KR102321658B1 - Implant surface coating composition comprising rosmarinus officinalis extract - Google Patents

Abstract

The present invention relates to a composition for coating the surface of an implant comprising a rosemary extract, and by improving the effect of differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts and mineralization of the jawbone and implant. By using the effect of inducing osseointegration between the surfaces of the body, it can be usefully used as a composition for coating the surface of an implant.
Furthermore, it is possible to provide an implant coated with the composition.

Description

Composition for coating an implant surface comprising rosemary extract {IMPLANT SURFACE COATING COMPOSITION COMPRISING ROSMARINUS OFFICINALIS EXTRACT}

The present invention relates to a composition for coating an implant surface comprising a rosemary extract, and more particularly, to a composition using the effect of inducing osseointegration between the jawbone and the implant body surface.

Implant is a collective term for devices implanted in the body to restore lost human tissue. In particular, dental implants are implanted in the jawbone where there is a tooth defect or where a tooth is extracted. It is a dental treatment procedure that restores the function of natural teeth by implanting a biocompatible implant body after increasing the volume through additional surgery.

Although these implants are made of a bio-ceramic material such as alumina or zirconia, most of them are currently made of a metal material, and among them, titanium (Ti) or an alloy thereof is the main implant material. Titanium (Ti) is the most frequently used material for implants because of its excellent physical and biological properties such as low density, mechanical strength and chemical resistance to body fluids. Therefore, the chemical properties and structure of the surface of titanium (Ti) are major factors influencing osseointegration.

Because the surfaces of titanium (Ti) and titanium (Ti) alloys are bioinert, when used for osseointegration, they can form fibrous tissue of varying thickness that encapsulates the implant and separates it from the surrounding environment. can

This lack of osseointegration is being solved through modification of the implant surface by forming a bioactive coating. Current research on implant surface modification is focused on making virtually inactive materials into bioactive materials, or rather affecting the type of protein that is easily absorbed into the surface after implantation. Classifications of surface modifications range from abiotic coatings such as carbides, fluorine, calcium, hydroxyapatite or calcium phosphate, to lipid monolayers or lipid bilayers, growth factors, proteins, and/or peptides that mimic the biological environment. They range from coatings to coatings.

Specifically, by coating the surface of a metal material with a calcium phosphate-based ceramic material such as hydroxyapatite (HA) or tricalcium phosphate (TCP) having sufficient strength while having human-friendly bioactive properties. A method for improving biocompatibility and improving osseointegration between the jawbone and the implant surface has been used, and in recent research, an implant with a magnesium ion-implanted titanium oxide layer prepared by anodization has been improved by mechanical cutting or cutting. It is known to promote osseointegration compared to the blast-treated implant (Korean Patent Application Laid-Open No. 2004-0046248).

However, even with the implant having the coating layer, osseointegration, which forms a bone at the interface between the implant body and the surrounding tissue, cannot be achieved quickly to a satisfactory level, and the surface of the implant manufactured by the anodization method is micron-scale. Although it has a porous structure of , it does not maintain the unique surface structure of the micron unit formed by the blasting process, and there is a problem that the coating layer may be dropped due to the relatively thick oxide layer of 0.5 to 10 μm.

In order to overcome these problems, a lot of research has not been done on a method of using a composition containing a natural extract, but the present applicant includes a natural extract, rosemary extract, as an active ingredient to increase human compatibility, and to increase the compatibility of the implanted implant. A coating composition capable of improving osseointegration between a surface and surrounding tissues has been invented.

US 10-1134977 B1 US 10-1463386 B1

It is an object of the present invention to provide a composition for coating an implant surface comprising a rosemary extract.

Another object of the present invention is to induce osseointegration between the jawbone and the implant body surface by improving the differentiation and mineralization effects of MC3T3-E1 preosteoblasts into osteoblasts. It is to provide a composition for coating the surface of the implant that can be.

Another object of the present invention is to provide an implant coated with the composition.

In order to achieve the above object, the composition for coating the implant surface according to an embodiment of the present invention is a coating composition comprising a rosemary extract as an active ingredient, and the coating composition is coated on the surface of the implant body to provide a jaw bone and an implanted body. It induces osseointegration between surfaces.

The composition enhances the differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body.

The differentiation and mineralization are regulated by the expression of non-collagenous genes and collagenous genes.

The composition increases the cell viability of MC3T3-E1 preosteoblasts.

The rosemary extract is rosmarinic acid.

The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.

The implant according to another embodiment of the present invention is coated with the composition.

Hereinafter, the present invention will be described in more detail.

As used herein, the term 'extract' has the meaning commonly used as a crude extract in the art, but broadly includes a fraction obtained by additionally fractionating the extract. That is, the plant extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract of the present invention.

The extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

As used herein, the term "comprising as an active ingredient" means an amount sufficient to affect a desired biological effect, such as, but not limited to, beneficial results, including, but not limited to, prevention, reduction, alleviation or elimination of signs or symptoms of a disease or disorder. is meant to include, and this amount is referred to as an “effective amount”. Accordingly, the total amount of each active ingredient of a composition or method is sufficient to exhibit a significant individual benefit. Accordingly, the effective amount will vary depending on the circumstances in which it is administered. An effective amount may be administered in one or more prophylactic or therapeutic administrations.

The term 'implant body' used in the specification of the present invention refers to a site serving as a root of a tooth, including zirconium, an alloy and/or oxide thereof, tantalum, an alloy and/or oxide thereof, hafnium, an alloy and/or oxide thereof, niobium, alloys and/or oxides thereof, chromium-vanadium alloys and/or bonded oxides, and other metal(s) such as stainless steel, other metal alloy(s) and/or metal oxide(s), for example titanium, One or more metals, such as, but not limited to, titanium alloys and/or titanium oxides, metal alloys and/or metal oxides.

The composition for coating the implant surface according to an embodiment of the present invention is a coating composition comprising a rosemary extract as an active ingredient, and the coating composition is coated on the surface of the implant body to form osseointegration between the jawbone and the implant body surface. ) to induce

The rosemary (Rosmarinus officinalis L.) is also referred to as rosemary or bitter gourd, and is an evergreen small shrub of the Lamiaceae family (Labiatae) whose leaves, shoots and flowers are edible. It is a perennial plant with needle-like leaves and is a herb native to the Mediterranean coast. It has green leaves and a characteristic scent. The flowers bloom in winter and spring and come in white, pink, purple, and blue colors. It has been widely used in Western traditional cuisine since ancient times, and is still used in almost all dishes in Italy. In particular, it is often used in meat dishes along with sage or thyme because the flavor is preserved even when heat is applied.

Main ingredients include beta-carotene, beta-sitosterol, bedulin acid, borneol, caffeic acid, caper, capnosol, carvacrol, carvone, caryophyllene, chlorogenic acid, diosmin, genkwanin, geraniol, hespei Dean, limonene, linalool, olenic acid, 1,8-cineyl, rosemarinol, rosmaric acid, salicylic acid, squalene, tannin, thymol, assolic acid, calcium, magnesium, manganese, phosphorus, potassium, zinc, vitamin B1 , vitamin B3 and vitamin C, and a wide variety of active ingredients exist.

In addition, rosemary is known to have various effects such as antibacterial activity, antioxidant activity, whitening activity, and antiviral activity due to the above components. In particular, the most widely used function currently industrially is the antioxidant effect of oils and fats, and oil-soluble antioxidant products using rosemary extract are being sold.

The osseointegration is an important factor determining the success of the implant procedure, and after the implant body made of titanium (Ti) or titanium (Ti) alloy is surgically implanted in the jawbone, osteoblasts are implanted It refers to the process of forming a morphological, physiological, and direct connection between the jawbone and the implanted body surface by proliferating on the surface of the implanted body. By this osseointegration, the implant body is firmly fixed to the jawbone.

When the composition containing the rosemary extract as an active ingredient is coated on the surface of the implant body, the effect of osseointegration between the implant body surface and the jawbone can be increased by the action of the active ingredient contained in rosemary. .

The composition enhances the differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body.

Osteoblasts are cells that have the ability to mineralize bone tissue by synthesizing and secreting bone matrix and depositing inorganic salts such as calcium and magnesium ions in the matrix. These are the cells that can be seen at the site of neogenesis.

On the other hand, preosteoblasts are cells that are differentiated into osteoblasts through a differentiation process into precursor cells of osteoblasts, and in the present invention, the preosteoblasts are MC3T3-E1 precursor osteoblasts. (preosteoblasts), but is not limited thereto, and may be used without limitation as long as it corresponds to the type of preosteoblasts that can act in the osseointegration process.

For successful implantation of the dental implant body, first, the proliferation of preosteoblasts is induced in the surrounding tissue of the missing tooth part and the surface of the implant body. Thereafter, the proliferated preosteoblasts are differentiated into osteoblasts through various signal transduction pathways, and the differentiated cells undergo osseointegration between the jawbone and the implant surface through mineralization. ) to induce

At this time, the composition comprising the rosemary extract as an active ingredient improves the differentiation and mineralization effect of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body, It can act to enhance osseointegration between implant surfaces.

The differentiation and mineralization are regulated by the expression of non-collagenous genes and collagenous genes.

Differentiation and mineralization of the MC3T3-E1 preosteoblasts into osteoblasts of the present invention are non-collagen genes ALP (alkaline phosphatase), BSP (bone sialoprotein), DMP-1 (dentin matrix protein-1), DSPP (dentin sialophosphoprotein), OCN (osteocalcin) and ON (osteonectin), and collagen genes (collagenous genes), Col I (type I collagen).

Specifically, in the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body, the expression rates of the ALP gene, OCN gene, and Col I gene increase.

The expression rate of the ALP gene increases in the initial stage of differentiation into osteoblasts, and is used as a biomarker for identification of the initial stage of differentiation.

Like the ALP gene, the Col I gene also has an increased expression rate at the initial stage of differentiation, leading to differentiation of MC3T3-E1 preosteoblasts into osteoblasts. However, the Col I gene has a characteristic that its expression rate is reduced in the course of the mineralization.

The OCN gene increases in expression rate at the end stage of differentiation into osteoblasts, and is utilized as a biomarker of the end stage of differentiation and bone matrix formation.

Meanwhile, the BSP gene, DSPP gene, DMP-1 gene and ON gene are genes related to the mineralization of bone matrix.

In the case of coating the surface of the implant body using the coating composition of the present invention, the active ingredient of the rosemary extract regulates the expression of the non-collagenous genes and the collagen genes, so that MC3T3-E1 is MC3T3-E1 on the surface of the implant body. It can promote differentiation and mineralization of preosteoblasts into osteoblasts.

The composition increases the cell viability of MC3T3-E1 preosteoblasts.

When the cell viability of the preosteoblasts is increased, the differentiation and mineralization effects of the preosteoblasts into osteoblasts are increased, so that the osseointegration between the surface of the implant body and the jawbone is increased. (osseointegration) can be effectively induced.

The composition can induce effective osseointegration by increasing cell viability in the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body.

Preferably, the rosemary extract may further include a natural extract selected from the group consisting of a mulberry leaf extract, a mulberry leaf extract, a wisteria leaf extract, an elm leaf extract, and mixtures thereof.

The Cudrania tricuspidata (Carr.) Bureau ex Lavallee) is a small tree belonging to the dicotyledonous nettle family, and contains vitamins A, B1, B2, C, dietary fiber, flavonoids, gamma aminobutyric acid, rutin, etc. , hypertension, diabetes, cholesterol improvement, bowel activity improvement, antioxidant action, anticancer action, etc. The root bark, bark, bark and leaves of Cudrania biloba contain various ingredients effective for the human body. It has been used as a medicine such as a treatment, antipyretic, anti-dry, expectorant, diuretic, hemostatic, antipyretic, etc., and is used for athlete's foot as an antifungal agent and for chronic indigestion caused by weakness of the digestive system.

The gulpi tree (Cornus kousa) is a deciduous small tree of the Astragalus family, and mainly grows in the Yangji or coastal waters at the foot of the mountains in Korea (South Chungcheong Province), Japan, Taiwan, and China. The leaves are odd pinnate leaves, and consist of 7 to 19 small leaves without petioles. The small leaves are oval lancet-shaped or egg-shaped lanceolate with a sharp tip and deep serrations on the edge. There are white hairs on both sides of the leaves, but they gradually disappear. There is also hair on the peduncle, but it gradually disappears. The flowers are yellowish green flowers, 5-8cm in male ear and 2-4cm in female ear, and mature female ear of female flower is in the shape of a pine cone. Ears of fruit are long oval, blackish-brown, and hairless. The bract does not fall off and is lanceolate, and the fruit is a winged nut and ripens in September to October.

The fruit bark (peel) of gulpi tree contains eugloon and the fruit contains tannins, so it is used in folk medicine as a phlegm, insecticide, and pregnancy emetic.

The Eupatorium japonicum Thunb. ex Murray is a perennial herb that grows in mountains and fields in Korea. The growing environment is independent of the fertility of the soil, and it grows in semi-shaded places and sunny places. The height is 70 to 150 cm, the leaves are opposite to each other in an egg-shaped long oval or oval shape, and the pointed end is 10 to 18 cm long and 3 to 8 cm wide. disappears Flowers are arranged in flat clusters at the ends of main stems to form small flowers. The fruits ripen in October to November, and the seeds have white feathers. It is used for ornamental purposes, the young shoots are edible, and the outpost including the roots is used for medicinal purposes.

Ayapin, which is contained in stamens, is used in oriental medicine for jaundice, colic, stroke, hypertension, postpartum abdominal pain, hematemesis, and pneumonia. In addition, it is known that the leaf stalk is a diuretic to remove edema and is good for fatigue recovery as a bathing agent. Furthermore, it is known that it has an antiviral action, a direct inhibitory action against an epidemic virus, indigestion, abdominal pain, and the like.

The elm tree (Ulmus davidiana var. Japonica) belongs to a deciduous broadleaf tree of the dicotyledonous plant Nettle family Elmaceae. It is distributed in Korea, Japan, Sakhalin, the Kuril Islands, northern China and Siberia, and grows to a height of 20m and a diameter of 60cm near a mountain stream or valley. There are several types of elm, but the shape of the leaves and the use of medicines are all the same. These elm trees have been used since ancient times as a diuretic, a remedy for sore throats or boils. As a medicine, the root bark of elm is used, which is known to contain flavonoids, saponins, tannins, or a large amount of mucilage.

When the rosemary extract further comprises a natural extract selected from the group consisting of a mulberry leaf extract, an oleifera leaf extract, a stalagmite leaf extract, an elm leaf extract, and mixtures thereof, the non-collagenous genes ) and the effect of regulating the expression of collagen genes can be further maximized, and as a result, differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body (mineralization) effect can be further improved. Furthermore, it is possible to maximize the effect of osseointegration between the jawbone and the implant body surface by increasing the cell viability in the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts. .

More preferably, the rosemary extract, Cudrania mulberry leaf extract, Gyulpi tree leaf extract, Staple tree leaf extract and Elm leaf extract are in a weight ratio of 1:0.3:0.3:0.5:0.5 to 1:1:1:1:1 It is used by mixing with MC3T3-E1 preosteoblasts and shows excellent expression control effect of non-collagenous genes and collagen genes within the above weight range. ) can maximize the effect of differentiation and mineralization, and can exhibit the effect of increasing cell viability in the process of differentiation of MC3T3-E1 preosteoblasts. However, when it is less than or exceeding the range value, there is a problem in that the effect is significantly reduced.

In the present invention, the rosemary extract is rosmarinic acid.

Rosmarinic acid is a compound that is a biochemical composition found in various plants such as rosemary, sage, basil, and mint. It is known as a substance that prevents spoilage due to oxidation of food. It is known that the main action has antioxidant action, anti-inflammatory action, anti-mutagenic action, anti-microbial action, anti-viral action, and anti-cancer action. In addition, after a meal, the blood condition temporarily thickens for nutrients to enter the blood, and rosmarinic acid decomposes maltose into glucose, thereby discharging the sugar dispersed in the blood to the outside of the body. It has been reported that it suppresses the rise in blood sugar levels and prevents hypertension, diabetes, and hyperlipidemia.

In addition, rosmarinic acid is effective for headache and is known as a good substance for the circulatory system. Furthermore, rosmarinic acid has a strong antiseptic action, is effective in preventing hair loss, helps in hair management, has a function of restoring dry and aged skin to normal, and promotes cell regeneration to reduce wrinkles serves to reduce Because of these functions, it is a material that is currently attracting attention as a very important ingredient in the cosmetic field.

The rosmarinic acid is represented by the following formula (1):

[Formula 1]

In the present invention, the extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof. The 'rosemary extract' refers to an extract obtained by extraction from rosemary.

The rosemary extract is a pulverized product of dried rosemary having a mixing ratio of 1:0.1 to 1:10 of an alcohol having 1 to 4 carbon atoms, such as water, ethanol, methanol, etc., a polar solvent such as dimethyl sulfoxide, or alcohol and water. Elution may be performed with a mixed solvent, and more specifically, ethanol may be used as an extraction solvent. At this time, the extraction temperature may be 10 ℃ to 100 ℃, preferably the extract extracted at 25 ℃.

The extraction solvent may be used in an amount of 2 to 50 times, preferably 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, and in particular, from 1 to 24 hours.

It may be a result obtained by concentrating the filtered extract under reduced pressure with a vacuum rotary concentrator, but as long as it is a rosemary extract that can exhibit the effect of inducing osseointegration between the jawbone and the implanted body surface of the present invention, it is limited thereto. It includes all extracts, diluted or concentrated solutions of extracts, dried products obtained by drying the extracts, or prepared or purified products thereof.

The Cuji mulberry leaf extract, Gulpi tree leaf extract, Staple tree leaf extract and Elm leaf extract can also be prepared using the same method as the rosemary extract.

The implant according to another embodiment of the present invention is coated with the composition.

As a method of coating the surface of the implant body, an aerosol deposition (AD) method, a sol-gel method, a plasma spraying method, an ion beam deposition method, a hydro thermal method There is a method using a reaction, but is not limited thereto, and includes all methods of coating the surface of the implant body that can be applied by those of ordinary skill in the art.

According to the composition for coating the implant surface containing the rosemary extract of the present invention, by regulating the expression of non-collagenous genes and collagen genes, osteoblasts of MC3T3-E1 preosteoblasts ( It is possible to induce osseointegration between the jawbone and the implant body surface by enhancing the effect of differentiation and mineralization into osteoblasts.

Furthermore, by providing an implant coated with the composition, it is possible to effectively induce osseointegration between the jawbone and the surface of the implant body.

1 is an experiment showing the effect of rosmarinic acid on cell viability in the process of differentiation of MC3T3-E1 preosteoblasts on the surface of a titanium plate (Ti discs) according to an embodiment of the present invention; It is the result.
2 is a process of differentiation of MC3T3-E1 preosteoblasts on the surface of titanium discs according to an embodiment of the present invention, non-collagenous genes and collagenous genes; FIG. It is an experimental result showing the effect of rosmarinic acid on the expression of genes.
Figure 3 shows the effect of rosmarinic acid on the mineralization action during the differentiation process of MC3T3-E1 preosteoblasts on the titanium plate (Ti discs) surface according to an embodiment of the present invention; The effect was shown through the Alizarin Red S staining method.
Figure 4 shows the effect of rosmarinic acid on the mineralization action during the differentiation process of MC3T3-E1 preosteoblasts on the titanium plate (Ti discs) surface according to an embodiment of the present invention; These are the results showing the effect through the alkaline phosphatase staining method.
Figure 5 shows the differentiation (differentiation) of MC3T3-E1 preosteoblasts on the surface of the titanium plate (Ti discs) according to an embodiment of the present invention, mineralization (mineralization) and bone formation (bone formation) through the action It is a block diagram showing the effect of rosmarinic acid on the osseointegration between the surface of the implant body and the jawbone.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Manufacture of titanium plates (Ti discs)]

The polished titanium plates (Ti discs) had a thickness of 2 mm and were cut to have diameters of 15 mm, 20 mm and 48 mm.

[Preparation Example 2: Preparation of rosemary extract (rosmarinic acid)]

After dissolving rosmarinic acid (Sigma, USA) in DMSO (dimethyl sulfoxide) as a solvent, it was stored at -20 °C to prepare a rosemary extract (rosmarinic acid).

Thereafter, the stock solution was diluted to the treatment concentration applied in the experiment before use.

[Preparation Example 3: Preparation of other extracts]

Cuji mulberry leaves were pulverized with a blender and then prepared into powder. 50% ethanol as an extraction solvent was added to the powder sample at a ratio of 1;10 (w; v), and then completely immersed, and extracted three times for 3 hours while refluxing at 80°C. The extract is Whatman No. 2 filtered with filter paper. The filtrate was concentrated under reduced pressure at 60° C. to prepare a Cuji mulberry leaf extract.

In the same manner as in the preparation of the Cuji mulberry leaf extract, a gulpi tree leaf extract, a stamen leaf extract and an elm leaf extract were prepared.

[Preparation Example 4: Preparation of mixed extract]

The rosemary extracts of rosmarinic acid (RA), mulberry leaf extract (BE), gulberry leaf extract (GE), staphylococcus leaf extract (DE) and elm leaf extract (NE) are shown in Table 1 below. Mixed extracts were prepared by mixing within the weight range.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 RA 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 BE - 65 - 65 - - - 65 65 - 65 20 30 65 100 120 GE - - 65 65 - - - 65 65 65 - 20 30 65 100 120 DE - - - - 75 - 75 75 - 75 75 40 50 75 100 130 NE - - - - - 75 75 - 75 75 75 40 50 75 100 130

(Unit: parts by weight)

[Experimental Example 1: Experiment on the effect of osseointegration between the jawbone and the implanted body surface]

1. Experimental method

Cell culture and culture under conditions treated with rosmarinic acid

Alpha containing 10% fetal bovine serum (FBS, WelGENE) and 1% antibiotic and antifungal solution (WelGENE), MC3T3-E1 cells, a cell line of preosteoblasts collected from the cranial tube of mice It was cultured in modified eagle's medium (α-MEM; WelGENE, KOR).

Thereafter, the cells were transferred to the surface of Ti discs, and the medium conditions were 5% fetal bovine serum (FBS), 10 mM beta-glycerol phosphate, and 50 μg/ Changed to a differentiation medium containing ㎖ of ascorbic acid (ascorbic acid), and cultured for 24 hours in conditions treated and not treated with 14 ㎍ / ㎖ of rosmarinic acid (rosmarinic acid).

Thereafter, the cells were placed in a humidified chamber and maintained at _{37° C. of 5% CO 2 .}

Cell viability assay

^{3 x 10 5} cells/ml of MC3T3-E1 preosteoblasts transferred to the surface of Ti discs were treated with 14 μg/ml of rosmarinic acid in a differentiation medium under conditions without and without treatment. incubated for 4 days, 7 days, and 10 days, respectively.

Then, MTT assay (Sigma-Aldrich, USA) was performed to test the cell viability of MC3T3-E1 preosteoblasts.

Extraction of total cell RNA and reverse transcription polymerase chain reaction (RT-PCR)

^{1 x 10 6} cells/ml of MC3T3-E1 preosteoblasts transferred to the surface of titanium plates (Ti discs) were treated with rosmarinic acid for 4 days, respectively, on differentiation medium under conditions without and without treatment. Cultured for 7 days and 10 days.

Total RNA of cells was extracted using RiboEXTM reagent (GeneAll, KOR), and DNA (cDNA) complementary to RT Premix (GeNet Bio, Korea) was synthesized using 1 μg sample of total RNA.

Thereafter, polymerase chain reaction (PCR) was performed in a thermocycler (Takara Bio Inc., Japan) by adding 1 μg of cDNA and a complementary primer.

A primer complementary to the nucleotide sequence of each of ALP, BSP, DSPP, DMP-1, OCN, ON, Col I and GAPDH was prepared and used for the polymerase chain reaction (PCR).

The annealing temperature of each primer and the number of cycles of the polymerase chain reaction (PCR) were 62° C., 35 cycles for ALP; 60°C, 35 cycles for BSP; For DSPP, 55°C, 35 cycles; 55°C, 35 cycles for DMP-1; 66°C for OCN, 27 cycles; 63°C for ON, 35 cycles; 50° C., 35 cycles for Col I; In the case of GAPDH, it was tested at 60°C and 30 cycles, and GAPDH was used as a control.

All products of the polymerase chain reaction (PCR) were electrophoresed on 1.2% agarose gel (Takara Bio Inc.) buffered with 0.5 X Tris-borate-EDTA, and stained with ethidium bromide (GeNet Bio, KOR).

Stained bands were expressed using Gel-Doc (Bio Rad Laboratories, USA), and band intensity was measured using Science Lab Image Gauge (Fuji Film, Japan).

Alizarin red S staining and alkaline phosphatase (ALP) staining assays

To confirm the formation of mineralized nodules, 1.5 x 10 ⁵ cells/ml MC3T3-E1 preosteoblasts transferred to the surface of Ti discs were treated with rosmarinic acid. It was cultured for 4 days, 7 days, and 10 days on differentiation medium under one condition and untreated condition, respectively.

After removing the medium, the cells were fixed in 4% paraformaldehyde (paraformaldehyde), and stained with 2% Alizarin red S (Alizarin red S, pH4.2) (Sigma-Aldrich) for 15 minutes.

For ALP staining, cells were fixed in 4% paraformaldehyde, rinsed 3 times with 1 X TBST, and then ALP substrate solution (NBT/BCIP Solution; Roche, USA) at 37°C for 1 hour. ) was treated.

Mineralized nodules were observed through a stereoscopic microscope (Stemi 2000 C, Carl Zeiss, GER).

Then, for quantitative analysis, 10% cetylpyridium chloride (Samchun chemical, KOR) was treated on 10 mM sodium phosphate (pH 7.0) to extract the stain for 15 minutes. Then, using a microplate reader (BioTek Instruments, USA), by measuring the absorbance at wavelengths of 562 nm and 540 nm, Alizarin red S staining (Alizarin red stain) and alkaline phosphatase staining (ALP stain) results were quantified.

Statistical Analysis

All experiments were repeated three times, and all data were calculated as mean and standard deviation determined using SPSS 12.0 (SPSS, USA). Statistical analysis was performed with independent samples t-test, and differences at p < 0.05 were considered statistically significant.

2. Experimental results

Effect of rosmarinic acid on cell viability of MC3T3-E1 preosteoblasts

During the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts on the surface of Ti discs, the cell viability of MC3T3-E1 preosteoblasts is acid), when treated with rosmarinic acid, increased 1.2 times on days 4 and 7, and increased 1.1 times on day 10 ( FIG. 1 ).

The above results show that rosmarinic acid is converted into MC3T3-E1 precursor bone in the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts, which are essential for osseointegration between the jawbone and the implant surface. It is demonstrated to increase the cell viability of cells (preosteoblasts).

Experiments on the effect of rosmarinic acid on the expression of non-collagenous genes and collagenous genes in the process of differentiation of MC3T3-E1 preosteoblasts

As a result of reverse transcription polymerase chain reaction (RT-PCR) analysis, the mRNA expression rates of ALP, BSP, DSPP, OCN and Col I in the process of differentiation of MC3T3-E1 preosteoblasts were treated with rosmarinic acid. Or, it was found to increase with the passage of time in all of the untreated cells (FIG. 2).

In particular, when treated with rosmarinic acid, it was confirmed that the expression rate was significantly increased compared to the case where rosmarinic acid was not treated ( FIG. 2 ).

Specifically, compared with cells not treated with rosmarinic acid, in MC3T3-E1 preosteoblasts treated with rosmarinic acid, respectively, the mRNA expression rate of BSP increased by 1.3 times on day 4, The mRNA expression rate of DSPP increased by 1.2-fold on the 7th day, and the mRNA expression rate of ALP increased by 1.1-fold during all periods of the experiment ( FIG. 2 ).

In addition, Col I was found in MC3T3-E1 preosteoblasts treated with rosmarinic acid and without rosmarinic acid, mRNA levels on the 7th day compared to the 4th and 10th days. It can be seen that the expression rate decreases. However, compared with the case where rosmarinic acid is not treated, it can always be confirmed that the expression rate is high when treated with rosmarinic acid (FIG. 2).

On the other hand, in the case of DMP-1, the mRNA expression rate in MC3T3-E1 preosteoblasts treated with rosmarinic acid was 1.1 on the 4th and 7th days compared to the cells not treated with rosmarinic acid. It can be seen that the increase is doubled. However, on the 10th day, the mRNA expression rate was decreased and maintained at a level similar to that of cells not treated with rosmarinic acid (FIG. 2).

In the case of ON, mRNA expression rates in MC3T3-E1 preosteoblasts treated with rosmarinic acid were 1.2-fold and 1.1 on days 4 and 10, respectively, compared to cells not treated with rosmarinic acid. It can be seen that the increase is doubled. However, on the 7th day, it can be confirmed that the mRNA expression rate is maintained at a level similar to that of cells not treated with rosmarinic acid ( FIG. 2 ).

According to the above results, rosmarinic acid regulates the expression of ALP, OCN and Col I genes to induce differentiation of MC3T3-E1 preosteoblasts into osteoblasts, and BSP, Corresponds to the results showing that MC3T3-E1 preosteoblasts effectively induce mineralization in the process of differentiation into osteoblasts by regulating the expression of DSPP, DMP-1 and ON genes. do.

Effect of rosmarinic acid on cell mineralization in the process of differentiation of MC3T3-E1 preosteoblasts

During the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts on the surface of Ti discs, rosmarinic acid was treated to MC3T3-E1 preosteoblasts. It can be seen that the mineralization effect and the activity of ALP are increased over time in both cases and cases where rosmarinic acid is not treated. However, it can be confirmed that the mineralization effect and the activity of ALP are higher when rosmarinic acid is treated as compared to the case where rosmarinic acid is not treated ( FIGS. 3 and 4 ).

Looking at the results of Alizarin Red S staining combined with calcium, rosmarinic acid was treated in MC3T3-E1 preosteoblasts compared to the case in which rosmarinic acid was not treated. In this case, it can be seen that the formation of calcified nodules increases 1.5-fold, 2.0-fold, and 1.3-fold on the 4th, 7th and 10th days, respectively (FIG. 3B).

On the other hand, looking at the results of alkaline phosphatase staining (ALP staining), compared to the case where rosmarinic acid was not treated, MC3T3-E1 preosteoblasts were treated with rosmarinic acid. It can be seen that the activity increases 1.2-fold and 1.3-fold, respectively, on the 4th and 7th days. However, it was found that the activity of ALP on the 10th day was similar to the case where rosmarinic acid was not treated (FIG. 4B).

In summary, when rosmarinic acid is treated with MC3T3-E1 preosteoblasts present on the surface of the implant body, ALP, OCN and By regulating the expression of the Col I gene, differentiation of MC3T3-E1 preosteoblasts into osteoblasts can be induced.

On the other hand, by regulating the expression of BSP, DSPP, DMP-1 and ON genes, it is possible to effectively induce mineralization in the process of differentiation of MC3T3-E1 preosteoblasts into osteoblasts. can

As a result, the effect of osseointegration between the jawbone and the implant surface can be improved by inducing bone formation of the lost or damaged bone portion through the above effect.

Synergistic effect by the combined treatment of rosemary extract (rosmarinic acid), currant mulberry leaf extract, gulberry leaf extract, stamen leaf extract and elm leaf extract

In order to test whether the effect of inducing osseointegration between the jawbone and the implant body surface is increased by the combined treatment of the extracts,

MC3T3 according to the treatment of a single treatment of rosemary extract, rosmarinic acid, and a mixed extract of rosmarinic acid, mulberry leaf extract, gulberry leaf extract, staphylococcus leaf extract and elm leaf extract in a certain weight ratio -Effect on cell viability of E1 preosteoblasts, increase in expression of non-collagenous genes and collagenous genes in the differentiation process of MC3T3-E1 preosteoblasts, The synergistic effects of differentiation and mineralization of MC3T3-E1 preosteoblasts were tested using the above experimental method and the like.

For relative comparison, the cell viability of MC3T3-E1 preosteoblasts in the case of single treatment (T1) with rosemary extract, rosmarinic acid, was set to index 5, and MC3T3-E1 precursor osteoblasts by the mixed extract The cell viability of (preosteoblasts) was evaluated as an index.

The index is rated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Cell viability of MC3T3-E1 preosteoblasts 5 4.2 5.1 4.9 5.5 5.5 3.9 4.5 5 3.8 4.8 6 8 9 8.8 4

According to Table 2, rosemary extract rosmarinic acid (rosmarinic acid), mulberry leaf extract, gulpi tree leaf extract, stamen leaf extract and elm leaf extract 1:0.3:0.3:0.5:0.5 to 1:1:1 When mixed in a weight ratio of 1:1:1, it was demonstrated that the cell viability of MC3T3-E1 preosteoblasts was superior in the differentiation process into osteoblasts.

On the other hand, when the mixing ratio of the rosemary extract, rosmarinic acid, mulberry leaf extract, gulberry leaf extract, stapes leaf extract, and elm leaf extract, is less than or greater than the above range value, MC3T3-E1 preosteoblasts ), it has been demonstrated that the cell viability is poor in the process of differentiation into osteoblasts.

Furthermore, the effect of increasing the expression of non-collagenous genes and collagen genes in the differentiation process of MC3T3-E1 preosteoblasts was confirmed through experiments, and rosemary extract was used for relative comparison. When rosmarinic acid was treated alone (T1), the expression increase effect of non-collagenous genes and collagen genes was set to index 5, and the expression increase effect by the mixed extract was indexed was evaluated as

The index is rated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Effect of increasing expression of non-collagen genes and collagen genes 5 4.5 3.5 5 5.2 4.7 4.7 3.9 6 4.9 5.1 4 8.9 9 8.8 4

According to Table 3, rosmarinic acid, which is a rosemary extract, a mulberry leaf extract, a gulpi leaf extract, a stamen leaf extract, and an elm leaf extract were 1:0.3:0.3:0.5:0.5 to 1:1:1. When mixed in a weight ratio of 1:1:1, the effect of increasing the expression of non-collagenous genes and collagen genes in the differentiation process of MC3T3-E1 preosteoblasts is better it can be seen that

On the other hand, when the mixing ratio of rosemary extract, rosmarinic acid, Cudrania mulberry leaf extract, gulberry leaf extract, stamen leaf extract, and elm leaf extract is less than or greater than the above range value, non-collagenous genes (non-collagenous genes) ) and it can be seen that the effect of increasing the expression of collagenous genes is low.

On the other hand, the synergistic effect of differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts was confirmed through experiments.

For relative comparison, the effect of differentiation and mineralization of MC3T3-E1 preosteoblasts in the case of single treatment with rosemary extract, rosmarinic acid (T1), was set to index 5, The synergistic effect of the mixed extract was evaluated as an index.

The index is rated on a scale from 1 to 10, and a higher value means better effect.

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 Synergistic effect of differentiation and mineralization of MC3T3-E1 progenitor cells 5 4.3 4.5 5 4.1 3.8 5.1 6.5 5.5 4.8 6 4.5 8.6 9.2 8.6 4.2

According to Table 4, rosmarinic acid (rosmarinic acid) as a rosemary extract, a mulberry leaf extract, a gulpi tree leaf extract, a stamen leaf extract and an elm leaf extract were 1:0.3:0.3:0.5:0.5 to 1:1:1. It can be confirmed that the synergistic effect of differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts is more excellent when mixed in a weight ratio of 1:1:1. there was.

On the other hand, when the mixing ratio of the rosemary extract, rosmarinic acid, mulberry leaf extract, gulberry leaf extract, stapes leaf extract, and elm leaf extract, is less than or greater than the above range value, MC3T3-E1 preosteoblasts ), it can be seen that the synergistic effect of differentiation and mineralization is poor.

Looking at the results of the above experiments, compared to the case where the rosemary extract, rosmarinic acid, was treated alone, the rosemary extract rosmarinic acid, Cudrania leaf extract, Gulpi tree leaf extract, Staphylococcus leaf extract and elm Cell survival in the differentiation process of MC3T3-E1 preosteoblasts when tree leaf extract is mixed in a weight ratio of 1:0.3:0.3:0.5:0.5 to 1:1:1:1:1 Effect, increase in the expression of non-collagenous genes and collagen genes in the process of differentiation of MC3T3-E1 preosteoblasts, MC3T3-E1 preosteoblasts It can be seen that the synergistic effect of differentiation and mineralization is further enhanced, and as a result, osseointegration between the jawbone and the surface of the implant body is improved.

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (7)

It is a coating composition comprising a rosemary extract, a mulberry leaf extract, a gulpi leaf extract, a stamen leaf extract and an elm leaf extract as active ingredients,
The coating composition is coated on the surface of the implant body to induce osseointegration between the jawbone and the implanted body surface.
A composition for coating an implant surface. According to claim 1,
The composition increases the differentiation and mineralization of MC3T3-E1 preosteoblasts into osteoblasts on the surface of the implant body.
A composition for coating an implant surface. 3. The method of claim 2,
The differentiation and mineralization are,
which is regulated by the expression of non-collagenous genes and collagenous genes
A composition for coating an implant surface. According to claim 1,
The composition increases the cell viability of MC3T3-E1 preosteoblasts.
A composition for coating an implant surface. According to claim 1,
The rosemary extract is rosmarinic acid
A composition for coating an implant surface. According to claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ether, chloroform, ethyl acetate, dimethyl sulfoxide, and mixtures thereof.
A composition for coating an implant surface. Coated with the composition according to any one of claims 1 to 6
implant.

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