WO2015009261A2

WO2015009261A2 - Product used in differentiation of human tooth stem cells

Info

Publication number: WO2015009261A2
Application number: PCT/TR2014/000274
Authority: WO
Inventors: Fikrettin Sahin; Selami DEMIRCI; Aysegul DOGAN; Pakize Neslihan TASLI
Original assignee: Yeditepe Universitesi
Priority date: 2013-07-16
Filing date: 2014-07-16
Publication date: 2015-01-22
Also published as: WO2015009261A3

Abstract

The present invention discloses a boron containing product used in differentiation of human tooth germ stem cells. Sodium pentaborate pentahydrate and other boron compounds are used in the stem cell differentiation of the present invention. The invention is a product which can be applied in soft and hard tissue and organ regeneration, repair, healing and cell production technologies.

Description

DESCRIPTION

PRODUCT USED IN DIFFERENTIATION OF HUMAN TOOTH STEM

CELLS Field of the Invention

The present invention discloses a boron containing product used in differentiation of human tooth germ stem cells. Background of the Invention

In the stem cell technology; which has become a source of hope for many diseases such as Parkinson's, Alzheimer's, diabetes and cancer; embryonic stem cells are used which are capable of transforming into all types of cells in the body. However this application could not be made clinically practicable because of the side effects, ethical problems and carcinogenicity (Strauer and Kornowski, 2003). In organ and tissue engineering, adult stem cells obtained from different parts of the body such as bone marrow, adipose tissue or umbilical cord blood can be used more effectively and widely as an alternative to embryonic stem cell (Phinney and Prockop, 2007).

It was observed that in addition to embryonic stem cells; mesenchymal stem cells can also transform into many types of cells mainly bone, cartilage, fat and muscle cells (Yamada et al., 2006). Although the stem cells isolated from bone marrow constitute the best primary MSC source that has been characterized to date, during the isolation process of these cells, there are requirements of painful surgeries as well as risks of contamination and transmission of bone marrow-related diseases. Besides the bone marrow, there are many MSC sources such as adipose tissue (Zuk et al., 2001), striated muscle (Young et al., 2001), amniotic fluid (De Coppi et al., 2007), placenta (Miao et al., 2006), dental pulp (Gronthos et al., 2000). In the state of the art applications, it was observed that human tooth germ Stem cell (hTGSC) (pulp and surrounding tissues) derived from third molar tooth of adults transformed into cartilage, bone, fat and nerve cell lines under in vitro conditions (Dogan et al., 2012; Yalvac et al., 2011). Furthermore, in order to meet the current requirement for stem cells without ethical problems, hTGSCs can protect the differentiation potentials without losing them (Kitagawa et al., 2007; Yalvac. et al., 2011). In in vivo tissue regeneration studies, in addition to use of multipotent stem cells, signal molecules controlling transformation are also required for a complete and effective regeneration.

Recently in tissue regeneration studies, use of systems formed with biodegradable polymers and mesenchymal stem cells whose differentiations are controlled under special culture conditions has become a point of interest (Meinel et al. 2004). Treating the stem cells with an inductive medium comprising special signals and incubating them in a suitable scaffold is one of the most preferred methods for programming and differentiating stem cells. In a well characterized scaffold system, factors inducing differentiation specific to the target tissue and the bioactive molecules affecting cell regeneration make the tissue and organ regeneration more effective (Rahaman et al. 2011).

According to the researches conducted with boron element on rats, it is determined that boron is a necessary material for providing optimum health conditions, normal embryonic development, bone growth, ability to produce immune response and proper operation of physicomotor system (Pan et al., 2010). Furthermore, it has been demonstrated by the studies conducted that boron taken into the body is extremely important for bone and teeth development (Hakki et al., 2012). Nielsen and Stoecker demonstrated with their study conducted on rats that boron deprived diets effectively reduce bone mass in the body (2009). Additionally, it was also reported that boron deficiency adversely affects bone development and in such case there may be abnormal bone development (Devirian and Volpe, 2003). Apart from all of these, it has been demonstrated that boric acid prevents periodontal inflammation and alveolar bone loss (Demirer et al., 2012). Hakki et al. showed that boric acid increases molecular mineralization in osteoblast cells and causes increase of the mRNA levels in the collagen type 1 and osteocalcine genes taking part in bone growth (2012). In spite of all these studies, there are no studies conducted in molecular level describing the effect of boron on dental and bone differentiations of stem cells. With the present study, the feature of sodium pentaborate pentahydrate increasing bone and dental differentiation on human tooth germ stem cells (hTGSC) is shown for the first time under in vitro conditions.

The International patent document no. WO2012129106 discloses that boron composite surface coatings are applied onto implantable devices for use in accelerating osseous healing.

Summary of the Invention

An objective of the present invention is to provide a boron containing product which increases bone and dental differentiation on human tooth germ stem cells (hTGSC).

Another objective of the present invention is to provide a boron containing product which can be applied in soft and hard tissue and organ regeneration. A further objective of the present invention is to provide a boron containing product which is effective in cell repair, healing and cell production.

Another objective of the present invention is to provide a boron containing product which increases the healing hard tissue and mineralization in diseases, damages and losses of hard tissues such as bones and teeth. A further objective of the present invention is to provide a boron containing product which effectively heals bone fractures in old people whose bone development is slow. Another objective of the present invention is to provide a boron containing product which can be used as an agent for increasing bone hardness and mass when treating diseases such as osteoporosis.

Detailed Description of the Invention

The present invention discloses a boron containing product used for differentiation of human tooth germ stem cells. Ulexite, colemanite, pandermite (Priceite), boric acid (H3B03), hydroboracite, probertite (NaCaB509.5H2O), borax pentahydrate, boron oxide, orthoboric acid, metaboric acid, borates, borophosphates, borosilicates, and preferably sodium pentaborate pentahydrate is used as the boron component.

"Product used in differentiation of bone stem cells" developed to fulfill the objectives of the present invention is illustrated in the accompanying figures wherein

Figure 1 is the view of mesenchymal stem cell characterization of hTGSCs (according to Flow Cytometry analysis, the cells include mesenchymal stem cell surface antigens and do not include hematopoietic cell surface antigens).

Figure 2 is the view of viability of hTGSCs in media with and without NaB ^g/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml, 100 μg/ml and 200 g/ml) * >0.05.

Figure 3: Determination of odontogenic differentiation: (a) ALP activity, (b) marking the cells with COLIA, Amelogenin and DMP 1 antibodies, (c) staining the cells with von Kossa. (d) determining the COLIA, DMP 1, AMBN and DSPP gene levels in the cells by quantitative real time PCR. NaB; group treated with NaB and odontogenic differentiation product. PK; group treated only with odontogenic differentiation product. NK; hTGSC. The sign # represents comparison with PK and the sign * represents comparison with NK (#,*p<0.05).

Figure 4: Determination of bone differentiation: (a) ALP activity, (b) marking the cells with COL1A and osteocalcine antibodies, (c) staining the cells with von Kossa. (d) determining the COL1A and osteonectin gene levels in the cells by quantitative real time PCR. NaB; group treated with NaB and bone differentiation product. PK; group treated only with bone differentiation product. NK; hTGSC. The sign # represents comparison with PK and the sign * represents comparison with NK (#,*p<0.05).

Experimental Study

Preparation of Boron Product

Obtaining the product that will be used for differentiation of bone stem cells comprises the steps of

- dissolving the boron component in a culture medium,

- preparing the main stock solution,

- filtering the stock product,

- preparing products at sub-concentrations,

- performing experimental studies with the sub-concentrations.

Accordingly, sodium pentaborate pentahydrate (NaB) solution was dissolved in the culture medium and the main stock product at a concentration of 0.1 mg/ml was prepared. After the stock product was filtered through a 0.2 μιη filter (Sartorius AG, Gottingen, Germany) and sterilized, 13 different concentrations were prepared as 5-10-20-50-75-100-150-200-250-300-400-500-700μg/ml (Dulbecco's modified Eagle's medium) (Invitrogen, Carlsbad, CA). Isolation and Characterization of hTGSC hTGSC was isolated from the third molar tooth of a 13 year old male subject obtained after the patient and his family signed the consent form received from Istanbul University (Istanbul, Turkey) Ethics Committee. Isolation and characterization of hTGSC was performed as previously stated in the literature (Tas et al, 2012). The stem cells were grown in Dulbecco's modified essential medium (DMEM) which contained 10% fetal bovine serum and 1% PSA (penicillin, streptomycin and amphotericin), in an incubator at a temperature of 37°C and in 5% C0₂ atmosphere. After the cells were trypsinized, they were treated for 1 hour with primer antibodies diluted in PBS (PBS; cat #10010, pH 7,4; Invitrogen). CD29 (cat #BD556049), CD34 (cat #SC-51540), CD45 (cat #SC-70686), CD90 (cat #SC-53456), CD105 (cat #SC-71043), CD133 (cat #SC- 65278), CD166 (cat #SC-53551) (Santa Cruz Biotechnology Inc, Santa Cruz, CA), and CD73 (cat #D 550256) (Zymed, San Francisco, CA) primer antibodies were used for characterization. The cells were washed with PBS three times for removing the excess unbonded primer antibodies in the medium. Then the cells were treated at 4°C for 1 hour with secondary antibodies conjugated with fluorescein isothiocyanate (cat. #SC-2989, 200μg/0.5mL; Santa Cruz Biotechnology). Only CD29 was not treated with a secondary antibody because it was in the form of a monoclonal antibody conjugated with phycoerythrin comprising chromophore. Flow cytometry analysis was performed by using Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, CA) system.

Cytotoxicity Experiment

13 different concentrations of sodium pentaborate pentahydrate (5-10-20-50-75- 100-150-200-250-300-400-500-700μg/ml) were prepared in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and 1% PSA (Biological Industries, Beit Haemek, Israel). 24 hours after being seeded in 96-well culture plates (Corning Glasswork, Corning, NY) at 5000 cells/well, hTGSCs were treated for 3 days with NaB products prepared at different concentrations. Cell viability was measured by using 3-(4,5-di-methyl-thiazol-2- yl)-5 -(3 -carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)- method (CellTiter96 AqueousOne Solution; Promega, Southampton, UK). ΙΟμΙ MTS product was added onto the cells within a ΙΟΟμΙ growth medium and they were incubated in dark for 2 hours. After the incubation process, cell viability was observed by performing absorbance measurement via ELISA plate reader (Biotek, Winooski, VT) device at 490 nm wavelength.

Differentiation of Mesenchymal Stem Cells

In order to observe the effect of sodium pentaborate pentahydrate on dental and bone differentiation, hTGSCs were treated with dental and bone differentiation media as previously described in the literature (Task et al, 2012). For dental and bone differentiation, hTGSCs were seeded into 24-well culture plates (Corning Glasswork) at 15,000 cells/well. 24 hours after seeding, dental and bone differentiation medium containing 20μg/ml NaB was prepared and given to the cells every other day for a period of 14 days and incubated at a temperature of 37°C and in 5% C0₂ atmosphere. DMEM containing 10% FBS (Invitrogen, Carlsbad, CA, USA), 0.1 mmol/L dexamethasone, 10 mmol/L β-glycerol- phosphate, 50 mmol/L ascorbate (Sigma Chemical Co., StLouis, MO, USA) was used as the bone differentiation medium; and DMEM containing 10% FBS (Invitrogen, Carlsbad, CA, USA), 10 nmol/L dexamethasone, 10 mmol/L β- glycerol-phosphate, 50 mmol/L ascorbate (Sigma Chemical Co., StLouis, MO, USA) was used as the dental differentiation medium.

Immunostaining Immunostaining was performed as previously stated in the literature (Dogan et al, 2012). Generally, the fixed cells were treated at 4°C overnight with primer antibodies (Santa Cruz Biotechnology) specific for bones and teeth. Anti- collagen-type- 1 (Santa Cruz, #59772) and anti-osteocalcine (Santa Cruz, #30044) primer antibodies were used for the cells used in bone differentiation; and collagen-type- 1 (Santa Cruz, #59772), dentine matrix protein 1 (DMP1) (Santa Cruz, #sc-73633) and Amelogenin (Santa Cruz, #sc-32892) primer antibodies were used for the cells used in dental differentiation. Then, the cells were washed with PBS and treated with secondary antibodies (AleaFluor-488 goat anti-rabbit or goat anti-mouse immunoglobulin G) (Invitrogen) at 4°C for 1 hour. Nuclei of the cells were stained with 4',6-di-amidino-2-phenyl-indole (DAPI: Applichem, Darmstadt, Germany) which was diluted to a ratio of 1 :1000. The stained cells were observed under a fluorescence microscope (Nikon Eclipse TE200: Nikon, Tokyo, Japan).

Real Time Polymerase Chain Reaction

After the process of differentiation, total RNA of the cells were isolated by using High Pure RNA-isolation kit (Roche, Indianapolis, IN). cDNA synthesis was performed from the obtained RNA by using High Fidelity cDNA-synthesis kit (Roche). The real time polymerase chain reaction (RT-PCR) was used for detecting the mRNA levels in the cells with SYBR Green staining method. The obtained cDNAs were mixed with primer and SYBR-Premix FERMENT AS to a final volume of 20μΙ,. The primers used for bone and dental differentiation are shown in Table 1. β-actin gene was used for normalization of the results. All of the RT-PCR experiments were performed by using iCycler RT-PCR system (Bio- Rad, Hercules, CA).

Table 1 - The primers used for bone and dental differentiation

PRIMERS SEQUENCES REFERENCES

AMBN 5'-GCGTTTCCAAGAGCCCTGATAAC-3' [24]

5'-AAGAAGCAGTGTCACATTTCCTGG-3' ON 5 '- ATGAGGGCCTGGATCTTCTT-3 ' [29] 5'-CTGCTTCTCAGTC AGAAGGT-3 '

DSPP 5'-CAGTAAGGATGAGTTAAATGCC-3'

5'-TACTTCTGCCCACTTAGAGCC-3'

DMP 1 5^*-CCCAGAGGCACAGGCAAATA-3' [23]

5'-TCCTCCCCAATGTCCTTCTT-3'

COL1A 5'-CCACGCATGAGCGGACGCTAA-3'

5 '- ATTGGTGGGATGTCTTCGTCTTGG-3 '

GAPDH 5'-TGTATCGTGGAAGGACTCA-3' [21]

5'-GCAGGGATGATGTTCTGGA-3'

Abbreviations: AMBN; Ameloblastic ON; Osteonectin, DSPP;

Destinsialophosphoprotein, DMP1; Dentine matrix protein 1, COL1A; collagen type 1, GAPDH; Glycer-aldehyde-3-phosphate-de-hydrogenase.

ALP Activity Assay

Alkaline phosphatase activity (ALP) of the cells was measured immediately after the differentiation process. The cells were seeded into 12-well culture plates at 25,000 cells/well. At the end of a 14-day process of bone and dental differentiation, the medium in the wells were withdrawn and 2% Triton-X- 100 (BioBasicInc, cat#9002-93-l) was added to the medium for decomposition of the cells. The lysate formed after the cells were collected was added to 96-well culture plates and ALP product (BioAssay Systems, USA, cat #DALP-250) was added thereon. 15 minutes later, measurement was performed at 405 nm wavelength in the ELISA (Biotek, Winooski, VT) plate reader.

Von Kossa Staining Von Kossa staining which is considered to be the indicator of dental and bone differentiation was performed to show calcium deposits. The fixed cells were stained in accordance with the procedure of von Kossa kit (BioOptica, Milano, Italy). The stained calcium deposits were observed under a light microscope (Yalvac et al., 2009). Experimental Results hTGSC Characterization

After the 3^rd passage, hTGSCs which are the primer cell culture showed a single layer, big and flat fibroplast-like morphology. hTGSCs characterized in the flow cytometry device according to the surface proteins were detected to be positive in terms of the cell membrane surface proteins CD29, CD 105, CD90, CD73 and CD 166 (Figure- 1) specific to the adult stems cells, and negative in terms of the surface proteins CD14, CD34, CD44, CD45 and CD133 specific to the blood cells. In the light of these results, it can be claimed that the isolated cells are mesenchymal stem cells.

NaB Toxicity Cell viability analyses of the cells subjected to different NaB concentrations were performed by MTS assay at hour 24, 48 and 72. The results showed that viability of the cells subjected to lC^g/ml and 2C^g/ml concentrations of NaB were higher. However toxic effects were observed for the cells subjected to high NaB concentrations (Figure 2).

Odontogenic Differentiation

The cells which were treated for 14 days with dental differentiation media containing and non-containing NaB were analyzed for determining differentiation. Alkaline phosphatase, which was the first parameter that was measured in this sense, is a cell membrane-associated enzyme and the primary indicator of mineralized tissues; and it is the most widely used marker particularly for dental and bone differentiation (Zou L. Et al. 2008; Serigano K. et al. 2010; Stucki U. et al. 2001). In addition, ALP activity commences intercellular mineralization in dental cells and bones (Gerstenfeld LC. et al. 1987). According to the ALP result, ALP activity of the group treated with 2(^g/ml NaB was found to be significantly greater than the ALP activity of the group (PK) treated only with the differentiation medium. Additionally, fluorescence photographs of the groups expressing dentine matrix protein, collagen type 1 and Amelogenin proteins, which are indicator of dental differentiation, were taken. High amount of marker protein synthesis was observed in the group treated with NaB. Based on these data, it was asserted that hTGSCs formed tooth-like cells. In the third stage, it was observed that mineralization increase in the cells was induced by NaB exposure. In the group treated with NaB, von Kossa staining, which is a mineralization indicator, was observed more intensely. Finally, mRNA levels of COL1A, DSPP, DMP1 and AMBN genes associated with dentine were determined. Significantly higher mRNA levels were observed in the group treated with NaB, in comparison to PK (Figure 3).

Osteogenic Differentiation

The cells which were treated for 14 days with bone differentiation media containing and non-containing NaB were analyzed for determining differentiation. According to the results of ALP activity, it was observed that the group treated with NaB had a significantly higher ALP activity when compared with PK (Figure 4a). The levels of osteocalcine and collagen type 1 proteins, which are important markers of osteogenesis, were determined to be higher in the group treated with NaB by taking the immunofluorescence photographs thereof. Then von Kossa staining was performed to show the intracellular mineralization and a more intense staining was detected in the group treated with NaB compared to the other groups. Finally, mRNA levels of collagen type 1 and osteonectin, which are the genes associated with osteogenesis, were determined quantitatively by real time PCR (Figure 4b).

Based on the results obtained, sodium pentaborate pentahydrate plays an important role in bone and dental differentiation in human tooth germ stem cells (hTGSC).

Applicability of the Invention Sodium pentaborate pentahydrate and the other boron compounds used in stem cell differentiation can be applied in hard and soft tissue and organ regeneration, repair, healing and cell production technologies.

The product of the present invention can be effectively applied in differentiation of osteogenic cell, odontogenic cell, dentine cell, cementoblast cell, osteoblast cell, periodontal ligament cell.

The product of the present invention can be produced and used in non-toxic biocompatible materials such as natural polymers, metal alloys, textile, synthetic, coating and mineral construction materials, and the product can be produced and used as a cosmetics product or a drug active ingredient.

Furthermore, the said product can also be provided in the form of a toothpaste, mouthwash solution, chewing gum, soluble tablet, soluble film, gel, biodegradable natural polymer, biodegradable synthetic polymer. References

Strauer BE and R Kornowski. (2003). Stem cell therapy in perspective. J Am Heart Assoc 107:929-934.

Phinney DG and DJ Prockop. (2007). Concise Review: Mesenchymal Stem/Multi-Potent Stromal Cells (MSCs): The state of transdifferentiation and modes of tissue repair- current views. Stem Cells 25:2896-2902.

Yamada Y, A Fujimoto, A Ito, R Yoshimi and M Ueda. (2006). Cluster analysis and gene expression profiles: a cDNA microarray system-based comparison between human dental pulp stem cells (hDPSCs) and human mesenchymal stem cells (hMSCs) for tissue engineering cell therapy. Biomaterials 27:3766-3781. Zuk PA, M Zhu, H Mizuno, J Huang, JW Futrell, AJ Katz, P Benhaim, HP Lorenz and MH Hedrick. (2001). Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7:211-228.

Young HE, TA Steele, RA Bray, J Hudson, JA Floyd, K Hawkins, K Thomas, T Austin, C Edwards, J Cuzzourt, M Duenzl, PA Lucas and AC Black. (2001). Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec (Hoboken) 264:51-62.

De Coppi P, G Bartsch Jr., MM Siddiqui, T Xu, CC Santos, L Perin, G Mostoslavsky, AC Serre, EY Snyder, JJ Yoo, ME Furth, S Soker and A Atala (2007). Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25: 100-106.

Miao Z, J Jin, L Chen, J Zhu, W Huang, J Zhao, H Qian and X Zhang. (2006). Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int 30:681-687.

Gronthos S, M Mankani, J Brahim, PG Robey and S Shi. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97:13625-13630. Dogan A, ME Yalvac, F Sahin, AV Kabanov, A Palotas and AA Rizvanov. (2012). Differentiation of human stem cells is promoted by amphiphilic pluronic block copolymers. Int J Nanomedicine 7:4849-4860.

Yalvac ME, A Yilmaz, D Mercan, S Aydin, A Dogan, A Arslan, Z Demir, II Salafutdinov, AK Shafigullina, F Sahin, AA Rizvanov and A Palota's. (201 1). Differentiation and Neuro- Protective Properties of Immortalized Human Tooth Germ Stem Cells. Neurochem Res 36:2227-2235.

Kitagawa M, H Ueda, S Iizuka, K Sakamoto, H Oka, Y Kudo, I Ogawa, M Miyauchi, H Tahara and T Takata. (2007). Immortalization and characterization of human dental pulp cells with odontoblastic differentiation. Arch Oral Biol 52:727-731.

Meinel L, V Karageorgiou, R Fajardo, B Snyder, V Shinde-Patil, L Zichner, D Kaplan, R Langer and G Vunjak-Novakovic. (2004). Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng 32: 112-122.

Rahaman MN, DE Day, BS Bal, Q Fu, SB Jung, LF Bonewald and AP Tomsia. (2011). Bioactive glass in tissue engineering. Acta biomater 7: 2355-2373.

Pan HB, XL Zhao, X Zhang, KB Zhang, LC Li, ZY Li, WM Lam, WW Lu, DP Wang, WH Huang, KL Lin and J Chang. (2010). Strontium borate glass: potential biomaterial for bone regeneration. J R Soc Interface 7: 1025-1031.

Hakki SS, N Dundar, SA Kayis, EE Hakki, M Hamurcu, U Kerimoglu, N Baspinar, A Basoglu and FH Nielsen. (2012). Boron enhances strength and alters mineral composition of bone in rabbits fed a high energy diet. J Trace Elem Med Biol (In-Press).

Nielsen FH. (2008). Dietary fat composition modifies the effect of boron on bone characteristics and plasma lipids in rats. Biofactors 3:161-171.

Devirian TA and SL Volpe. (2003). The physiological effects of dietary boron. Crit Rev Food Sci Nutr 43:219-231.

Demirer S, MI Kara, K Erciyas, H Ozdemir, H Ozer and S Ay. (2012). Effects of boric acid on experimental periodontitis and alveolar bone loss in rats. Arch Oral Biol 57:60-65. Tash PN, S Tapsin, S Demirel, ME Yalvac, S Akyuz, A Yarat and F Sahin. (2012). Isolation and Characterization of Dental Pulp Stem Cells from a Patient with Papillon-Lefevre Syndrome. J Endod 39:31-38.

Yalvac ME, M Ramazanoglu, AA Rizvanov, F Sahin, OF Bayrak, U Salli, A Palotas and GT Kose. (2009). Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neovascularization, osteo-, adipo-and neurogenesis. The Pharmacogenomics J 10:105-113.

Zou L, X Zou, L Chen, H Li, T Mygind, M Kassem and C Bunger. (2008). Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern. J Orthop Res 26:56-64.

Serigano K, D Sakai, A Hiyama, F Tamura, M Tanaka and J Mochida. (2010). Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model. J Orthop Res 28: 1267-1275.

; Stucki U, J Schmid, CF Hammerle and NP Lang. (2001). Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans. Clin Oral Implants Res 12: 121-127.

Gerstenfeld LC, SD Chipman, J Glowacki and JB Lian. (1987). Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol 122:49-60.

Claims

Method of obtaining a product that will be used for differentiation of bone stem cells comprising the steps of

- dissolving the boron component in a culture medium,

- preparing the main stock solution,

- filtering the stock product,

- preparing products at sub-concentrations,

- performing experimental studies with the sub-concentrations.

2. Boron product obtained with the method according to Claim 1.

Boron product according to Claim 2 wherein the sub-concentrations are 5- 10-20-50-75-100-150-200-250-300-400-500-70(^g/ml.

Product according to Claim 2 and 3, wherein the boron compound is ulexite, colemanite, pandermite (Priceite), boric acid (H3B03), hydroboracite, probertite (NaCaB509.5H2O), borax pentahydrate, boron oxide, orthoboric acid, metaboric acid, borates, borophosphates, borosilicates, and preferably sodium pentaborate pentahydrate.

Product according to Claim 4 comprising boron derivatives at a therapeutically effective amount.

Boron product according to Claims 2 to 5 which can be applied in soft and hard tissue and organ regeneration, repair, healing and cell production technologies.

The product according to Claims 2 to 6 which can be applied in differentiation of osteogenic cell, odontogenic cell, dentine cell, cementoblast cell, osteoblast cell, periodontal ligament cell.

13

8. The product according to Claims 2 to 7 which can be produced as natural polymers, metal alloys, textile, synthetic, coating and mineral construction materials or as a cosmetics product or a drug active ingredient.

9. The product according to Claims 2 to 8 which can be provided in the form of a toothpaste, mouthwash solution, chewing gum, soluble tablet, soluble film, gel, biodegradable natural polymer, biodegradable synthetic polymer.

14