WO2015187110A2

WO2015187110A2 - Coating dental and orthopedic implant surfaces with bioactive material

Info

Publication number: WO2015187110A2
Application number: PCT/TR2015/000248
Authority: WO
Inventors: Ercan Durmus; İlhami CELIK
Original assignee: Ercan Durmus; Celik İlhami
Priority date: 2014-06-06
Filing date: 2015-06-04
Publication date: 2015-12-10
Also published as: WO2015187110A3

Abstract

The invent is related to the bioactive surfaced implants produced by means of adsorbing organic bioactive material, which are biocompatible and augmenting implant attachment with tissues, stimulating bone production.

Description

DESCRIPTION

COATING DENTAL AND ORTHOPEDIC IMPLANT SURFACES WITH

BIOACTIVE MATERIAL

TECHNICAL AREA

PRIOR ART

Orthopedic, oral and maxillary disorders affect millions of the patients, who need long-term therapy. In relation to the changes in life expectancy and life style, and technological achievements use of medical implant incredibly increased in the last few decades. Force transfer from artificial implant to host tissues is at vital importance in the attachment of implants to the tissues. Force transfer between artificial implant and host tissues has vital importance in the attachment of bone implants to the host tissues. Any incompliance between these increases bone resorption. More strong bone tissue is formed around a well-fixed implant material and attachment force of the implant material increases. Because of these reasons, an implant material implanted into the bone tissue must be nontoxic, non-immunogenic and non-carcinogenic. On the other hand, the main satisfactory success criteria in long-term performance of endoosseous implants are biocompatibility and osteoinduction. Titanium (Ti) and its alloys meet most of these requirements, moreover a thin and stable oxide layer, which gives its bioinert feature, comprises on the surface the material.

Although surface bioinertia is preferred and desired features of force- bearing implant surfaces, biocompatibility and attachment capacity are more powerful of the materials with surfaces stimulating host tissues. Therefore, extensive scientific studies have been carried out in order to acquire bioactive features to the surfaces of dental and orthopedic materials in last years. Recently, "biocompatibility" even has been re-defined as "suitable response creating capacity of a material inserted into the body to a specific stimulant". Therefore, to the surfaces of currently being used implants, adsorption of specific proteins interacting specifically with host cells has been applied, in addition to adsorption of non-specific proteins, in order to stimulate host tissues for desired biological reactions.

Such kind of coatings are degraded in a controlled manner without changing general features of the material and converting from passive state to active, with time. Osteintegration of the dental and orthopedic implants is formation of direct structural and functional attachment between live osseous tissue and force-bearing material.

Although titanium (Ti) and its alloys, which are the most preferred materials in the production of most widely used dental and orthopedic implants, are biological inert materials, they cannot directly attach to the bone and other tissues. Whereas, one of the most important results expected following insertion of the endoosseous prostheses is establishment direct contact between the material and live bone tissue, conjunction of live bone with implant surface. Therefore, interacting surface features of dental and orthopedic implants are very important in the success of implant. Topography and chemical features of the surface determine performance of the implant by playing vital role in biocompatibility and attachment. Thus, surface modification of of dental and orthopedic implants is necessary for osteointegration. Modification of surfaces of the implants and prostheses plays significant role in the control of cellular reactions to the material surface and stimulation of osteoproduction. Fort that reason, the most important objectives pursued in surface modification of dental implant and orthopedic prostheses are increasing surface roughness and bioactive surface area. In the improvement of the surface topography, increasing the surface roughness has become the first target. Increased surface roughness enlarges contact area between bone cells with implant and prostheses. In this case, tissue and implant attachment is augmented by increasing interaction surface between implant and host tissues get enlarged. Roughening of the surface is performed by means of the mechanical roughness and acid etching, because that mechanical roughness is mostly carried out with micro and nano materials, molecular and cellular attachment could be increased at nano scales.

Surfaces of the first-generation dental implants and orthopedic prostheses surfaces were mechanically processed. Although this process improves osteointegration properties of the implant, it resulted in decrease of mechanical stability of the implant depending on bone remodeling starting a few weeks after implant placement. After four weeks, stability of the implant increases during the 16 weeks following this stage. Although the recovery period as long as 4 months, implant-host tissue contact area increased by 85 %, and placed implants functioned more than 12 years, in the implants subjected to mechanical roughening.

In second-generation implants, acid etching, surface processing techniques such as controlled oxidation and anodization, nano surface processing with LASER and phosphate coating with hydroxyl apatite have been applied. The main objective in third generation implants is shortening healing period and augmenting osteointegration via giving bioactive feature to the surface by means of processing the surface with more advanced processes. Thus, long-term interface force increases in LASER-etched implants at nano scale.

Bioactive surfaces increases attachment to bioactive elements in addition to their activity in protein adhesion and this accelerates early wound healing processes. Another way improving the surface properties of implant material is the surface coating with bio compatible, attachment increasing and bioactive materials. Biological response against to coating material is time-dependent and must ensure complete integration to the host tissue.

Hydroxyl apatite (HA) surface coating via plasma straying, which one of the surface coating methods of dental and orthopedic implants, is widely used surface coating method. Although, osteointegration of the implants coated with this technique is better than non-coated materials, the method has some disadvantages. In addition to differences in coating density, non-uniformity of coating thickness and chemical differences of the coating limit the clinical success of the implants coated with the method. Early dissolution or delamination between implant-HA, which is concerned as among the factors limiting the success of plasma spraying coated implants might show that the attachment between implant-HA is weaker than that of the bone-HA. Early dissolution of the HA coating depends on high porosity in addition to the surface crystallinity. Delamination is largely depend on coating thickness. Bioglass, fluoroapatite and tricalcium phosphate compounds and bioinert ceramics such as alumina have been used for surface coating of implants. With their high osteoconductivity, the bioactive ceramic-coated implants gained preferential use in the case of the bone amount, quality is lower, and additional bone attachment is required. In the bioactive ceramic surface modification, nano surface forming technology is recently applied. Nano surfaces formed on the dental and orthopedic implants augment the osteoblastic adhesion depending on the changes in protein-surface interaction, in comparison to those of the conventional methods. This is a critical stage of the osteointegration process.

Nano surface features can change conformation of the cell adhesion proteins to the surface. Thus, nano surface forming method is an important tool in changing protein interactions between the surface and implant material. Increasing wettability, changes in the surface chemistry and differences in biomechanical milieu and osteoblastic differentiation capacity are the parameters which nano surfaces are effective.

Clinical advantages of moderately roughened are much more than those of more roughened HA coated dental and orthopedic implants.

Acid treatment alone and acid treatment with sanding are the techniques used in the surface treatment. In sanding techniques, silica, resorbable ceramics, alumina and titanium oxide are frequently used.

Additionally, acid treatment is important in removing remnants on the surface. The aim of the acid treatment is to increase surface profile and removing remnants on the material. In the acid treatment, hydrofluoric, nitric, sulfuric acids alone or their combinations are used. With acid treatment, total surface positivity is obtained, depending on electrochemical differences Another method used in the improvement of the surfaces of implant and prostheses is fluoride coating. Te aim of the fluoride coating is activating osteogenesis. Results of the scientific researches have shown that fluoride coating activate osteoblast differentiation and proliferation. During the fluoride coating, titanium fluoride is formed by substitution of fluoride with oxygen atom bound to titanium. When fluoride modified titanium placed into the bone, surfaces meet phosphates at neutral pH. In this way, phosphate binds to the bone, because of formation a covalent bond by substitution of oxygen with fluoride.

Intensive studies have been carried out to gain hydrophilic property to surfaces of the dental and orthopedic implants made of titanium. The initial bone apposition, initial cellular response and dynamic wettability of chemically cleansed micro and macro structured titanium implant surfaces significantly increase. Changes in physicochemical properties of the surface are known to augment via protein adsorption and integrin mediated cell attachment mechanisms. Thus, protein adhesion increases to titanium dioxide (T1O2) and following bone apposition proceeds. Osteoblasts proliferate more rapidly and alkaline phosphatase activity of these cells enhances on such surfaces.

Another technique in improving surface characteristics of implant and prostheses materials is chemical anodization. The method cause significant changes in the surface microstructure and chemistry. Because that titanium oxide layer gets thickened and porous microstructures formed in the oxide layer, early implant response of the host is strengthen.

Orthopedic and dental implant surfaces gain bioactivity with coating the surface with bioactive substances. Among these substances, human bone morphogenetic protein (hBMP), Type I collagen, bioactive peptides, polymers are significant.

The bone morphogenetic proteins (BMPs) are the most powerful and the most promising molecule family, because that they have very high osteogenic activity. In particular, BMP-2 has found use as a bone regulator in maxillofacial surgery as supportive therapeutic agent. Adsorbing capability of BMPs onto titanium and its alloys enabled them to immobilize on dental and orthopedic implants.

Tip I collagen is one of the main components of the bone and extra cellular matrix of the connective tissue around the bone and used in the coating surfaces of dental and orthopedic implants. Because, Type I collagen, which is the most abundant protein of the body, plays significant roles in tissue regeneration and restoration. In the bone, the Type I collagen, which its big majority is produced by osteoblasts, serves as tissue scaffold in bone remodeling. Because of this feature, it is also accepted that Type I collagen can play a biomimetic agent role on the coated surface.

In order to biomimetic surface featuring and stimulating cell adhesion, adsorption of intercellular matrix small peptides of the connective and bone tissues is a promising method. Different cellular pathways involved in the tissue repair possibly activated using these peptides. The most promising peptide is RGD (arginine, glycine, aspartic acid peptide). RGD is a constituent of many extra cellular matrix proteins and has a high affinity to the integrin family. Thus, it plays significant roles in adhesion of osteoblastic cells to extra cellular matrix. Although the subject requires further experimental work, osteointegration of implants that this peptide adsorbed is considered to be more powerful. A linear amino polysaccharide, chitosan, which is obtained by deacetylation of chitin abundant the shells of shellfish, has a large medical usage potential, because that it is not toxic and bioactive.

It accelerates wound healing by supporting blood clotting. Its osteogenic activity is considered to occur via accelerating osteoblast attachment and proliferation, and increasing extra cellular matrix synthesis. Results of experimental animal studies support this conclusion. For the reasons given above, chitosan might be a competitor to porous coatings to enhance the osteointegration.

In recent years, one of the surface modification method developed to increase the success of dental and orthopedic implants is adsorbing the molecules controlling bone remodeling, onto implant surface. Among the substances used for this purpose, bisphosphonates, pamidronate and zoledronate are the most promising agents.

There are different advantages and disadvantages of each of the methods given above, and intensive efforts done to develop new implants with more perfect bioactive surfaces. Eggshell calcium and shell membrane proteins, as well as chicken uterine fluid are promising great hope in surface coating and to acquire bioactive features.

BRIEF DESCRIPTION OF THE INVENTION

The invention is related to the implants with bioactive surfaces, which prepared by adsorbing organic bioactive material augmenting biocompatibility and attachment to the host tissues, stimulating osteoproduction. Production of dental and orthopedic implants with bioactive surfaces will be possible by adsorbing bioactive material to dental and orthopedic implant surfaces.

The aim of the products prepared by this method to provide early loading facility of dental and orthopedic implants by positively affecting early wound healing and augmenting osteointegration. Coating the dental implants with given procedure has also been expected to prevent bacterial infections by strengthening attachment to the soft tissues and preventing deepening of gingival sulcus.

Hydroxyl apatite (HA, Ca10(PO4)6(OH)2) is the most important component of the tooth and bone. Because of bioactive and osteoconductive properties, different forms of HA are widely used in coating metallic prostheses for bone restoration. Natural materials such as, coral shell, insect shells, fish bone have been used as calcium source because of their crystal structure and biocompatibility properties. Eggshell has an important place among the natural calcium sources with its 94% calcium carbonate, 1% calcium phosphate, 4% organic matter 1 % magnesium content.

HA also can be synthesized from eggshell calcium carbonate with no more complex methods. Eggshell membranes are also used as a biomaterial source. In many tissue types, crystallization occurs under strictly conditions. Proteins play key roles in the control of mineralization. Some of the protein molecules wrap the crystals, while the others entrapped inside of the crystal. There is not enough information on how these crystals are being trapped, roles in the crystallization and activity in determination of crystal feature.

Although protein coating applied onto the implant surface does not contain calcium phosphate, it plays scaffold or matrix roles in the mineralization of extra cellular matrix. This advantageous feature provides to the implant or prosthesis the opportunity to act as a template in the bone tissue. On the other hand, the feature also supports precipitation of calcium and phosphate salts in the implant-placed region through meeting the need bone substitute material of the implant

Proteinaceous coating form a composite with calcium phosphate crystals under in vivo conditions and this feature might lead to a biomimetic coating with superior mechanical properties than those of the conventional coatings.

It is believed that proteins act as a force for biomimetic coating by pushing to accumulate calcium and phosphate crystals. Moreover, proteinaceous coating increases attachment of the cells onto the implant surface and augments biocompatibility and attachment properties to the bone tissue.

The coated material may be inorganic, metallic, polymeric organic origin. Implant may be hard, flat or complex-shaped. Implant may have flat, straight or complex shaped surface.

Stainless steel, titanium, nickel, cobalt, niobium, molybdenum, zirconium, tantalum and their combinations are coated for orthopedic applications. Surfaces of many orthopedic prostheses and dental implants used in the dentistry are coated. Coating materials may be ceramics such as, alumina and zirconium, calcium oxide (CaO), bioactive glass comprised of silicone dioxide (SiCte) and phosphorus penta oxide (P205), hydroxyl apatite and tricalcium phosphates. Mechanical roughening, LASER application, chemical processes are applied in order to get surface roughening prior to the coating. The most widely used acids in the chemical process, hydrofluoric, hydrochloric, sulfuric, nitric and perchloric acids. Treatment of the material with oxidizing substances such as, nitric acid, peroxy halogen acids, hydroxyl peroxides or hydrogen peroxide is beneficial because that fresh oxide layer is forming on the surface. The treatment increases surface area and augments adhesion force. The material surface will be coated should be quite clean. For this aim, cleaning with degreasers (acetone, alkyl alcohols) and following washing with deionized water are applied. Cleansing of the material with ultrasonic shaker following mechanical and/or chemical treatment is very important for removing small particles.

In the chemical coating, a coating layer is formed on the material surface with the dipping into calcification solution. Calcium phosphate salts better precipitate in the weak acidic solutions than in the neutral or basic solutions. At high pH levels, when the system reached super saturation limit, non- homogenized calcium phosphate nuclei are formed on the substrate surface, in metastable liquids, crystal growth is at super saturation. At higher saturation, homogenous nucleation (crystal nucleus formation) event is dominant. The discovery of significant effect of pH on crystallization allowed forming calcium phosphate layers, in a controlled manner.

Water used in calcification solution preferentially should be deionized, if possible ultra pure, and filtered through 0.2 pm filter. Calcium source should have 2.5-25 mM concentration, preferentially between 1 -10 mM. Magnesium concentration should be between 0.1 -20 mM, preferentially between 1 .5-10 mM. Recommended carbonate concentration is 0-50 mM, preferentially between 0- 42 mM. Ionic strength should be between 0.10-2 M, preferentially between 0.15- 1.5 M. Coating temperature should be between 5-80°C, preferentially between 5-50°C, coating thickness between 0.5-100 pm, preferentially between 0.5-50 pm. If the thickness is lower than 5 pm, blue-red colorization occurs. Thicker coatings cause to gray or white colorization. Presence of the proteins in coating medium helps to mineralization. Proteins can be involved into the coating solution prior to dissolution of various ions, during dissolution and after dissolution. Protein concentration of the coating solution should be between 0.001-10 g/l, preferentially between 0.01-1 g/l. Electronegative ions are more convenient at the precipitation point. The most suitable protein type is the protein that dissolving 1g in one liter of the coating solution at neutral pH.

It is advantageous to use the proteins with disulphide bridges. In this regard, albumin, casein, gelatin, lysozyme, fibronectin, fibrin and chitosan are concerned as the most proper proteins. Principally, the proteins with isoelectric point below 7 and conformed by the amino acids with negative charge can be used. Examples of such amino acids are alanine, aspartic acid, cysteine, glutamine, glycine, isoleucine, leucine, methionine, proline, phosphorine, serine and valine. Particularly preferred proteins are synthetic proteins such as, polylisine, polyalanine and polycysteine. Additionally, biologically active proteins and growth factors such as bone morphogenetic protein (BMP) fibroblast growth factor (FGF), transforming factor can be advantageously used.

Eggshell is a composite of especially various minerals, especially calcium carbonate, water-soluble and water-insoluble proteins. Mineral component is mainly calcium carbonate (CaC03) in the form of calcite. Calcite is the most stable polymorph of CaC03. Inorganic salts compose of 91 ,87% of eggshell. 98.4 % of inorganic salts of CaC03 , 0.8% magnesium carbonate, tricalcium phosphate constitutes 0.8% .CaC03 constitutes 98.4%, magnesium carbonate 0.8%, tricalcium phosphate 0.8% of inorganic salts. Magnesium found in carbonate salt (Mg) at a rate of 0.8% in the eggshell is trace elements of both bone and teeth plays important roles in bone metabolism. In the deficiency of magnesium, fractures and bone losses increase. Thus, Mg element increases stiffness of hydroxyl apatite when the eggshell origin mineral content used as a calcium source in the coating dental and orthopedic implants with calcium phosphate and calcium carbonate salts. Eggshell calcification is the fastest and the highest rate calcium deposition biomineralization and calcium carbonate deposition rate is about 0.33 g/hour. The remaining content of inorganic section contains less amount of phosphor (P), magnesium (Mg), sodium (Na), potassium (K), zinc (Zn), manganese (Mn), iron (Fe) copper (Cu). The eggshell contains relatively high amounts of strontium (Sr). Flour (F), selenium (Se), copper (Cu), chrome (Cr) and strontium (Sr) levels change depending on the animal's feed. Strontium (Sr) has anabolic effect in humans and augments tooth enamel. Although the effective doses are not certain, strontium taken 170 mg daily increases bone density in 10 days period. Hydroxyl apatite when taken at suitable amounts increases bending strength of the bone.

The eggshell has some bioactive substances playing significant roles in bone modeling and remodeling. Among them ovotransferrin, ovocleidin-17, ovocleidin-116, osteopontine, ovalbumine, ovocalyxin-21 , ovocalyxin-25, ovocalyxin-32, ovocalyxin-37, clusterin and lysozyme are significant. These bioactive substances are the bioactive molecules starting calcite crystal nucleation and regulating its morphology and precipitation rate.

Ovocleidin-17 facilitates calcite crystal aggregation and changes crystal morphology at 500 pg/ml concentration. Ovotransferrine reduces crystal size and cause to crystal elongation at 500 pg/ml concentration. Another protein, ovocalyxin-32 is found in outer layers of the palisade layer, in the vertical and cuticle layers, and related to ending mineralization. Ovocalyxin-36 plays primary role in the control of egg shape, ovocleidin-116 in the control of the mineralization. A phosphorylated glycoprotein, osteopontine (OPN) is found at high amounts in both bone and kidney. Because that osteopontine (OPN) increases adhesion of the osteoblasts to the matrix and binds to hydroxyl apatite, it has significant roles in bone modeling and remodeling. Osteopontine (OPN) located in core regions of proteinaceous fibers of the eggshell membrane. Eggshell osteopontine (OPN) blockades growth of the calcite crystal in vitro. Clusterine located in the matrices of mamillary and palisade layers. This substance possibly plays a chaperone role in stability of the proteins and preventing precipitation, synthesized under stress conditions. Glycosaminoglycans (GAGs), the most important constituents of ground substance of the connective tissue are also found at large amounts in the organic matrix of eggshell and plays significant physiological roles including control of electrolytes and water retention. In the chicken eggshell, hyaluronic acid is found at a rate of 48% and 52% galactosaminoglycans (GAAGs).

Chondroitine sulfate-dermatan sulfate copolymers are mostly found in GAAGs. One of the GAGs, keratan sulphate plays role at nucleation stage of crystal growth in the eggshell formation. Ovoglycane, a dermatan sulfate chain is a poly anionic GAG. It shows high calcium affinity and regulates crystal growth. Hyaluronic acid, one of the GAGs is widely used as humidifier and in the treatment of arthritis.

Cartilage protecting and antiatherogenic effects of chondroitine sulfate have been shown in experimental animals. Transforming growth factor-1 (TGF- 1 ), calcitonine and progesterone are also found in the eggshell, although they are at lesser amounts.

Recently, potential anti microbial proteins, such as histones and beta- defensins have been isolated form the acid soluble matrix proteins of the eggshell. Additionally, anti bactericidal substances preventing growth of E. coli D31 and P. Aeruginosa in the eggshell. These anti bacterials also inhibit gram- positive bacteria such as, B. Subtilis and S. Aureus. This activity originates from cuticle and external eggshell protein extracts. Lysozyme, which is a bacterial wall destroying enzyme with 14.4 kDa molecular weight and 129 amino acid length, shows its activity by catalyzing 1 ,4-beta-bonds between N-acetyl muramic acid with N-asetil-D 5 glucosamine residues and between chitodextrins N-acetyl-D-glucosamine residues. With its anti bacterial properties, lysozyme has widely been used in toothpaste and mouthwashes in the preventing against bacteria causing periodontitis and oral mucosal infections. Oral and topical lysozyme application is effective in the prevention from herpes simplex and against HIV virus. Lysozyme also plays significant roles in the mineralization during eggshell formation because that it plays a role in the determination of calcite crystal morphology, in addition to its anti bacterial activities.

Transferrines are also found in the eggshell. One of them, ovotransferrine has negative effect on the bacteria because that it binds to iron when found at 1 mg/ml and higher concentrations. Ovotransferrine locating at close to eggshell pores shows its antibacterial activity at these sites. Ovocalyxin-32 (OCX-32) is another anti bacterial substance and found in cuticle and external eggshell layers. This substance inhibits growth of B. Subtilis and inhibits carboxypeptidase activity. OCX- 36 is found in the eggshell matrix. This protein has similarity with lipopolysaccharide binding proteins that increase the cell membrane permeability and defensive PLUNC protein family.

Anti bacterial activities of potential anti microbial proteins such as, histones and avian beta-defensins, those are the members of the acid-soluble eggshell matrix fraction, have been shown against to Bacillus cereus ve S. Aureus. 70% of eggshell membrane is organic matter, 10% is inorganic matter and 20% is water. The membranes contain 16% nitrogen, 2% saccharide, 1 .35% lipid. Ratio of neutral lipids to complex lipids is 86.14. 63% of the complex lipids is sphingomyeline and 12% is phosphotidylcholine.

Eggshell membrane structure is fibrous structure forming complex nodes. Fibrous membrane proteins give a semi permeable feature to the eggshell membrane. This network has a great importance in retaining microbes before entry into the egg. With this feature, the eggshell membranes form a large surface area for stromal cell adhesion.

70% of eggshell membrane is organic matter, 10% is inorganic matter and 20% is water. The membranes contain 16% nitrogen, 2% saccharide, 1 .35% lipid. Ratio of neutral lipids to complex lipids is 86: 14. 63% of the complex lipids is sphingomyeline and 12% is phosphotidylcholine. Multt layered water insoluble fibrous proteins form membranes. Fibrous membrane proteins oriented as to give semi permeable feature to the membrane. Eggshell membranes contain high amounts of arginine, glutamic acid, methionine, histidine, cystine and proline amino acids. In the membranes, hydroxyproline, hydroxylysine and desmosine amino acids are also found. The eggshell membranes contain high amounts of arginine, glutamic acid, methionine, histidine, cystine and proline amino acids. Hydroxy proline, hydroxylysine and desmosine amino acids are also at higher amounts. Cystine is a sulphur containing amino acid and essential for healthy skin, hair, bone and connective tissue. There are high amounts of cystine amino acid in the structure of alpha keratin, which is found at high amounts in nail, hair and skin, and found in the oral mucosa.

Because that cysteine plays role in the formation of collagen containing tissues, improves skin elasticity. Cysteine supplementation accelerates burns and other wounds, increases joint flexibility in arthritis. Each cystine molecule is formed by two cysteine molecules, which contains sulphur and close resemble with cystine. In fact, cystine is more stable form of cysteine. There is high amount of cystine in alpha-keratin, which is a significant constituent of nail, skin and hair. Because that cysteine plays role in the formation of collagen containing tissues, it increases skin flexibility and included into ant-ageing formulas found on the market. Cysteine supplementation accelerates burns and other types of wounds, increases joint flexibility in arthritis. Cysteine content of eggshell membranes is two fold of cuticle layer.

This finding evidences that eggshell membranes contain collagen. Thus, majority of inner and outer eggshell membrane content is constituted of Type- Type-V, Tip-X collagens and I. It is known that desmosine, isodesmosine and a non-elastin protein as a cross-linker are found eggshell membranes. Calculations have revealed that 10% of eggshell membrane protein is collagen and it is a high-value collagen. Presence of collagen in the eggshell membranes is very important from the point of its potential uses.

5-hydroxylysine content of eggshell membranes shows that the membranes contain collagen. Thus, majority of inner and outer eggshell membranes are constituted of Type-I, Type-V and Tip-X collagens. Desmosine, isodesmosine and a non-elastin protein as a cross-linker are also found in the eggshell membranes. Hyaluronic acid and its enzymatic hydrolyzates have been found as 0.1 -2% with colorimetric method. By weight basis the rate is 5- 10% (practically between %1 -5) with ELISA. In the membrane, 2-5% (w/w) hexosamine, 0.3-3% (w/w) chondroitine, 5-30% (w/w) collagens are found. Collagens comprise 35% of the eggshell membranes on the wet weight basis, and concerning hydroxyproline amino acid content as 4.5%. The most majority of collagens is Type-I, less amounts of Type-V and Type-X collagens are also found. Type-X collagen is known as a collagen controlling mineralization.

Glucosamine content of the eggshell membranes is found to be 10%, chondroitine rate is 9%, on the wet weight basis.

Eggshell membranes contain bacteriolytic enzymes such as, N-acetyl glucoseaminidase. Some constituents of the eggshell membranes decreases thermal resistance to high temperatures of pathogenic bacteria (Salmonella enteritis, Escherichia coli 0157.H7, Listeria monocytogenes and Staphylococcus aureus).

The eggshell membranes contain acid glycoseaminoglycans (GAGs). Dermatan sulphate and chondroitine sulphate are the most important ones of them. Sulphated proteins are also found in the eggshell membranes. Glycoproteins contain hexosamine, hexose and fucose.

High amounts of hyaluronic acid are also found in the eggshell membranes. Other constituents of the eggshell membranes are ovotransferrine, desmosine ve isodesmosine, lysil oxydase and lysozyme. Oral and injectable preparations containing eggshell membrane constituents, such as GAGs, chondroitine sulphate, hydrolyzed or natural collagen, sodium hyaluronate, ascorbate chelate of manganese and L-malic acid have been used in the prevention and therapy of connective tissue and skin diseases. The composition containing those substances increases synthesis of chondrocytes and wound healing, effective in sustaining the tissue health. Also, suspension of collagen and glucosaminoglycane (GAG) containing preparations also on the market for topical use.

In the eggshell membrane, there is lysil oxidase enzyme (EC 1.4.3.13) in amine oxydase form, which has the quinocofactor lysine tyrosylquinone (LTQ) and copper containing in its active region. This enzyme plays role in the development and restoration of the connective tissue. In the food originated copper deficiency, connective tissue damage occurs in the gingival tissues as in other body regions. Although collagen, glucosamine, chondroitine sulphate and hyaluronic acid, those are found in the eggshell membrane, can also be obtained from other sources, the rates of these substances are lower and also additional processes should be applied in order to desired purity.

Hyaluronic acid, glucosamine, chondroitine and collagen constituents can be relatively easily isolated from processed and unprocessed eggshell membranes. The most important drawback of the chicken eggshell membrane is limited amount of membrane can be obtained from egg. It is clear that use of ostrich eggshell will be more productive in biomaterial production. The ostrich eggshell membranes are also consisted of collagen fibers as the chicken eggshell membranes. The ostrich external eggshell membrane is composed of fibrous protein layers, oriented perpendicularly to each other.

Ostrich egg is averagely 1 ,5 kg in weight, 16 cmX12 cm in dimensions. Mechanical resistance of mineralized eggshell is 55 kg/cm². Among the avian eggs, mechanical features of the ostrich eggshell are excellent depending on its crystal structure. Calcite in different layers has different microstructure. This feature is gained during shell formation by polymers playing role in the eggshell formation and quite similar to the process in tooth and bone modeling.

Inorganic matrix of the ostrich eggshell is mainly constituted of calcium carbonate (96-97%) in calcite form, 1.9% is calcium phosphate and 0.7% is tricalcium phosphate. Chicken eggshell calcite is also used as raw material in HA synthesis. Hydroxyl apatite is found in the eggshell. Organic matrix about 4% and mainly constituted of glycoproteins and proteoglycans. In the organic matrix, 520 protein types have been found.

Most of them play role in crystal nucleation, crystal growth and control of crystal form. The most abundant proteins are ovocleidin-17, ovocleidin-1 16 and ovocalyxin-36. Type-X, which controls crystal growth is also found.

Carbon (C)/Calcium (Ca) rate of ostrich eggshell is 0.6 in crystal layer; two fold of this in cone layer. Magnesium rate is 3% crystal layer and 1% in cone layer. Avian egg is formed in acellular milieu of the avian uterus (shell gland), which is super saturated with calcium (Ca) and bicarbonate ions and with proteins, which concentrations change depending on the stage of eggshell formation.

The eggshell membranes contain bacteriolytic enzymes, such as N- acetyl glucoseaminidase. Some of the eggshell constituents decrease viability of Gram-positive and Gram-negative pathogenic bacteria (Salmonella enteritis, Escherichia coli 0157.Ή7, Listeria monocytogenes and Staphylococcus aureus), to high temperatures, thermal resistance.

Eggshell membranes also contain glycosaminoglycans (GAG). Dermatan sulphate and chondroitine sulphate are the most important of them. Sulfated glycoproteins are also found in the eggshell membranes. Glycoproteins contain hexosamine, hexoses and fucose. There are high amounts of hyaluronic acid in the eggshell membranes. Other constituents of the eggshell membrane are ovotransferrine, desmosine and isodesmosine, lysil oxydase and lysozyme. Oral and injectable preparations containing eggshell membrane constituents, such as GAGs, chondroitine sulphate, hydrolyzed or natural collagen, sodium hyaluronate, ascorbate chelate of manganese and L-malic acid have been used n the prevention and therapy ot connective tissue and skin diseases. The composition containing those substances increases synthesis of chondrocytes and wound healing, effective in sustaining the tissue health.

Eggshell membranes also contain bacteriolytic enzymes, such as lysozyme and N-acetyl glucoseaminidase. Some of the eggshell constituents decrease viability of Gram-positive and Gram-negative pathogenic bacteria (Salmonella enteritis, Escherichia coli 0157:H7, Listeria monocytogenes and Staphylococcus aureus), to high temperatures, thermal resistance.

The eggshell membrane proteins in the natural form do not dissolve in water. However, water-soluble protein products can be prepared form hydrolized eggshell membrane. The biocompatibility biofilm prepared from these proteins is better than membrane type- I collagen. Positive effects of these membrane proteins on the cell proliferation have been observed in the cell culture. Growth rate of human normal skin fibroblasts in eggshell membrane coated dishes increases with the increase of the protein concentration. Although collagen, glucosamine, chondroitine sulphate and hyaluronic acid, those are found in the eggshell membrane, can also be obtained from other sources, the rates of these substances are lower and also additional processes should be applied in order to desired purity.

Hyaluronic acid, glucosamine, chondroitine and collagen can be produced relatively easier techniques from non-processed or mechanically processed eggshell membrane. Because that eggshell membrane don contain DNA, its antigenicity is low. Viral agent transmission risk can be completely removed. Thus, transmission risk of zoonotic diseases via eggshell and eggshell membrane is relatively low, when compared to the risk of the transmission of zoonotic diseases, such as mad cow disease (bovine spongious encephalopathy, BSE) and other prion diseases from the bovine, and HIV from human origin. Adsorption of eggshell membrane proteins onto implant surface might Positively affect mineralization with bioactive substances positively affecting on mineralization and increase attachment of the material to the bone and biocompatibility, in addition to contribution to the wound healing process.

DETAILED DESCRIPTION OF THE INVENTION

The invent is related to the coating of dental and orthopedic implant surfaces calcium carbonate and calcium phosphate with eggshell origin, adsorption of bioactive eggshell membrane and the uterine fluid proteins.

The invent is comprised of the following steps:

Separation of the eggshell and eggshell membrane:

1.1. Separation of the eggshell and eggshell membrane with mechanical and chemical methods;

Fort his purpose, the eggs are drilled in the blunt ends and discarded, washed, filled with 1% acetic acid solution for 10 minutes. The eggshell membranes are mechanically removed. The removed membrane and fragments washed three times with deionized water and dried overnight at 50°C, under 300-mbar vacuum. The prepared products are stored in deep freezer until use.

1.2. Separation of the eggshell and eggshell membrane with acetic acid treatment;

For his purpose, the eggshell is partially demineralized. 5 ml of 20% acetic acid is used per 250 mg eggshell. The partial demineralization of the membrane performed shaking at +4°C. When dissolution completed, membranes are from the calcified eggshell. The separated eggshell and membrane fragments are washed at least three times with deionized water, and dried overnight at 50°C under 300-mbar vacuum. The prepared products are stored in deep freezer until use. 1.3. Separation of the eggshell and eggshell membrane with EDTA

(ethylene diamine tetra acetic acid) treatment;

In this method, 0.02 % sodium azide as anti bacterial agent is added into 0.5M EDTA (tetra sodium alt), pH is adjusted to 7.2 and subjected to partial demineralization at 4°C for two days. The calcified eggshell is separated from the membranes. The separated eggshell and membrane fragments are washed at lest three times with deionized water and dried overnight at 50°C, under 300-mbar vacuum. The prepared products are stored in deep freezer until use. 1.4. Mechanical separation of the eggshell and eggshell membrane powder:

The eggshell is grounded into a powder with the membrane. The powder discarded into a cylindrical water tank and the membrane fragments are separated from calcified shell fragments by floating via giving pressurized air from bottom of the tank, and shell particles precipitate. Floating membrane fragments are collected. The separated eggshell and membrane fragments are washed at lest three times with deionized water, and dried overnight at 50°C under 300-mbar vacuum. The prepared products are stored in deep freezer until use.

Isolation of water-soluble membrane proteins: .1. Isolation of water-soluble membrane proteins by treating membrane powder with 3-mercaptopropionic acid;

600 mg of the membrane powder prepared by one of the items 1.1 , 1.2, 1.3 and 1.4 is added into 20 ml of 20% 1 ,25 N aqueous 3-mercaptopropionic prepared in 10% acetic acid at room temperature (22°C). Temperature of the mixture is brought to 90°C, stirred for 12 hours until membrane dissolving completely. The mixture is cooled to the room temperature. Insoluble particulate material is precipitated by high speed-centrifugation. Supernatant is separated and its pH adjusted to 5 with 5M NaOH. The precipitate is filtered, washed with absolute methanol, and dTied overnight at 50°C under 300-mbar vacuum. The prepared product is stored in deep freezer until use. .2. Isolation of water-soluble membrane proteins by treating NaOH- absolute ethanol mixture;

The membrane powder prepared by one of the items 1.1 , 1.2, 1.3 and 1.4 is kept in 3 parts 1.5 N NaOH and 1 part of absolute ethanol at 50° for 3 hours. When dissolution is completed, the liquid phase is evaporated. The remaining solid fraction is the membrane protein fraction. This fraction is washed with absolute methanol, and dried overnight at 50°C under 300-mbar vacuum. The prepared product is stored in deep freezer until use. .3. Isolation of water-soluble membrane proteins by treating performic acid; 1g of the membrane powder prepared by one of the items 1.1 , 1.2, 1.3 and 1.4 is treated for 24 hours with performic acid prepared by mixing 10 ml of hydrogen peroxide, 90 ml of formic acid, at 25°C. The solution is filtered through glass filter is thoroughly washed and treated with 10 mg, 3200 U/mg pepsine prepared in 5%5 acetic acid at 25°C for 48 hours. Enzymatic reaction is stopped by addition of 0.2 mg of pepstatin. The solution is centrifuged, supernatant is dialyzed against to water and remaining part lyophilized. The prepared product is stored in deep freezer until use.

.4. Isolation of water-soluble membrane proteins by pepsine-acetic acid treatment;

The membrane powder prepared by one of the items 1.1, 1.2, 1.3 and 1.4 is extracted with 1 % pepsine prepared in 30 volumes of 0.5M acetic acid, at 4°C for 48 hours. Following extraction, supernatant of the solution is taken after centrifugation at 10.000 rpm at 4°C, 3M NaCi is added, and centrifuged again. The sediment at the bottom is dialyzed against 0.1 acetic acid and the protein is enriched by lyophilization. The prepared product is stored in deep freezer until use.

Processing the dental and orthopedic implant surfaces with chemical method:

3.1. Titanium alloy material is grinded with 1200-gride silicon carbide (SiC) abrasive. The material is stirred in ether-acetone mixture (1:1 , v/v) for half an hour, then washed at least three times with ultrasound shaker, and following in 5 M OH solution at 60°C for 24 hours with continuous shaking. The material washed in deionized water by continuous shaking for 1 hour is sinterized at 600°C in a porcelain pot for 1 hour by 3°C/minute heating and cooling cycles. The prepared product is stored in refrigerator until use. Surface coating of titanium alloy dental and orthopedic implants:

4.1. Surface coating of titanium alloy dental and orthopedic implants with hydroxy I apatite (HA) by using 10X simulated body fluid solution:

For his purpose, 10X simulated body fluid is used which it's chemical contents is given in the Table 1. The chemicals are dissolved in 1900 ml of the deionized water in the order given in the table. After completely dissolution of the last added chemical, the volume is completed to 2000 ml. pH level of this solution is 4.35-4.40. Titanium alloy implant or prosthesis material, which was previously subjected to surface treatment, is dipped into coating solution prepared by taking enough volume of 10X simulated body fluid and adjusted its pH value to 6.50 at room temperature (22°C) with NaHCOa addition. For each 2 cm² surface of the implant prosthesis material 25 ml the coating solution is used. The materials are coated 20-65-pm thick with hydroxyl apatite by keeping in the fresh coating solutions for 2 hours of each, a total of 6 hours, at room temperature. At the end of the process, the coated material is washed thoroughly, and dried under 300-mbar vacuum at 110°C in the oven for 5 hours. The coated material is sterilized with ethylene oxide, and stored in a cool and dry place until use. Surface coating is performed by using the solution given in the table below (Table 1).

Table 1. Constituents of the 10X simulated body fluid stock solution.

Substance Addition Amount Concentration order (mM)

NaCI 1 116.8860 1000

KCI 2 0.7456 5

CaCI₂'2H20 3 7.3508 25

MgCI₂*6H₂0 4 2.0330 5

NaH2P04 5 2.3996 10 Surface coating of titanium alloy dental and orthopedic implants with hydroxyl apatite (HA) with eggshell origin;

Organic materials in the eggshell fragments and eggshell powder, which were prepared by one of the items 1.1 , 1.2, 1.3 and 1.4 are removed by treating with hypochlorite, washed at least three times with deionized water. The material is dried under 300-mbar vacuum at 1 10°C for 5 hours in the oven, and following Ca-EDTA complex is formed by mixing 0.1 M ethylene diamine tetra acetic acid (EDTA). To the mixture, 0.06 M Na2HPC is added slowly by continuous mixing for 30 minutes, and the pH value of the mixture is increased to 13 with 0.06 M a2HPC supplementation. Previously surface-processed titanium alloy implant or prostheses material as in the item 3 is dipped into the solution and kept in a microwave oven (600 W and 2.45 GHz) for 10 minutes. For each 2 cm² surface of the implant prosthesis material 25 ml the coating solution is used. The process is repeated until veachmg a\ the deseed coating thick ess o1 h drox ^ apatite (HA) surface coating. At he end of the process, the coated material is dried at 110°C for 5 hours under 300-mbar in the vacuum oven. The coated material is sterilized with ethylene oxide and stored in a cool and dry place until use.

Surface coating of titanium alloy dental and orthopedic implants with calcium carbonate with eggshell origin;

Organic materials in the eggshell fragments and eggshell powder prepared by one of the items 1.1 , 1.2, 1.3 and 1.4 are removed by treating with hypochlorite and ground into a fine powder. The eggshell powder washed at lest three times with deionized water is dried at 110°C in vacuum oven under 300-mbar for 5 hours. The powder material is decomposed in a pot for 2 hours at temperatures starting from 400°C to 900°C with 5°C increments. 800 mM calcium hydroxide (Ca(OH)2) is prepared from the achieved CaO and its temperature is brought to 40°C. To this solution, titanium alloy dental and orthopedic implant materials prepared as the item 3.1 are dipped and coated with CaC03, by bringing the pH value of the mixture is to 9 with sodium bicarbonate (NaHCC ) and supplementing 240 m phosphoric acid (H3PO4) to this solution at rate of 15 ml/minute under continuous stirring. For each 2 cm² surface of the implant prosthesis material 25 ml the coating solution is used. Following, the materials are washed at lest three times in deionized water, dried in the vacuum oven at 50°C overnight, sterilized with ethylene oxide, packed and stored in a cool and dry place. Surface coating of titanium alloy dental and orthopedic implants with calcium carbonate/water-soluble eggshell membrane protein with eggshell origin;

Titanium alloy dental and orthopedic implant materials previously subjected to surface processing as the item 3.1 are coated with composite containing CaO prepared as in the method explained in item 4.3 of the "Detailed Explanation of the Invention" and constituted of 400 mM Ca(OH)₂, 120 mM phosphoric acid (H3PO4) and 3% eggshell protein dissolved in 10% acetic acid. Temperature of previously prepared Ca(OH)2 is brought to 40°C. To this solution, previously surface-processed implant material is dipped, the eggshell membrane dissolved in 10% acetic acid and prepared with one of the methods as in the items 2.1 , 2.2, 2.3 and 2.4 as to form 3% of the total volume of the solution. For each 2 cm² surface of the implant prosthesis material, 25 ml the coating solution is used. By adding phosphoric acid (H3PO4) drop by drop at a rate of 15 ml/minute to the solution containing the materials, whose PH is adjusted to 9, the implant material surfaces are coated with CaC03. Following, the material is washed at lest three times in deionized water, dried in the vacuum oven at 50°C overnight, sterilized with ethylene oxide, packed and stored in a cool and dry place. Surface coating of titanium alloy dental and orthopedic implants with nano hydroxyl apatite (HA) ;

Titanium alloy dental and orthopedic implant materials previously subjected to surface processing as the item 3.1 are coated with hydroxil apatite. For his aim, 1 M calcium carbonate tetra hydrate (Ca(N03)2 4H₂0) and 0.6 M potassium dihydrogen phosphate (KH2PO- solutions are prepared separately. The calcium carbonate tetra hydrate solution is agitated well, and it is added into the potassium dihydrogen phosphate solution, drop by drop. pH value of this mixture is adjusted to 11 , ammonium hydroxide is added drop by drop under continuous shaking. Titanium alloy implant and prostheses materials are dipped into this solution, kept for 6 hours under continuous stirring and following leaved to a 24 hours resting period. For each 2 cm² surface of the implant and prosthesis material 25 ml the coating solution is used.

Following, the material is washed at lest three times in deionized water, dried in the vacuum oven at 40°C overnight. Following, the materials are sinterized at 400°C in an oven with 10°C/minute heating and cooling cycles. The material is sterilized with ethylene oxide, packed and stored in a cool and dry place, until use. Surface coating of titanium alloy dental and orthopedic implants with nano hydroxyl apatite (HA)Zwater-soluble eggshell membrane protein/alginate composite;

Eggshell membrane protein prepared with one of the methods as in one of the items 2.1 , 2.2, 2.3 and 2.4 is dissolved in 10% acetic acid as eggshell protein to be 3%. To this solution, 1 CaC and 0.1 N phosphoric acid (H3PO- are added drop wise Ca/P ratio as to be1.66. In this case, 13.4 g CaC ·6Η2θ is added at the end of the process. To this mixture, sodium alginate [(C6H206Na)_n] is supplemented to guarantee weight ratio to be 9:1-2:1. pH value of the solution is adjusted to 7 with addition NaOH during continuously agitating. To this solution, titanium alloy dental and orthopedic implant materials, which received surface treatment with one of the methods 4.1 , 4.2, and 4.3, surface coating applied by continuous agitation for 48 at room temperature. At the end of the coating, the material is dried in the vacuum oven (under 300-mbar vacuum) at 110°C for 5 hours. The materials are packed, sterilized and stored in a cool and dry place until use.

Coating the water-soluble eggshell membrane protein adsorbed titanium alloy dental and orthopedic implant surfaces with eggshell calcium carbonate/water-soluble eggshell membrane protein composite;

Titanium alloy dental and orthopedic implant material received surface treatment as in item 3 ave dipped into 3% eggshell membrane protein solution prepared as in one of the items 2.1 , 2^*2, 2.3 ve 2.4 and kept for 24 hours under 300-mbar vacuum. For each 2 cm² surface of the implant and prosthesis material, 25 ml the coating solution is used. Then, the material is kept in 1 -ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) solution (10mg/ml) under 300-mbar vacuum for 12 hours, transferred through 70% and 99.5 % alcohols three times of each and dried at 37°C under 300-mbar vacuum for 24 hours. Then, the material is dipped into 250 ml of 0.2 sodium tri- metaphosphate (STMP), stirred at room temperature for 24 hours, after the pH value adjusted to 11.5. At the end of the period, the material is washed three times in distilled water, for half an hour in each, kept in saturated Ca(OH)2 at 37°C for 24 hours.

The material stirred in deionized water, then dipped into mineralization solution containing 1.5 mM calcium (as in the CaCte form) and 0.9 mM phosphate (KH2P04 form) in 20 mM HEPES buffer solution (pH 7.0). For each 2 cm² surface of the implant and prosthesis material, 25 ml the coating solution is used. The material is kept for 1-4 days with continuous shaking at 37°C, by changing the solution every 2 days. At the end of the period, the material taken is out of the coating solution, washed with deionized water and dried under 300-mbar vacuum at 37°C for 24 hours. The material is sterilized with ethylene oxide, packed and stored in a cool and dry place until use.

Adsorbing the eggshell membrane protein onto the titanium alloy dental and orthopedic implant surfaces, which coated with different materials:

5.1. Adsorbing the eggshell membrane protein by glutaraldehyde cross-linking onto the titanium alloy dental and orthopedic implant surfaces, which coated with different materials;

To the solution, which is prepared by dissolution of 3% of the eggshell membrane protein prepared with one of the methods in the items 2.1 , 2.2, 2.3 and 2.4, titanium alloy dental and orthopedic implant material surface-coated with one of the methods in items 4.1 4.2, and 4.3 is dipped, kept for 24 hours under 300-mbar vacuum. For each 2 cm² surface of the implant and prosthesis material, 25 ml the coating solution is used. At the end of the period, the materials are kept under 300-mbar vacuum in 2.5% glutaraldehyde solution for 24 hours. Then, the material is taken out of the coating solution, washed with deionized water and dried at 50°C overnight, sterilized with ethylene oxide, packed and stored in a cool and dry place until use.

5.2. Adsorbing the eggshell membrane protein by 1-ethyl-3-(3- [dimethyl amino] propyl) carboimide (EDC) cross-linking onto the titanium alloy dental ve orthopedic implant surfaces, which coated with different materials; To the solution, which is prepared by dissolution of 3% of the eggshell membrane protein prepared with one of the methods in the items 2.1 , 2.2, 2.3 and 2.4, titanium alloy dental and orthopedic implant material surface-coated with one of the methods in items 4.1. , 4.2, and 4.3 is dipped, kept for 24 hours under 300-mbar vacuum. For each 2 cm² surface of the implant and prosthesis material, 25 ml the coating solution is used. Then, the material is kept in 1-ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) solution (10mg/ml) at 37°C, under 300-mbar vacuum for 12 hours, and stirred in 70% and 99.5% alcohols, three times in each, and dried at 37°C under 300-mbar vacuum for 24 hours. The material is sterilized with ethylene oxide, packed and stored in a cool and dry place until use. dsorbing the eggshell membrane protein/gelatin by 1-ethyl-3- (3-[dimethyl amino] propyl) carboimide (EDC) cross-linking onto the titanium alloy dental and orthopedic implant surfaces, which coated with different materials',

Titanium alloy dental and orthopedic implant materials, which surface-coated with one the items in 4.1., 4.2, and 4.3, are dipped into the solution prepared with 0.05 M acetate buffer (pH 6), by dissolving %3 eggshell membrane protein prepared with one of the methods in the items 2.1 , 2.2, 2.3 and 2.4 and 3% gelatin (eggshell membrane protein: gelatin :1:1) and kept at 37°C under 300-mbar vacuum for 24 hours. For each 2 cm² surface of the implant and prosthesis material, 25 ml the coating solution is used. The material taken out of the solution is dried under 300-mbar vacuum at 37°C for 24 hours. Then, the material Is kept in -ethyl-3-(3-[dimethyl amino] propil) carboimide (EDC) in solution (10mg/ml) at 37°C under 300- mbar for 12 hours and stirred in 70% and %99.5 alcohols three times in each and dried under 300-mbar vacuum at 37°C for 24 hours. The material is sterilized with ethylene oxide, packed and stored in a cool and dry place until use. Coating the surface of the hydroxyl apatite coated titanium alloy dental and orthopedic implant with water-soluble eggshell membrane protein and adsorption of recombinant human bone morphogenetic protein (rhBMP) onto the protein layer;

All procedures are carried out under sterile conditions. 4 mg of recombinant human bone morphogenetic protein (rhBMP) dissolved in 5 ml of 5 mM glutamic acid (pH: 4.5), 2.5% glycine, 0.5% sucrose and 1 ml of 0.01% Tween-80 containing buffer. And kept at -80°C.

MES (N-morpholino 2-ethane sulfonic acid) at pH 5 might also be used as buffer. pH of the solution containing 3mg/ml gelatin dissolved in 3% acetic acid and 3 mg/ml eggshell membrane protein prepared as in one of the items 2.1 , 2.2, 2.3 ve 2.4 is adjusted to 7 with 1 M sodium hydroxide. To this solution, 50 pg rhBMP (Wyeth,

Cambridge, MA, USA) dissolved in buffer is added. To the prepared solution, titanium alloy (Ti6Al4V) dental and orthopedic implant material coated with one of the methods in items 4.1., 4.2, ve 4.3 is dipped and kept in vacuum oven , under 300-mbar at 37°C for 48 hours. Then, the material is transferred to 0.1 M phosphate buffer at pH 7.0. The material is stored at -80°C until use. Adsorbing chicken uterine fluid proteins by cross-linking with 1 - ethyl-3-(3-[dimethyl amino] propil) carboimide (EDC) on the surface of titanium alloy dental and orthopedic implants coated with various materials;

An incision is made in the body wall through ventral midline of laying hens killed 6-19 hours after 50 pg prostaglandin F2a injection to each laying hen and uterus is eviscerated. After taking out the egg, which is being shaped, a plastic tube inserted into oviduct and uterine fluids are emptied by massaging uterine with hands. Uterine mucosa is exposed through a longitudinally located incision, and scratched with a scalpel. Tissue scrapings is taken into Tris HCI buffer solution (0.0625 M, pH 6.8) homogenized with mechanical homogenizer. The uterine fluid and tissue homogenate are filtered through a membrane filter (0.2 μιη), and filtrate is diluted 1.0 mM with Tris 1.0 mM HCI buffer (0.0625 M, pH 6.8) containing proteinase inhibitor (benzamidine HCI, 2.5 mM; £-amino-2-caproic acid, 50 mM; /\/-etilmalei-imide, 0.5 mM; phenyl methyl sulfonyl fluoride (PMSF)at rate of 1 :1 , and frozen in liquid nitrogen, stored in deep-freezer until use. Dental and orthopedic implant material prepared with one of the methods in the items 4.1 , 4.2, 4.3, 4.4, 4.4 and 4.5 is dipped into the previously prepared 25 ml of uterine fluid for each 2 cm² surface area of the material, kept under sterile conditions overnight under 300-mbar vacuum oven at 40°C. Following, the material is dried under 300-mbar vacuum at 37°C for 24 hours. Then, the material kept in 1-ethyl-3-(3-[dimethyl amino] propil) carboimide (EDC) solution (10mg/ml) at 37°C under 300-mbar vacuum for 12 hours, stirred in 70% and 99.5% alcohols three times each, dried under 300-mbar vacuum at 37°C for 24 hours. The material sterilized with ethylene oxide, packaged and stored in cool and dry until use.

Adsorbing chicken uterine fluid proteins by cross-linking with glutaraldehyde on the surface of titanium alloy dental and orthopedic implants coated with various materials;

Dental and orthopedic implant material, which is prepared with one of the methods in the items 4.1 , 4.2, 4.3, 4.4, 4.4 and 4.5, is dipped into the 25 ml of uterine fluid previously prepared with the method in item 5.5 and determined for each 2 cm² surface area of the material, the material is kept under sterile conditions overnight under 300-mbar vacuum oven at 40°C. Following, the material is dried under 300-mbar vacuum at 37°C for 24 hours. The material is kept in 2.5% glutaraldehyde under 300-mbar vacuum for 12 hours, stirred in 70% and 99.5% alcohols three times each, dried under 300- mbar vacuum at 37°C for 24 hours. The material sterilized with ethylene oxide, packaged and stored in cool and dry until use. 5.6. Coating surface of alginate/gelatin/eggshell membrane protein scaffold of hydroxyl apatite pre-coated titanium alloy dental and orthopedic implants;

8 g of alginate powder is dissolved in 90 ml of deionized water through stirring with magnetic stirrer. To this solution, 8 g of the bovine gelatin is added. The mixture is homogenized by shaking at

50.8°C. To this solution, eggshell protein prepared with the methods in one of the items 2.1 , 2.2. and 2.3 and 2.4 and dissolved in 10% acetic acid is added as to be 3% of total solution (w/w). Pre-coated titanium alloy dental and orthopedic implant material with one of the methods in the items 4.1., 4.2, ve 4.3, 4.4 ve 4.5 is dipped into the solution, kept under 300-mbar vacuum for 24 hours. 25 ml solution is used per 2 cm² of the prosthesis material, At the end of the period, the material is dipped into the solution prepared by supplementing with 0.05M calcium chloride as to be %0.25 of alginate in the scaffold and glutaraldehyde as to be %0.25 of alginate in the scaffold, kept under 300-mbar vacuum for 24 hours.

Following, the materials washed at least three times with deionized water are dried in vacuum oven at 50°C overnight, sterilized with ethylene oxide, packed and stored in a cool and dry place until use.

Upon completion of the steps given above, the dental and orthopedic implants with bioactive surface, which was formed by adhesion of the biocompatible, powerfully attaching, osteoproductive bioactive material, will be ready to use.

Claims

C L A I M S

The claim is related to a surface coating method of dental and orthopedic implant surfaces with hydroxyl apatite (HA) with eggshell origin, and characterized by following steps;

— Formation of calcium-ethylene diamine tetra acetic acid (Ca-EDTA) complex with eggshell powder produced at least one of the mechanical and chemical procedures applied for production of eggshell and eggshell membrane in the powder form, treating with acetic acid, EDTA (ethylene diamine tetra acetic acid), mechanical procedures,

— Increasing the pH value of Ca-EDTA solution to 13 with NaOH addition,

— Dipping the dental and orthopedic implant with chemically processed surface in to the solution,

— Coating with hydroxyl apatite the dental orthopedic implant by keeping 600 W and 2.45 GHz microwave oven for 10 minutes,

— Drying the coated dental and orthopedic implant in vacuum oven (under 300-mbar vacuum) at 11 OX for 5 hours,

— Storing the coated dental and orthopedic implant in a dry and cool place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with eggshell calcium carbonate, and characterized by following steps;

— Calcium oxide production with thermally decomposition of eggshell fragments or eggshell powder produced one of the mechanical and chemical methods with acetic acid, EDTA (ethylene diamine tetra acetic acid) treatment in a platinum pot at temperature starting form 400°C to 900°C with 5°C/minute increments,

— Preparing 800 mM Ca(OH)2 from previously prepared calcium oxide (CaO) bringing its temperature to 40°C, — Dipping the chemical surface-processed dental and orthopedic implant into (Ca(OH)₂) solution heated to 40°C,

— Bringing pH of the Ca(OH)2 solution containing dental and orthopedic implants to 9 with sodium hydroxide, with continuous stirring,

— Coating the material surface with CaC03 by supplementing 240 mM phosphoric acid (H3PO4) at 15 ml/minute speed to the solution, with pH 9, and containing dental and orthopedic implants,

— Drying the implant material cleansed with deionized water at

least three times, following coating at 50°C, in the oven overnight,

— Sterilizing with ethylene oxide,

— Packaging under sterile condition,

— Storing in a cool and dry place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with composite of the eggshell calcium carbonate/water-soluble eggshell membrane protein, and characterized by following steps;

— Chemical processing dental and orthopedic implant surfaces,

— Preparing 400 mM Ca(OH)2 solution from calcium oxide (CaO) with eggshell origin,

— Preparing 120 mM phosphoric acid (H3P0₄) solution,

— Isolation of water-soluble eggshell membrane protein with one of the given methods, treating with 3-mercaptopropionik acid, treating with NaOH and absolute ethanol mixture, treating with performic acid, treating with pepsine-acetic acid,

— Preparing 3% eggshell membrane protein solution by dissolving water-soluble membrane protein in 10%acetic acid,

— Bringing temperature of previously prepared Ca(OH)2 to 40°C,

— Dipping the surface-processed dental and orthopedic implant into the solution, — Adding 3% water-soluble membrane protein solution dissolved in 10%acetic acid into the solution containing dental and orthopedic implant,

— Adjusting ph of he solution containing dental and orthopedic implant to 9,

— Cleansing the material with deionized water at least three times,

— Drying of the orthopedic and dental implants 50°C in the oven overnight,

— Sterilizing with ethylene oxide,

— Packaging under sterile condition,

— Storing in a cool and dry place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with nano hydroxyl apatite, and characterized by following steps;

— Chemical processing dental and orthopedic implant surfaces,

— Preparation of 1 M calcium carbonate tetra hydrate (Ca(N03)2 4H2O) and 0.6 M potassium dihydrogen phosphate (KH2PO4) solutions with deionized water,

— Adding the well agitated calcium carbonate tetra hydrate solution drop by drop into potassium dihydrogen phosphate solution,

— Increasing pH to 11 by adding ammonium hydroxide drop by drop, while shaking well the solution,

— Dipping the surface-processed dental ve orthopedic implant in to the solution with pH 11 (25 ml for each 2 cm² surface of he material) with continuous shaking, keeping for 5 hours,

— Following, keeping 24 hours for resting,

— Cleansing 3 times the completely coated material in deionized water with continuous shaking, — Drying under 300-mbar in vacuum oven at 40°C for 24 hours,

— Sintering the material at 400°C for 1 hour with 10°C/minute increments, and cooling with 10°C/minute increments,

— Sterilizing with ethylene oxide,

— Packing under sterile conditions,

— Storing in a cool and dry place until use.

— Removing organic material by treating with sodium hypochlorite and grinding into a fine powder the eggshell produced with one of the mechanical and chemical procedures applied for production of eggshell and eggshell membrane in the powder form, treating with acetic acid, EDTA (ethylene diamine tetra acetic acid), mechanical procedures,

— Cleansing the eggshell powder with deionized water three times,

— Drying in vacuum oven under 300-mbar at 1 10°C for 5 hours,

— Subjecting to thermally decomposition in platinum pot for 2 hours starting from 400°C to 900°C with 5°C/increments,

— Preparing 800 mM Ca(OH)2 solution from produced CaO by decomposition and bringing its temperature to 40°C,

— Dipping the dental and chemical surface-processed orthopedic implant material into Ca(OH)2 solution (25 ml coating solution for each 2 cm² surface of implant and prostheses), and bringing pH of the solution to 9 at 40°C, with sodium hydroxide,

— Coating surface of the material with CaCO3 by adding 240 mM H3P04 at 15 ml/minute speed,

— Following cleansing at least 3 times with deionized water,

— Drying at 50°C in the oven overnight,

— Sterilizing with ethylene oxide,

— Packing under sterile conditions, — Storing in a cool and dry place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with eggshell calcium carbonate/water- soluble eggshell membrane protein composite, and characterized by following steps;

— Chemical processing the surface of dental and orthopedic implant surfaces,

— Preparing 400 mM Ca(OH)2 from eggshell originated CaO,

— Dipping the previously surface-processed dental and orthopedic implant into Ca(OH)2 solution at 40°C,

— Preparing the coating solution by adding water-soluble eggshell membrane protein, which was isolated with one of the given methods; 3-mercaptopropionic acid treatment, NaOH and absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment, dissolved in 10% acetic acid as to make 3% of total volume,

— Dipping the dental and orthopedic implants into the coating solution as to 25 ml coating solution for each 2 cm² surface of implant and prostheses and adjusting pH to 9,

— Coating the material surface with CaC03 by adding drop wise H3PO4 into the coating solution at a rate of 15 ml/minute,

— Following cleansing at least 3 times with deionized water,

— Drying at 50°C in the oven overnight,

— Sterilizing with ethylene oxide,

— Packing under sterile conditions,

— Storing in a cool and dry place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with nano hydroxyl apatite (HA), and characterized by following steps;

— Chemical processing the surface of dental and orthopedic implant surfaces, — Preparing 1 M calcium carbonate tetra hydrate (Ca(N03)2 4H2O) and 0.6 M potassium dihydrogen phosphate (KH2PO4) solutions with deionized water,

— Adding the well agitated calcium carbonate tetra hydrate solution into potassium dihydrogen phosphate solution drop by drop,

— Increase to 11 of the pH of the well agitated solution by adding drop wise ammonium hydroxide,

— Dipping the dental and orthopedic implants into the solution as to 25 ml coating solution for each 2 cm² surface of implant and prostheses and keeping with continuous stirring for 5 hours,

— Following, keeping 24 hours for resting,

— Cleansing 3 times the coated material in deionized water with continuous shaking,

— Drying the coated dental and orthopedic implants under 300-mbar in vacuum oven at 40°C for 24 hours,

— Following, sintering the material at 400°C for 1 hour with 10°C/minute increments, and cooling with 10°C/minute increments,

— Sterilizing with ethylene oxide,

— Packing under sterile conditions,

— Storing in a cool and dry place until use.

The claim is related to a surface coating method of dental and orthopedic implant surfaces with nano hydroxyl apatite/water- soluble eggshell protein/alginate composite, and characterized by following steps;

— Dissolving water-soluble eggshell membrane protein, which was isolated with one of the given methods; 3-mercaptopropionic acid treatment, NaOH and absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment, in 10% acetic acid as to make 3% of total volume, — Adding, 1 M CaC and 0.1 N H3PO4 solution drop wise into this solution (as to about 13.4 g of CaCh ·6Η2θ supplemented to per liter of the solution at the end of the process),

— Adding sodium alginate [(C6H206Na)n] , the weight ratio as to be 9:1- 2:1 to the mixture,

— Adjusting to 7 of pH value of the solution with NaOH with continuous stirring,

— Surface coating of the chemically surface-processed dental and orthopedic implant by dipping into the solution and keeping for 48 hours at room temperature with continuous stirring,

— Drying the material in vacuum oven (under 300-mbar vacuum) at 1 10°C for 5 hours,

— Packing under sterile conditions,

— Storing under cool and dry conditions until use.

The claim is related to a surface coating method of water-soluble eggshell protein adsorbed dental and orthopedic implant with eggshell calcium carbonate water-soluble eggshell membrane protein composite, and characterized by following steps;

— Chemical processing dental and orthopedic implant surface,

— Dissolving at a rate of 3% in 10% acetic acid of the water-soluble eggshell membrane proteins isolated with one of the methods 3- mercaptopropionic treatment, NaOH-absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment,

— Dipping the dental and orthopedic implant (25 ml solution for per 2 cm² implant surface) and keeping for 24 hours under 300-mbar vacuum,

— Then, keeping in 1-ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) solution (10mg/ml) at 37°C under 300-mbar vacuum for 12 hours,

— Stirring in 70%70 and 99.5% ethyl alcohols respectively,

— Drying under 300-mbar vacuum at 37° for 24 hours, — Following, dipping into 250 ml of 0.2 M sodium tri-metaphosphate (STMP),

— Adjusting the pH value of the solution to 1 1 and shaking for 24 hours at room temperature,

— At the end of the period, stirring in deionized water at least three times, for half an hour in each washing,

— Keeping in saturated Ca(OH)2 at 37°C for 24 hours,

— Stirring in deionized water,

— Dipping the prosthesis material into mineralization solution prepared (25 ml solution is used for each 2 cm² implant surface) by mixing 1.5 mM calcium (in CaCh form) and 0.9 mM phosphate (in KH2P04 form) containing 20 mM HEPES buffer solution (pH 7.0),

— Keeping the material at 37°C for 1-4 days, by refreshing the solution every other day,

— Cleansing the coated material in deionized water, after removing of the coating solution,

— Drying at 37°C under 300-mbar vacuum for 24 hours,

— Sterilizing in ethylene oxide,

— Packing the material under sterile conditions,

— Storing in a cool and dry place under use.

The claim is related to adsorbing the water-soluble eggshell protein by glutaraldehyde cross-linking to the previously surface-coated dental and orthopedic implant surface with different methods, and characterized by following steps;

— Preparing a solution by dissolving the water-soluble eggshell membrane proteins isolated with one of the methods; 3- mercaptopropionic treatment, NaOH-absolute ethanol treatment, perform^'ic acid treatment, pepsine-acetic acid treatment, in 10% acetic acid at a rate of 3%, — Dipping into the solution (25 ml solution is used per 2 cm² surface of the implant and prosthesis material) the chemically treated dental and orthopedic implants with one of the chemical methods,

— Keeping under 300-mbar vacuum for 24 hours,

— Keeping the material under 300-mbar vacuum in 2.5% glutaraldehyde solution for 24 hours,

— Following, cleansing three times the coated material in deionized water,

— Drying at 50°C in the oven overnight,

— Sterilizing with ethylene oxide,

— Packing the material under sterile conditions,

— Storing in a cool and dry place under use.

The claim is related to adsorbing the water-soluble eggshell protein by 1-ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) cross- linking to the previously surface-coated dental and orthopedic implant surface with different methods, and characterized by following steps;

— Preparing a solution by dissolving the water-soluble eggshell membrane proteins isolated with one of the methods; 3- mercaptopropionic treatment, NaOH-absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment, in 10% acetic acid at a rate of 3%,

— Dipping into the solution (25 ml solution is used per 2 cm² surface of the implant and prosthesis material) the chemically treated dental and orthopedic implants with one of the chemical methods,

— Keeping under 300-mbar vacuum for 24 hours,

— Then, keeping in 1 -ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) solution (10mg/ml) at 37°C under 300-mbar vacuum for 12 hours,

— Stirring in 70%70 and 99.5% ethyl alcohols respectively,

— Drying under 300-mbar vacuum at 37°C for 24 hours, — Sterilizing with ethylene oxide,

— Packing the material under sterile conditions,

— Storing in a cool and dry place under use.

The claim is related to adsorbing the water-soluble eggshell protein/gelatin by 1-ethyl-3-(3-[dimethyl amino] propil) carboimide (EDC) cross-linking to the previously surface-coated dental and orthopedic implant surface with different methods, and characterized by following steps;

— Preparing eggshell membrane protein/gelatin solution by using water- soluble eggshell membrane proteins isolated with one of the methods given below; treatment with 3-mercaptopropionic acid prepared in 0.05 M acetate buffer (pH 6) treatment, NaOH-absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment, in 10% acetic acid at a rate of 3% and 3 % gelatin (eggshell membrane protein/gelatin= 1 :1),

— Keeping under 300-mbar vacuum for 24 hours,

— Drying under 300-mbar vacuum at 37°C for 24 hours,

— Stirring the dental and orthopedic implants in 70%70 and 99.5% ethyl alcohols respectively,

— Drying under 300-mbar vacuum at 37°C for 24 hours,

— Sterilizing with ethylene oxide,

— Packing the material under sterile conditions,

— Storing in a cool and dry place under use. The claim is related to surface coating with water-soluble eggshell protein onto hydroxyl apatite coated dental and orthopedic implant surfaces and adsorption of recombinant human bone morphogenetic protein (rhBMP) as the top layer, and characterized by following steps;

— Under sterile conditions, dissolving 4 mg of recombinant human bone morphogenetic protein (rhBMP) in the solution constituted of 1 ml buffer (MES (N-morpholino 2-ethane sulfonic acid at pH 5 can be used) containing 5 ml of 5 mM glutamic acid (pH 4.5), 2.5%glycine, 0.5 % sucrose and 0.01% Tween-80, and storing at -80°C,

— Adjusting pH to 7 with 1 M NaOH of the eggshell membrane protein/gelatin solution prepared by using 3 mg/ ml of water-soluble eggshell membrane proteins isolated with one of the methods given below; treatment with 3-mercaptopropionic acid prepared in 0.05 M acetate buffer (pH 6) treatment, NaOH-absolute ethanol treatment, performic acid treatment, pepsine-acetic acid treatment, and 3 mg/ml of gelatin,

— Adding into this solution 50 g/ml of rhBMP dissolved in the buffer,

— Dipping the dental and orthopedic implants surface-processed with one of the chemical surface processing methods,

— Keeping under 300-mbar vacuum in the vacuum oven 37°C for 48 hours,

— Following, transferring the dental and orthopedic implants into 0.1 M phosphate buffer with pH 7.0,

— Storing the prepared material at -80°C until use.

The claim is related to adsorbing chicken uterine fluid proteins by 1- ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) cross-linking to the previously surface-coated dental and orthopedic implant surface with different methods, and characterized by following steps; — Evisceration of uterus through an incision made through ventral midline of laying hens and ed 6-19 hours after 50 pg prostaglandin F2a injection,

— Emptying uterine fluids via plastic tube inserted into the oviduct by massaging uterine wall with hands after taking out the egg which is being shaped,

— Homogenization of the uterine mucosal scrapings taken into Tris HCI buffer solution (0.0625 M, pH 6.8), with a mechanical tissue homogenizer,

— Filtering the uterine fluids and tissue homogenate through membrane filter (0.2 μιη) and is diluting the filtrate with 1.0 mM with Tris 1.0 mM HCI buffer (0.0625 M, pH 6.8) containing proteinase inhibitor (benzamidine HCI, 2.5 mM; £-amino-2-caproic acid, 50 mM; N- ethylmalei-imide, 0.5 mM; phenylmethanesulfonyl fluoride (PMSF), — Freezing the homogenate in liquid nitrogen and storing in deep-freezer until use.

— Keeping dental and orthopedic implant material prepared with one of the chemical surface processing methods in the previously prepared 25 ml of uterine fluid for each 2 cm² surface area of the material, under sterile conditions overnight under 300-mbar vacuum oven at 40°C.

— Then, drying the material under 300-mbar vacuum at 37°C for 24 hours,

— Keeping the material in 1-ethyl-3-(3-[dimethyl amino] propyl) carboimide (EDC) solution (10mg/ml) at 37°C under 300-mbar vacuum for 12 hours,

— Stirring in 70% and 99.5% alcohols three times each,

— Drying under 300-mbar vacuum at 37°C for 24 hours,

— Sterilizing with ethylene oxide,

— Packaging under sterile conditions,

— Storing in a cool and dry place until use. The claim is related to adsorbing chicken uterine fluid proteins by glutaraldehyde cross-linking to the previously surface-coated dental and orthopedic implant surface with different methods, and dental and orthopedic implant coated with above method, and characterized by following steps;

— Keeping overnight the dental and orthopedic implants processed with one of the chemical methods in chicken uterine fluid solution (25 ml solution per 2 cm² implant surface area) under sterile conditions and 300-mbar in vacuum oven at 40°C,

— Following, drying under 300-mbar at 37°C for 24 hours,

— Keeping in 2.5% glutaraldehyde solution at 37°C under 300-mbar for 12 hours,

— Stirring in 70% and 99.5% alcohols three times each,

— Sterilizing with ethylene oxide,

— Packaging under sterile conditions,

— Storing in a cool and dry place until use.