TECHNICAL FIELD

The present invention belongs to bone tissue engineering technical field, relates to a bio- compliant product which is suitable for use in repair and/or treatment of bone breakages and/or bone diseases that are genetic or that occur afterwards and which enables realization of this repair or treatment processes in short durations.

In another perspective, the present invention provides a method for the production of a bio- compliant product which is suitable for use in repair and/or treatment of bone breakages and/or bone diseases that are genetic or that occur afterwards and which enables realization of this repair or treatment processes in short durations.

PRIOR ART

In animals and humans, bone is the hardest one among the tissues that form the body. Bone is the tissue that functions as a support for organisms. Bones have a substantially hard structure since they are substantially rich in terms of calcium, this condition leads to brittleness of bones.

Damages or breakages in bone tissues can take place due to genetic structure or events (accidents, impacts) that occur afterwards. Particularly in the advancing ages, bone tissues become more prone to damages or breakages in animals and humans. Said proneness substantially depends on feeding, living styles and genetic characteristics of persons.

Proneness to breakages or damages in bone tissues can also result from genetic characteristics; the most common genetic bone diseases are for instance osteoporosis, rheumatoid arthritis, rachitism.

The treatment of bone breakages has three phases. These phases are called as reactive phase where the cells that shall repair the breakage are collected at the breakage region, repair phase where the proteins produced by osteoblasts and condroblasts begin to harden by precipitation of calcium mineral thereon, and where the structure which provides fixation of the breakage called as ‘soft callus’ occurs, and re-development phase where the bone continues to return to its prior form. These phases must be realized respectively for eliminating or repairing the damages formed by bone diseases or the events that develop afterwards.

Some methods are developed in the technical field for realization of treatment phases of bone breakages. The application of said methods shows change depending on damage intensity of bone diseases or the events that occur afterwards. In case of bone breakages, since the bone is essentially automatically healed, the medical intervention and treatment are focused on supporting of the bone and at the same time, providing of the best conditions (immobilization) to the injured bone for optimum healing. The basic rule is the re-placement of broken parts and fixation thereof such that they do not move until healing. This process is named as “reduction”. Re-positioning of the bone without surgery is “closed reduction”.

Surgical intervention may be needed particularly for the treatment of breakages resulting from genetic-based bone diseases. The kind of the needed treatment depends on whether the breakage is “open” or “closed” or the place and the intensity of the breakage. For instance, a broken bone of the backbone is treated in a different manner from a broken leg bone or a broken hip. Generally, these surgical operations that are in the form of bone transfer are recently realized by placement of the bio-compliant parts that enable shortening of disease healing processes to the damaged bone tissues. For this reason, research and development specialists make studies which enable realization of healing phases formed by bone damages at the optimum values and which enable shortening of treatment durations and in a manner not forming any toxic effect for the biological organism.

In the related technical field, it is considered that products must be developed that enable treatment of bone diseases, repair and formation of new healthy bone tissue, and production methods of these products must be developed in rapid, low-cost and industrially applicable manners, and new technological information must be obtained in these subjects.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a bio-compliant product which provides treatment and/or repairing for bone diseases and breakages. The present invention relates to a bio-compliant product which enables realization of the phases, needed for healing of bone damages that are genetic or that occur afterwards, in an optimum manner.

In another perspective, the present invention relates to a method for production of bio- compliant product that enables realization of the phases, needed for healing bone damages that are genetic or that occur afterwards, in an optimum manner.

In another perspective, the present invention relates to gaining antibacterial and anti- oxidative characteristics to a bio-compliant product that enables realization of the phases, needed for healing of bone damages that are genetic or that occur afterwards, in an optimum manner.

DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject matter belongs to bone tissue engineering technical field, relates to a bio-compliant product which is suitable for use in repair or treatment of bone breakages or bone diseases that are genetic or that occur afterwards and which enables realization of this repair or treatment processes in short durations, and is explained with references to examples without forming any restrictive effect only in order to make the subject more understandable.

The subject matter product is placed to the regions, where damaged bones in viable organisms exist, by means of a surgical operation, and afterwards, it provides realization of healing processes of bone damages and provides repairing of the damaged region. Accordingly, there are some characteristics expected from the product; the most important one is that the product must be bio-compliant. In the invention, while “a bio-compliant product” is being mentioned, the natural or synthetic materials used for fulfilling or supporting the functions in viable tissues in human body are mentioned. Another important characteristic is that the bio-compliant product placed to the damaged bone region must enable realization of the phases which provide treatment and repair of said damaged region.

In accordance with all these arguments, the subject matter product comprises components that have bio-compliant characteristics and that provide treatment and repair of bone diseases. Accordingly, the subject matter product is a composite material. The composite material comprises at least one matrix and at least one supportive and/or at least one supplementary auxiliary component.

In the invention, the term “composite material” describes the materials formed by combining two or more different materials for collecting the best characteristics thereof in a single material at the macro-level.

In the invention, the term “matrix” describes the component where the composite material forms the main component and where the other components are encircled together and in the desired form.

Accordingly, the subject matter product comprises hydroxyapatite component as the matrix component. “Hydroxyapatites” are bio-ceramics used frequently since they increase bioactivity and mechanical characteristics of bioactive glass, alumina and calcium phosphate composite material. Hydroxyapatite forms 70% of the inorganic phase of natural bone, and is used in various biomedical areas like tissue engineering, controlled drug release. Hydroxyapatite provides a strong connection for accommodating hard tissues since it is chemically similar to the inorganic component of the bone tissue. As also known in the art, hydroxyapatite has disadvantages like late degradation, single bone induction performance, low mechanical resistance; and functioning of hydroxyapatite as a composite material component in a single manner is not preferred in the art due to these disadvantages. Accordingly, the present invention owners have considered that the hydroxyapatite exists as matrix component in the composite material in order to use technical advantages thereof.

The subject matter product comprises at least one supportive and/or supplementary component. The subject matter product comprises a natural and/or synthetic polymer material as at least one reinforcing and/or supportive component. Preferably the polymer material can be a hydrogel.

The subject matter product comprises at least one polymer material as the supportive and/or supplementary component; it comprises sodium alginate compound as said polymer material. Sodium alginate is a natural polymer material as also known in the art. Again as known in the art, sodium alginate can be preferred as component in bone tissue engineering. The present invention owners expect that the sodium alginate natural polymer material functions as a supplementary/supportive component in the composite material and provide contribution to improvement of bio-compliancy and muco-adhesion characteristics of the composite material. Since sodium alginate is a natural polymer material, it is supported that and not prevented that the subject matter product has low toxicity and high bio-compliancy characteristics.

One of the innovative characteristics of the present invention is that the subject matter product comprises manganese element. “Manganese” is one of the most apparent trace elements that exist in bone, and exists approximately at a value 1.7-3 ppm in bone. Manganese has an important duty in arrangement of the metabolism of bones and muscles. Moreover, manganese deficiency leads to weakening of osteoblast activities and delay of osteogenesis and this leads to bone deformation, growth inhibition, reduction in movement coordination and even bone loss. Manganese element partially takes part in carbohydrate synthesis metabolism of muco-polysaccharides and provides contribution to the growth and development of the bone. Manganese provides adhesion of cells like integrins to each other among the molecules that function in cell adhesion. Manganese increases stimulation of protein osteocalcin that binds calcium to bones, moreover, it increases alkaline phosphatase activity and improves collagen type I production and provides extra-cellular matrix of the bone to be shaped again. The present invention owners have decided that manganese element must exist in the product due to said characteristics.

It has been detected that manganese element also provides contribution to the antimicrobial characteristics of the final product. Antimicrobial activity tests have been applied to final product samples and the antimicrobial activity of the manganese element is searched. The samples which are to be examined are added into 5 ml Luria-Bertani liquid growth medium, and the bacteria culture shall be left to incubation at 37°C for 24 hours. After 24 hours, the series shall be diluted, and afterwards, spreading culturing shall be realized to Tryptic Strain Agar growth medium, and colony counting shall be realized after incubation at 37°C for a night. Inhibition % proportion shall be calculated by comparing with bacteria control that does not include sample therein. Test shall be made on gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria. These bacteria have been selected since they are prone to lead to multiple infections during implantation and surgical treatments. As seen in Table 1 , antibacterial test has been made by using Staphylococcus aureus bacteria which is a gram positive bacteria. As a result of this test, it is seen that the bacteria inhibition is 0% in frameworks that do not have Mn, bacteria inhibition proportion is 100% in bone frameworks that have 10% Mn proportion, and bacteria inhibition is 88% in bone frameworks that have 5% Mn proportion. Antibacterial characteristic is provided by adding Mn element to the structure. At the continuation of the study, the antibacterial test is planned to be made on Escherichia coli which is a gram negative bacteria. Moreover, Mn proportion shall be determined also by taking into consideration that it does not lead to toxic effect in the cells in the study to be made.

Table 1 : Results of the antibacterial test made by using gram positive bacteria of bone frameworks comprising 5-10% Mn element pressed as a result of pre-study.

The subject matter product is a composite material for providing the desired characteristics; and comprises hydroxyapatite as the matrix component and comprises sodium alginate and manganese as the supplementary/supportive component.

In another perspective, the present invention relates to a method for production of the product comprising hydroxyapatite as the matrix component and comprising sodium alginate and manganese as the supplementary/supportive component. Accordingly, in the invention, the operation principle of 3 dimensional printers is used as the method, the following processes are realized respectively;

- Preparing bio-inks for use in 3D printers,

- Obtaining the product in 3D printers,

- Realizing sintering processes for protecting the product and for providing additional mechanical characteristics.

Preparation of the bio-ink

In the present invention, 3D printer technology is the process of production of a designed virtual object from materials like polymer, composite and resin by subjecting said materials to thermal or chemical process. Accordingly, the most critical step of the present invention is obtaining the bio-ink to be used in 3D printers. The bio-ink must have a form that is suitable to be processed in 3D printer, and the final product to be obtained must have the desired mechanical and bioactive characteristics. Accordingly, the existence percent of the components that exist in the bio-ink is substantially important. Accordingly, the invention owners have made studies about the percent that has to exist in the product for each component. Accordingly, preferably water is used as solvent for providing homogenous structure to the components in obtaining bio-ink. The technical solution and advantages provided by using water as solvent are known in the art, but the protection scope of the present invention is not delimited with the kind of the solvent. As a result of advancing of technology, the usage of different solvents can also be possible in obtaining the subject matter bio-ink. Accordingly, hydroxyapatite/water (g%/ml) between solvent and hydroxyapatite which is the matrix component is at a value between 90% and 120%. Preferably, while the proportion of hydroxyapatite/water is 100% and above, the most optimum values are between 105% and 115%.

Another important issue is the existence proportion of hydroxyapatite and sodium alginate in preparation of the bio-ink. Sodium alginate must exist at the weight proportions in a manner providing muco-adhesive characteristic in the final product, at the same time, it has limited existence proportion for obtaining bio-ink with the desired form together with the solvent and hydroxyapatite. The invention owners have determined that sodium alginate/hydroxyapatite (gram/gram%) is at a value between 0.06% and 0.1 %. This value is preferably between 0.075% and 0.09%.

The technical solutions and advantages provided by manganese element to the final product have been mentioned in the lines beforehand. Accordingly, manganese, that is for providing the desired performances, exists at a value between 2% and 10% by weight in accordance with total bio-ink weight. It is very important that manganese element that is at a value between 2% and 10% by weight exists in the bio-ink. While it is possible that the desired technical effects can be observed under said value, it has been detected by means of the studies that manganese can have toxic effect above the given value. Accordingly, for preparing the bio-ink, the following process steps are provided:

- The determined proportions of sodium alginate are added into pure water, and mixing process is realized with the help of a stirrer such that a homogenous mixture shall occur therein,

- Specific proportions of hydroxyapatite component are added to the obtained mixture, and the mixing processes are realized until the mixture is obtained again,

- Specific proportions of manganese element are added to the obtained mixture, and the mixing processes are realized until the mixture becomes homogenous.

Processing the obtained bio-ink in 3D printers and obtaining the pre-product In this process step, pre-products are obtained by utilizing 3D technology of the bio-ink samples obtained in a prior process step. Accordingly, determination of specific parameters in 3D printers for the obtained bio-ink has become the study subject. Accordingly, 3D technology has been used by using 3D printer parameters given in Table 2.

Table 2: Parameter values used in 3D printer in the subject matter method

Sintering processes

In the present invention, the sintering process is applied for combining powder particles of pre-product samples, obtained by means of 3D technology, with the help of thermal energy. As a result of application of sintering processes, the pre-product becomes a final product.

The invention owners have worked on the required parameters for the sintering process, and they have shared these under this title for the addition of the obtained results to the literature. Accordingly, the parameters to be used for realization of the sintering processing are given in Table 3.

Table 3: Degree and duration parameters that can be used for the sintering processes In a preferred application of the present invention, sintering parameters arranged as b) given in Table 3 are used. Accordingly, the pre-products have been sintered for 1 hour at 350°C, for 1 hour at 650°C and for 2 hours at 1200°C.

Another innovative characteristic of the present invention is that the coating process is realized on the obtained final product in order to obtain and/or improve antibacterial and anti- oxidative characteristics. It is very important that said coating is bio-compliant material. The subject matter product is coated with cardamom oil.

In the present invention, the coating material, namely cardamom oil can be coated onto the final product by means of various coating methods but preferably electro-spraying method is used in the present invention. Accordingly, the invention owners have determined suitable coating process parameters or electro-spraying method. The following processes are realized respectively,

- Determined proportions of PLA (polylactic acid) is dissolved preferably in DCM,

- Cardamom oil, that is at a value between 50% and 100%, is added to the obtained solution, and mixing is realized until a homogeneous mixture is formed.

Preferably, PLA is dissolved in DCM at a value between 1% and 10% by weight. In the most preferred application, PLA is dissolved at a value between 2% and 6% by weight.

The obtained solution is sprayed onto the final product, where sintering processes are applied, by means of electro-spraying method. Accordingly, the device used in electrospraying process has flow speed at a value between 15 and 25 pl/min and has a voltage value between 10 kV and 20 kV.

Tests

Cell culture

MC3T3-E1 mouse osteoblast cells have been obtained from Atihm University Metallurgy and Material Engineering Department (Ankara, Turkey) for realizing cell culture studies.

DMEM/F-12 (Modified Pteridium Medium of Dulbecco/ Nutrient Mixture F-12), FBS (Fetal Bovine Serum), L glutamine, penicillin/streptomycin, BSA (Bovine Serum Albumin) and Phosphate buffered saline (PBS) tablets have been purchased from Amresco (Solon, USA). (99% purity, volume/volume), 3-(4,5-dimethyl-2-tiazol)-2,5 diphenyl-2H-tetrazolium bromide (MTT) powder, 0.25% Trypsin/EDTA solution (weight/volume), glutaraldehyde, dimethylsulfoxide (DMSO), hexamethyldisilan (HMDS) (99% purity, volume/volume), BCIP/NBT tablets shall be obtained from Sigma-Aldrich (St Louis, USA). Moreover, dyeing kits have been purchased from Alizarin Red-S and Alkali Phosphates (ALP) Sigma-Aldrich (St Louis, USA).

Biological characterizations of raw and sintered 2% Mn, 4% Mn and 6% Mn (by weighthydroxyapatite have been realized by means of MC3T3-E1 mouse osteoblast line. In this experiment, two different hydroxyapatite morphologies, namely powder and framework have been used. At the beginning, the two groups have been sterilized for 45 minutes under ultraviolet light. While the powder samples are being placed to 96-well plates, the frameworks have been placed to 24-well plates. MC3T3-E1 cell with concentration of 3x10 ³ cell/ml concentration has been cultured to all plates.

The samples have been incubated for 21 days (at 37°C 5% CO ₂ ), and the growing medium DMEM/F-12 has been prepared by using FBS (10% volume/volume), penicillin streptomycin solution (1% volume/volume), L-glutamine (1% volume/volume). The biological characterization of the prepared material has been realized by MTT test for determining cell viability, Alizarin Red Dyeing for determining calcium accumulation, and Alkaline Phosphate Activity for determining differentiation. Additionally, the adhesion, growth and augmenting of the cells cultured to the prepared samples have been examined by means of SEM.

MTT test

The viability of the osteoblast MC3T3-E1 cells sintered and cultured onto raw HA bone frameworks and powder pellets has been realized with MTT determination on 1 ^st , 7 ^th , 14 ^th and 21 ^st days. After incubation (5% CO ₂ , 37°C), the medium has been removed, and the samples have been washed with PBS solution three times. After the rinsing process, the samples that exist on the 24-well plates have been transferred to a new medium with 96 wells comprising 90 pL new medium and 10 pL MTT solution. MTT solution has been added to well plaques, and afterwards, all samples have been incubated 3 hours more. When the incubation duration finishes, MTT solution is removed and 200 pL DMSO has been added for dissolving formazan crystals. Finally, the growth medium is taken from the wells, and the absorbance values of the solutions have been measured at 540 nm by means of micro-plate reader.

Cell morphology analysis by means of SEM Cell adhesion and growth have been imaged by means of SEM on the produced sintered and raw HAP bone frameworks. The images have been taken in 7 ^th and 21 ^st days of cell culturing. The cultured samples have been taken from the incubator, and the medium has been removed, and the cells have been washed with PBS solution three times. For fixing the cells, 10% formalin solution has been added to the wells, and has been kept in a dark medium for 20 minutes at room temperature. Then, formalin solution has been removed from the medium and the cells have been washed with PBS solution three times again. Then, the samples have been rinsed in ethanol solutions (30%-100%) such that each one is washed for two minutes. The samples have been transferred to the new 24-well plate in a careful manner, and have been subjected to process with 100% hexamethyldisizan solution for 5 minutes. Finally, all samples have been transferred to SEM holders and has been coated with Au-Pt and the images thereof have been taken at 2500X and 5000X.

Alizarin red dyeing

The characterization of the calcium accumulation of the samples where alizarin red dyeing cell has been cultured has been realized on 7 ^th , 14 ^th and 21 ^st days. In order to obtain the dyeing solution, 2 grams of alizarin red have been dissolved in 10 ml pure water, and the solution pH has been adjusted to 4.1-4.3 and the solution has been preserved in dark medium until it is used. After incubation, the growing medium (5% CO2, 37°C) has been removed, and all samples have been washed three times with PBS solution. After the rinsing process, 10% formalin solution has been added for fixing of the cells and has been kept in the dark for 30 minutes. Formalin solution has been removed from the medium in a careful manner, and the cells have been washed with pure water. Then, dyeing solution shall be added onto the samples and shall be waited for 30 minutes in a dark medium. When the waiting duration ends, the samples have been centrifuged at 200 rpm. Finally, in order to provide calcium accumulation and in order to characterize mineralization level, the absorbance values of the solutions have been measured at 405 nm by means of micro-plate reader.

Alkaline phosphatase activity

ALP activity of the cultured HAP frameworks has been examined on 7 ^th , 12 ^th and 21 ^st days of the culture in order to determine the differentiation. In order to prepare the substrate solution, nitro blue tetrazolium and 5-bromo-4 chloro-3-indolil phosphate tablet has been dissolved in 10 mL pure water. Then, this prepared solution has been waited for 2 hours at room temperature in a dark medium. The samples that exist in the culture have been removed from the incubator and have been put to the medium, and the cells have been washed with PBS solution for three times. 10% formalin solution shall be added to the samples and has been kept for 60 seconds for fixing the cells. Then, the formalin solution has been removed and the cells have been washed three times with PBS solution. Finally, substrate solution has been added to the wells, and has been incubated for 10 minutes at room temperature and measurement has been taken at 405 nm by means of the micro-plate reader.

The subject matter product belongs to bone tissue engineering technical field, and as a result of the tests made, it has been detected that the subject matter product is suitable for use in bone diseases that are genetic or that occur afterwards and/or in repair of breakages and/or in treatment of breakages, and these repair or treatment processes take place in short durations.

The protection scope of the present invention is set forth in the annexed claims and cannot be restricted to the illustrative disclosures given above, under the detailed description. It is because a person skilled in the relevant art can obviously produce similar embodiments under the light of the foregoing disclosures, without departing from the main principles of the present invention.