a structural protein of the connective tissue produced by fibroblasts, it is mostly found in the extra cellular matrix of the connective tissues. Proteolysis of it is also performed by fibroblasts. It provides elasticity to the elastin containing tissues, such as veins, skin, lungs and other elastic tissues. Because that this elasticity is tolerated by collagen and tissue integrity is ensured, the tissues mentioned above are have also collagen fibers.

Actin; Actin is a globular structural protein of the cell. Helical polymer of actin forms actin microfilaments. Actin microfilaments are structural components of the eukaryotic cell skeleton, which has a 3 dimensional meshwork. They provide mechanical support, determine cell shape, and enable cell movement.

Agar; Agar is a kind of gelatin, which is obtained from algae. The word "gel" has a Malay Language origin and comes from the word "agar-agar". Agar has a wide use in microbiological tests, dental medicine, electrochemistry, etc.

Silk; Silk is a soft, shiny fiber, which was produced by silkworm. Silk is very strong and biologically compatible with tissues.

Polysaccharides; Polyhydroxy aldehydes and polyhydroxy ketones with a general formula of CnH₂nOn are called as "carbohydrates". If a carbohydrate molecule is consisted of only one aldehyde or ketone group is called as "monosaccharide". Long chains of monosaccharide units, which are are connected by oxygen bridges are called as polysaccharides. Dextran; When glucose units bind in alinear form, the resulting biopolymer is dextran. Dextran dissolves in the water to form a colloid. It is used instead of the blood plasma in the treatment of shock. Natural polymer polynucleotides; The biopolymers bearing, storing codes of life and translating the codes into proteins are defined as nucleic acids. All nucleic acids are formed by units, which are called as nucleotides. Each nucleotide molecule is constituted of a nitrogenous base, a pentose sugar, and phosphoric acid. Both DNA and RNA are polynucleotide chains. A nucleotide polymer, ribonucleic acid (RNA) is a nucleic acid. Each nucleotide molecule is formed by a nitrogenous base, a pentose sugar, and a phosphoric acid molecule. RNA plays significant roles in biological processes, especially in many steps of the genetic code transfer from DNA into protein, via translation. Although its close resemblance with DNA, RNA has some in detailed structural differences. RNA is single-stranded, whereas DNA is double-stranded. Nucleotids of RNA contain ribose, whereas DNA contain deoxynbose ( a kind of ribose missing an oxygen atom). In the RNA, uracyl is found instead of thymine in the DNA and bases of RNA are chemically modified. RNA is synthesized from related DNA codes by RNA-polymerase and transcript is transformed by processing other enzymes. Some of these RNA-processing enzymes have their own RNAs.

Natural polymers are the essential substances in the biomaterial use. Because that they are close similar or resemble to macromolecules of the body, they do not cause unwanted results, such as toxic and inflammatory reactions when placed into the body. However, compositional changes and forming difficulties due to deteriotion at high temperatures are some of their disadvantages. Biodegradation of the biomaterials via enzymatic activity in the environment is one of their advantages. Early applications of the natural biodegradable materials are the use of threaded silk protein, processed animal bowels and potato starch in the wound healing. Due to elastomeric based behaviour, perfect biocompatibily and oxygen permeability of some protein- based polymers used in the tissue regeration, manufacture of biomaterial products used in biosensors and controlled drug delivery systems. Artificial biodegradable biomaterials (Poly phosphazene, aliphatic polyester, polyalkyl-2-cyanoacrylate, poly anhydride, poly ortho esters) are biologically degradable and resorbable by nearby tissues. Therefore, when using biomaterials it should be considered that the material must have a biodegradable chemical structure by the body. Degradation products should not be toxic and removed without damaging the cells.

Polyphosphazenes have a number of advantages as carrier molecules. A variety of polyphosphazene derivatives with different features could be synthesized by substitution of different active groups. In recent years, polyphosphazenes, which are hydrolyzed into low molecular weight products, may be synthesized.

By binding the high molecular weight proteins, such as adenosine deaminase (ADA) to poly ethylene glycol, new biodegardable biomaterials with extended half-life and low immunogenicity can be produced.

Protein crosslinkers; This group of chemicals can be used to convert the prepared gel into the dental cement. Some of these chemicals are Dimethyl adipimidate (DMA), Dimethyl Suberimidate (DMS), reversible and thio-cleavable bifunctional reagents, Bis (2-[succinimidoxycarbonyloxy]ethyl) Sulfone (BSOCOES), N-(p-Maleimidophenyl) Isocyanate (PMPI), Sulfosuccinimidyl-2- (m-Azido-o Nitrobenzamido) ethyl 1 ,3'-Dithiopropionate (SAND), C6- Succinimidyl 4-Hydrazinonicotinate Acetone Hydrazone (C6-SANH), Disuccinimidyl Suberate (DSS), 3,3'-Dithiobis SulfoSuccinimidyl Propionate (DTSSP), N-(Y-MaleimidoButyryloxy succinimide ester (GMBS), Ν-(κ- Maleimidoundecanoyloxy) sulfosuccinimide (Sulfo-KMUS) ester.

Calcium oxide (CaO) is a white colored, abrasive and solid chemical, which gives an alkaline calcium hydroxide solution with water, and has a wide range of industrial use. Industrially, calcium oxide is produced via removing carbon dioxide from calcium carbonate at high temperatures.

Calcium chloride is one of the ionic calcium salts, and it is solid at room temperature. It can be directly produced from limestone and as a by-product of Solvay method. Because of its hygroscopic feature, it should be stored in tightly closed containers. In the solution, calcium chloride Serves as calcium ion source and since calcium compounds can suspend, it causes to precipitation. Functions and features of calcium chloride are regulation of acidity, preservation of the freshness of fruits and vegetables, chelating metal ions.

Sodium hydroxide is a white colored, hyroscopic substance. Its chemical formula is NaOH. It easily dissolves in the water and form a solution giving a soapy slippery feeling.

Salicylic acid; It is a white colored, crystallized organic substance, with the Formula of [O-C6H4(OH)COOH], and produced by reacting phenol with carbon dioxide. Its melting point is 159 °C. In the medicine, salicylic acid is used in inhibiting callus formation, wart (papilloma) treatment, aspirine and paint production. Sodium salt is an analgesic and used is rheumatism therapy. Naturally, it is found in Gaultheria procumbens in methyl salicylate form with scented geranium plant odor.

Preservative additives; These substances are used in order to protect the cement ingredients, until use. An ideal preservative should have a simple chemical composition, heat-stable and easily accessible in the laboratory. Sodium fluoride, sodium azide, mercury chloride, potassium oxalate, heparin, iodo-acetate, chloramphenicol, cycloheximide, phosphate buffer are have largely been used as preservatives.

Because that phosphate buffer has many features of and ideal buffer solution, it has a wide use. However, it has some disadvantages such as inhibitory, activatory and metabolite roles in many biological reactions due to its high activity. It binds multivalent cations and prepicitates them. Although buffering capacity of the phosphate buffer has been reported between pH 5.8- 8.0, it is relatively weaker below 7.5.

7. Preparation of the cement;

7.1. The regenerative cement is prepared by adding tricalcium aluminate, sodium aluminate, activated carbon and water into the the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated 3-mercaptopropionic acid treatment. 7.2. The regenerative cement is prepared by adding tricalcium aluminate, sodium aluminate, activated carbon and water into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with NaOH and absolute methanol treatment.

7.3. The regenerative cement is prepared by adding tricalcium aluminate, sodium aluminate, activated carbon and water into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with performic acid treatment.

7.4. The regenerative cement is prepared by adding tricalcium aluminate, sodium aluminate, activated carbon and water into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with salicylic acid treatment.

7.5. The regenerative cement is prepared by adding protein polymers into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with 3-mercaptopropionic acid treatment.

7.6. The regenerative cement is prepared by adding protein polymers into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with NaOH and absolute methanol treatment.

7.7. The regenerative cement is prepared by adding protein polymers into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with performic acid treatment.

7.8. The regenerative cement is prepared by adding protein polymers into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with salicylic acid treatment.

7.9. The regenerative cement is prepared by adding 1.5 mM calcium chloride (CaCI₂), 0.9 mM potassium dihydrogen phosphate (KH₂PO₄) and 20 mM HEPES buffer solution (pH 7.0) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with 3-mercaptopropionic acid treatment.

7.10. The regenerative cement is prepared by adding 1.5 mM calcium chloride (CaCI₂), 0.9 mM potassium dihydrogen phosphate (KH₂PO₄) and 20 mM HEPES buffer solution (pH 7.0) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with NaOH and absolute ethanol treatment.

. The regenerative cement is prepared by adding 1 .5 mM calcium chloride (CaCI₂), 0.9 mM potassium dihydrogen phosphate (KH₂PO₄) and 20 mM HEPES buffer solution (pH 7.0) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with performic acid treatment.

. The regenerative cement is prepared by adding 1 .5 mM calcium chloride (CaCI₂), 0.9 mM potassium dihydrogen phosphate (KH₂PO₄) and 20 mM HEPES buffer solution (pH 7.0) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with salicylic acid treatment.

. The regenerative cement is prepared by adding calcium oxide (CaO) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with 3-mercaptopropionic acid treatment.

. The regenerative cement is prepared by adding calcium oxide (CaO) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with NaOH and absolute ethanol treatment.

. The regenerative cement is prepared by addition of calcium oxide (CaO) into the gel material, which is prepared by coating the eggshell powder with the membrane protein, which is isolated with performic acid treatment.

. The regenerative cement is prepared by adding calcium oxide (CaO) into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with salicylic acid treatment.

. The regenerative cement is prepared by adding protein polymers and preservative substances into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with 3- mercaptopropionic acid treatment. 7.18. The regenerative cement is prepared by adding protein polymers and preservative substances into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with NaOH and absolute ethanol treatment.

7.19. The regenerative cement is prepared by adding protein polymers and preservative substances into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with performic acid treatment.

7.20. The regenerative cement is prepared by adding protein polymers and preservative substances into the gel material, which is prepared by coating the eggshell powder with the membrane protein isolated with salicylic acid treatment.

The additions of substances into the gel during cement preparation do not adversely affect biological advantages of the cement.

The product described in this invent contains bioactive substances triggering calcium mineralization, controlling both crystal shape and growth, and having bacteriostatic effect. It also contains high amount of calcium in the form of calcium carbonate, strontium (Sr) and magnesium (Mg). The cement is prepared by mixing the eggshell powder containing gel with hardener substances, which do not impair the biological properties of the gel. The regenerative dental cement, whish is the subject of this patent, is believed to solve previously mentioned problems.

The product, which is prepared by shell powder and following gel preparation process, contains strontium (Sr) that substitutes with calcium ions in the hydroxyapatite crystal and increase resistance of the crystal to acid demineralization. Incidence of dental caries decreases, when strontium content of superficial enamel is high. Strontium supplemented oral cleaning gel increases enamel mineralization in vivo. Strontium has an anabolic effect on the human bone, augments tooth enamel. Therefore, use of the eggshell and membrane powder will help in the control of oral bacterial activity. Moreover, there are bacteriostatic substances, such as lysozyme and acetyl glucosaminidase, which are effective on both Gram-negative E. coli D31 in the eggshell. These substances also prevent proliferation of Gram-positive B. subtilis and S. aureus. This activity arise form the lysozome enzyme found in inner shell membrane, and protein extracts of the cuticle and external layer of the shell. Transferrins are also found in he eggshell. Ovoransferrin binds iron found at 1 g/ml concentrations in the milieu. Trasferrin which closely locates to shell pores, shows its antibacterial activity in these regions. It weakens inflammation by decreasing number of the pathogens.

Claims

1. The claim is related to preparation of the regenerating cement characterized by addition of tricalcium aluminate, sodium aluminate, activated carbon and water into the gel containing the eggshell membrane protein-coated shell powder.

2. The claim is related to preparation of the regenerating cement characterized by addition of protein polymers into the gel containing the eggshell membrane protein-coated shell powder.

3. The claim is related to preparation of the regenerating cement characterized by addition of 1 .5 mM calcium chloride (CaCI₂₎ and 0.9 mM potassium dihidrogen phosphate (KH₂P04) containing and 20 mM HEPES buffer (pH 7.0) into the gel containing the eggshell membrane protein-coated shell powder.

4. The claim is related to preparation of the regenerating cement characterized by addition of calcium oxide (CaO) into the gel containing the eggshell membrane protein-coated shell powder.

5. The claim is related to preparation of the regenerating cement characterized by addition of calcium oxide protein crosslinkers and preservative substances into the gel containing the eggshell membrane protein-coated shell powder.

6. The claim is related to preparation of the gel containing eggshell membrane protein-coated shell powder explained as in the item 1 , item 2, item 3, item 4 and item 5, and characterized by containing the shell membrane protein isolated with NaOH and absolute ethanol treatment.

7. The claim is related to preparation of the gel containing eggshell membrane protein-coated shell powder explained as in the item 1 , item 2, item 3, item 4 and item 5, and characterized by containing the shell membrane protein isolated with performic acid treatment.

8. The claim is related to preparation of the gel containing eggshell membrane protein -coated shell powder explained as in the item 1 , item

2, item 3, item 4 and item 5, and characterized by containing the shell membrane protein isolated with salicylic acid treatment.

9. The claim is related to preparation of the gel containing the eggshell membrane protein-coated shell powder explained as in the item 1 , item

2, item 3, item 4 and item 5, and characterized by containing at least two different sized eggshell particles.

10. The claim is related to preparation of the gel containing the eggshell membrane protein-coated eggshell powder explained as item 1 , item 2, item 3, item 4 and item 5, and characterized by containing sthe hell membrane protein isolated with tyndalization without destroying proteins.

11. The claim is related to preparation of the eggshell powder explained as in the item 1 , item 2, item 3, item 4 and item 5, and characterized by obtaining from eggshell, which is cleaned with pressurized ultra high pure water at temperature under 58°C.

12. The claim is related to preparation of the eggshell powder explained as in the item 1 , item 2, item 3, item 4 and item 5, and characterized by fixing in 2.5% glutaraldehyde for 24 hours.

13. The claim is related to preparation of the eggshell powder explained as in the item 1 , item 2, item 3, item 4 and item 5, and characterized by containing cuticle, calcite and at least one of the eggshell membranes.

14. The claim is related to preparation of the eggshell powder explained as in the item 2 and characterized by containing at least one of collagen, elastin, silk, poysaccharides, dextrane and natural polymer nucleotides.

15. The claim is related to addition of protein cross linkers, which are mentioned in the item 5 and characterized by containing at least one of Dimethyl Dadipimidate (DMA), Ddimethyl Suberimidate (DMS), reversible and thio-cleavable bifunctional reagents, Bis (2- [succinimidoxycarbonyloxy]ethyl) Sulfone (BSOCOES), N-(p- Maleimidophenyl) Isocyanate (PMPI), Sulfosuccinimidyl-2-(m-Azido-o Nitrobenzamido) ethyl 1 ,3'-Dithiopropionate (SAND), C6-Succinimidyl 4- Hydrazinonicotinate Acetone Hydrazone (C6-SANH), Disuccinimidyl Suberate (DSS), 3,3'-Dithiobis SulfoSuccinimidyl Propionate (DTSSP), N-(y-MaleimidoButyryloxy succinimide ester (GMBS), Ν-(κ- Maleimidoundecanoyloxy) sulfosuccinimide (Sulfo-KMUS) ester.

16. The claim is related to addition of a preservatie substance, which is mentioned in the item 5 and characterized by containing at least one of sodium floride, sodium azide, mercury chloride, potassium oxalate, heparine, iodo-acetate, chloramphenicol, cycloheximide, phosphate buffer.

17. The claim is related to the materials, which are mentioned in the item 1 , item 2, item 3, item 4 and item 5, and characterized by being stored in air-tight containers and maintaining sterility until use.

18. The claim is related to tricalcium aluminate, which is mentioned in the item 1 and characterized by the presence of 2-5% in the mixture of the regenerative dental cement.