JP2008295420A - Human periodontal ligament cell line, osteoblast dfferentiated from the cell line and artificial bone produced from the osteoblast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a human periodontal ligament cell line, to provide an osteoblast dfferentiated from the cell line, and to provide an artificial bone produced from the osteoblast. <P>SOLUTION: Provided are the human periodontal ligament cell line originated from the stem cell of a human periodontal ligament tissue and has a differentiation ability to osteoblast, and the osteoblast differentiated from the cell strain. The human periodontal ligament cell line increases the density of the cells in response to the number of culture days, and remarkably increases the activity of ALP in response to the increase. The human periodontal ligament cell produces type I collagen, osteopontin, and osteocalcin in large amounts, and exhibits active responsibility to cell proliferation factors such as transforming growth factor-β1, basic fibroblast growth factor, insulin-like growth factor-1, and epithelial cell proliferation factor. This artificial bone is produced from such the osteoblast. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a human periodontal ligament cell line, an osteoblast differentiated from this cell line, and an artificial bone produced from the osteoblast.

  Periodontal disease is said to affect about 80% of adults in Japan, and as the disease progresses, the alveolar bone is lost, resulting in tooth loss. Current treatment of periodontal disease is aimed at removing plaque, tartar, and defective granulation that cause inflammation and restoring new periodontal tissue. However, treatments aimed at removing the cause of inflammation can prevent the progression of periodontal disease, but cannot recover the tissue once lost.

  In recent years, production of cell lines by immortalization of mouse and human periodontal ligament cells has been attempted by cell engineering techniques such as gene transfer such as SV40 large T antigen, and several successful cases have been reported. (Patent Document 1).

  In general, it is understood that a cell line produced by such a cell engineering technique continues to maintain the characteristics (such as the degree of differentiation) of cells into which the gene has been introduced.

  However, when the cell into which the gene is to be introduced is in a state of being differentiated to some extent, it has been difficult to dedifferentiate again into a stem cell like state by manipulating the culture conditions after the introduction.

Here, with regard to periodontal ligament cells derived from mice and rats, successful cases of immortalized cell lines were sometimes published in academic journals, but human tissue-derived cells were recently published in Hiroshima University. There was no group announcement (Non-Patent Document 1). However, since the cells created by these gene introductions all maintain the level of differentiation of the introduced cells, there was a drawback that the characteristics of undifferentiated stem cells could not be reproduced by subsequent culture operations. .
JP 2002-262862 A T. Kaneda et.al .Characteristics of periodontal ligament subpopulations obtained by sequential cyclic digestion of rat molar periodontal ligament.Bone 38 (2006) 420-426

  Therefore, the present inventors have developed a novel human periodontal ligament cell line that maintains characteristics similar to those of primary cultured periodontal ligament cells containing undifferentiated stem cells, osteoblasts differentiated from this cell line, and artificial materials produced from this osteoblast. The purpose is to provide bone.

  Thus, as a result of intensive studies in view of the above problems, the present inventors have devised the cell freezing method without depending on gene transfer (a slightly higher DMSO concentration), and repeated freeze-thawing. The human periodontal ligament cell line close to the normal periodontal ligament having the ability to differentiate from an undifferentiated state until it is capable of calcification was established by subcultured surviving cells, and the present invention was completed.

  The human periodontal ligament cell line according to claim 1 of the present invention is characterized by having the ability to differentiate into osteoblasts.

  The human periodontal ligament cell line according to claim 2 of the present invention is characterized in that, in claim 1, the human periodontal ligament cell line is derived from stem cells of human periodontal ligament tissue.

  In the human periodontal ligament cell line according to claim 3 of the present invention, the receipt number is FERM AP-21302 in claim 1 or 2.

  In the human periodontal ligament cell line according to claim 4 of the present invention, in any one of claims 1 to 3, the cell density increases according to the number of days of culture, but the alkaline phosphatase activity significantly increases accordingly. It is characterized by that.

  The human periodontal ligament cell line according to claim 5 of the present invention is characterized by producing a large amount of type I collagen and / or osteopontin and / or osteocalcin in any one of claims 1 to 4.

  The human periodontal ligament cell line according to claim 6 of the present invention is characterized in that in any one of claims 1 to 5, the cell has a vigorous response to a cell growth factor.

  The human periodontal ligament cell line according to claim 7 of the present invention is the human periodontal ligament cell line according to claim 6, wherein the cell growth factor is transforming growth factor-β1 and / or basic fibroblast growth factor and / or insulin-like growth factor- I and / or epidermal growth factor.

  The human periodontal ligament cell line according to claim 8 of the present invention is characterized in that in any one of claims 1 to 7, cyclooxygenase-2 is expressed.

  The osteoblast according to claim 9 in the present invention is characterized by being differentiated from the human periodontal ligament cell line according to any one of claims 1 to 8.

  The artificial bone according to claim 10 of the present invention is produced from the osteoblast according to claim 9.

  The method for using osteoblasts according to claim 11 of the present invention is characterized in that the osteoblasts according to claim 9 are embedded in a base material for calcification.

  The method for using osteoblasts according to claim 12 of the present invention is characterized in that the osteoblasts according to claim 9 are transplanted into calcified bone tissue and calcified.

  The method of using the osteoblast according to claim 13 in the present invention is characterized by using the calcified osteoblast according to claim 9.

  Effectiveness and safety screening conducted in fields related to the development of periodontal tissue regeneration therapy, pharmaceutical companies related to the development of drugs for the treatment of periodontal disease, manufacturers related to the development of biomaterials used for dental implants, etc. Can be general purpose.

  According to the human periodontal ligament cell line of the present invention and the osteoblast differentiated from this cell line, in the development of a therapeutic agent for periodontal disease, human periodontal ligament is used for safety and efficacy tests each time. Need not be collected and cultured. Similarly, the number of animals sacrificed by animal experiments can be reduced.

  Similarly, in the development of biomaterials / base materials such as dental implants, screening costs and time can be greatly reduced.

  And unlike periodontal ligament cell lines by gene transfer so far, it covers a wide range of differentiation, so it is useful for the study of calcification control of periodontal ligament cells and tissues. It is also expected to lead to the development of therapeutic agents for periodontal tissue regeneration.

  Furthermore, in facilities that have a set of normal culture equipment, this technology facilitates the establishment of long-life span human periodontal ligament cells and human osteoblast cell lines. Can greatly contribute to development.

  Hereinafter, the present invention will be described in detail.

  The human periodontal ligament cell line in the present invention has the ability to differentiate into osteoblasts.

  This human periodontal ligament cell line is derived from stem cells of human periodontal ligament tissue.

  In addition, this human periodontal ligament cell line PDL-tk1 cell found by the present inventor was obtained from the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, June 6) Deposited as receipt number FERM AP-21302 on the 1st of the month.

  In the human periodontal ligament cell line of the present invention, the cell density increases according to the number of culture days, but the alkaline phosphatase (ALP) activity is significantly increased accordingly.

  Here, alkaline phosphatase (ALP) is widely accepted as a histochemical / biochemical marker for osteoblasts, and the increase in activity thereof has been promoted as the degree of differentiation as osteoblasts or osteoblast-like osteoblasts. Indicates that However, its function is not clearly understood. Probably because congenital hypophosphatase presents with osteomalacia, it is thought to be involved in local calcium phosphate formation and crystallization, and as a result, in tissue calcification.

  For the above reasons, selection of cells having a relatively high ALP activity is meaningful for differentiation from fibroblasts having a very low activity. In fibroblasts, ALP activity does not increase even if the cell density increases as the number of days of culture increases.

  The human periodontal ligament cell line in the present invention produces a large amount of type I collagen (collagen) and / or osteopontin and / or osteocalcin.

  Here, 90% of the bone matrix protein consists of type I collagen. According to the established theory, calcium phosphate is deposited around type I collagen fibers produced by osteoblasts to form hydroxyapatite and form new bone. Therefore, type I collagen is thought to play an important role in the process of calcification, and its large production is interpreted as setting the basic conditions for calcification.

  Similarly, osteopontin is one of the proteins produced by osteoblasts, but is known to deposit in the extracellular matrix prior to calcification. Therefore, it is counted as one of the markers of osteoblasts, but there are many unclear points about the details of its role.

Osteocalcin is said to be a marker indicating the degree of osteoblast differentiation, but is a protein expressed in mature osteoblasts. It is thought to play an important physiological role in bone metabolism, such as regulating local calcification and controlling movement of Ca ²⁺ between bone and body fluids.

  The human periodontal ligament cell line in the present invention exhibits vigorous responsiveness to cell growth factors.

  Here, the growth factor (growth factor) is also called a growth factor or a growth factor, and is a general term for endogenous proteins that induce proliferation and differentiation of specific cells in an animal body. It works in the regulation of various cytological and physiological processes, and acts as a signal substance between cells by specifically binding to receptor proteins on the surface of target cells.

  The cell growth factors include transforming growth factor-β1 (transforming growth factor-β1: TGF-β1), basic fibroblast growth factor (bFGF), insulin-like growth factor-I ( One of insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF).

  Here, transforming growth factor-β1 (TGF-β1) plays many important roles such as regulation of cell proliferation and differentiation, promotion of bone formation, promotion of production of collagen and fibronectin, and hematopoietic process. It is a dimeric protein with a molecular weight of about 25 kDa. TGF-β binds to a receptor on the cell membrane and transmits a signal to the cell nucleus via an intracellular signal transduction molecule.

  Basic fibroblast growth factor (bFGF) is a potent angiogenic factor (peptide) that is widely distributed in the human body belonging to the fibroblast growth factor (FGF) family, and is used for angiogenesis (angiogenesis) and arteries. Features that promote formation (arteriogenesis: arterial vasodilation, remodeling). It is also involved in nerve and bone formation.

  Here, fibroblast growth factor (FGF) consists of a total of 20 or more families, such as basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF). FGF is produced not only by epithelial cells but also by macrophages, and fibers at the time of wounding are involved in cell proliferation and angiogenesis.

  IGF (insulin-like growth factor) is a polypeptide having a sequence highly similar to that of insulin, and there are IGF-I and IGF-II having different structures. In cell culture, it induces mitogenic reactions like insulin. IGF-II is thought to be the first growth factor required for early development, whereas IGF-I expression is seen at a later stage.

  Insulin-like growth factor-I (IGF-I) is secreted mainly as a result of stimulation by growth hormones in the liver. Most cells of the human body, especially muscle, bone, liver, kidney, nerve, skin and lung cells are affected by IGF-I. In addition to insulin-like effects, IGF-I regulates cell growth (especially nerve cells) and development, and DNA synthesis.

  Epidermal growth factor (EGF) is a 6.045 kDa protein consisting of 53 amino acid residues and three intramolecular disulfide bonds. It binds to epidermal growth factor receptor (EGFR) present on the cell surface as a ligand and plays an important role in regulating cell growth and proliferation. EGF binds to a specific receptor on the cell surface with high affinity, thereby stimulating the protein tyrosine kinase activity of the receptor. The tyrosine kinase activity of the receptor initiates a signaling cascade that ultimately leads to DNA synthesis and cell proliferation.

  The periodontal ligament cell line of the present invention expresses cyclooxygenase-2.

  Here, cyclooxygenase (COX) is known as a rate-limiting enzyme for biosynthesis of prostaglandin (PG) from arachidonic acid. In 1991, the existence of two types of isozymes was revealed at the mRNA level, and the conventionally known enzyme is called Cox-1, and the newly discovered subtype is called Cox-2.

  Cox-1 is always expressed in most cells as an essential enzyme, and is considered to play a role of “housekeeping” for maintaining the stability of the living body.

  On the other hand, Cox-2 is a prostaglandin related to local inflammation that is expressed as an inducing enzyme in cells involved in inflammation such as monocytes, fibroblasts, and synovial cells, and is induced by inflammatory cytokines. It is a production enzyme.

  As described above, the two types of isozymes have a large difference in the regulatory function and appearance mode as an enzyme. The structural differences between Cox-1 and Cox-2 are the channel width and the amino acids constituting the active site. In the active site of Cox-1, there is an isoleucine residue at position 523 of the amino acid, whereas there is a valine residue at the same position in the active site of Cox-2.

  The osteoblast of the present invention is differentiated from such a human periodontal ligament cell line.

  And the artificial bone of this invention is produced from such an osteoblast.

  And the usage method of the osteoblast in this invention is the method of embedding such an osteoblast in a base material and making it mineralize.

  And it is the method of transplanting to such a bone tissue which lacked such osteoblast, and making it mineralize.

  Moreover, it is the method of using what calcified such an osteoblast.

  EXAMPLES The present invention will be described in detail below with reference to examples of the present invention, but the present invention is not limited to these examples.

(Experimental materials and methods and their significance)
1. Separation of periodontal ligament cells from extracted teeth The purpose and significance of the research for the present invention were explained to patients with potential adaptation, and extraction was performed after obtaining consent. The tooth obtained by convenient removal was washed with a physiological saline containing an antiseptic solution and an antibacterial agent, and then the soft tissue attached thereto was peeled off with sterilized scissors or razors. Further, the cells were cut into pieces and subjected to an enzyme treatment such as collagenase as necessary to obtain cell clusters or isolated cells. These were transferred to a normal culture dish and cultured in a culture solution containing fetal bovine serum (FBS).

  Here, the culture dish is a general plastic product (manufactured by Coster) used for culturing commercially available adherent cells, and one having a diameter of 60 mm was used.

2. Cryopreservation of periodontal ligament cells Logarithmically growing cells were treated with trypsin / EDTA solution (Dulbecco's phosphate buffer containing 0.05% trypsin and 0.53 mM EDTA) in the same manner as in normal subculture. The cells were exfoliated and suspended in 80% FBS + 20% DMSO (dimethyl sulfoxide) at a cell density of 0.8-1.2 × 10 ⁶ cells / mL. This was placed in a freezing tube, and stored at low temperature in steps of 30 minutes at ice temperature, 1 hour at −20 ° C. and 4 hours at −80 ° C., and finally immersed in liquid nitrogen.

Here, the concentration of DMSO used is usually such that cells in logarithmic growth phase are suspended in a growth medium containing 10% DMSO at a density of 0.1-1 × 10 ⁷ cells (cells) / mL and subjected to freezing. The present invention is suspended in FBS containing 15-20% DMSO and subjected to freezing with the cell density slightly reduced. DMSO is expected to have an effect of protecting cells from the formation of ice crystals, but on the other hand, it is also pointed out to have cytotoxicity and ability to induce cell differentiation against blood cells. In the present invention, increasing the DMSO concentration is likely to induce cell mutation, and as a result, it seems to lead to an extension of the life span. Therefore, it seems that the quality of FBS and DMSO has a subtle effect on the success or failure.

3. Method of thawing periodontal ligament cells Frozen cell tubes removed from liquid nitrogen were thawed in a 40 ° C. bath in a short time. The cell suspension was diluted 10-fold with DMEM medium, stirred gently, and then centrifuged (800 rpm, 5 minutes). The precipitated cells were cultured according to a usual method. The medium was replaced for the purpose of removing residual DMSO after 4-8 hours.

4). ALP Activity Measurement Method Cells cultured on a culture plate or dish were fixed with a 10% neutral formalin solution for 10 minutes. This was repeated twice and then rinsed 4 times with Dulbecco's phosphate buffer (PBS). Then, after sufficiently removing water, ALP activity was quantified colorimetrically using a kit called Laboassay ALP (registered trademark) (Wako). As a result, the ALP activity of the obtained PDL-tk1 cells increased according to the number of culture days (FIG. 1).

  Here, ALP is widely accepted as a histochemical / biochemical marker of osteoblasts, and the increase in activity indicates that the degree of differentiation as osteoblasts or osteoblast-like osteoblasts is advanced. However, its function is not clearly understood. Probably because congenital hypophosphatase presents with osteomalacia, it is thought to be involved in local calcium phosphate formation and crystallization, and as a result, in tissue calcification.

  For the above reasons, selection of cells having a relatively high ALP activity is meaningful for differentiation from fibroblasts having a very low activity. In fibroblasts, ALP activity does not increase even if the cell density increases as the number of days of culture increases.

5. Physiological significance and merits of type I collagen, osteopontin and osteocalcin Procollagen type I carboxylterminal peptide (PIP) (type I collagen (collagen type I) The amount of precursor) was increased according to the number of culture days (FIG. 2).

  The obtained PDL-tk1 cells produced osteopontin (FIG. 3).

  In addition, osteocalcin, which is a typical marker protein of mature osteoblasts, was produced from an early stage of culture (FIG. 4).

  Thus, the obtained PDL-tk1 cells produced a large amount of type I collagen, osteopontin and osteocalcin.

  Here, 90% of the bone matrix protein consists of type I collagen. According to the established theory, calcium phosphate is deposited around type I collagen fibers produced by osteoblasts to form hydroxyapatite and form new bone. Therefore, type I collagen is thought to play an important role in the process of calcification, and its large production is interpreted as setting the basic conditions for calcification.

  Similarly, osteopontin is one of the proteins produced by osteoblasts, but is known to deposit in the extracellular matrix prior to calcification. Therefore, it is counted as one of the markers of osteoblasts, but there are many unclear points about the details of its role.

Osteocalcin is said to be a marker indicating the degree of osteoblast differentiation, but is a protein expressed in mature osteoblasts. It is thought to play an important physiological role in bone metabolism, such as regulating local calcification and controlling movement of Ca ²⁺ between bone and body fluids.

6). Expression of cyclooxygenase-2 The obtained PDL-tk1 cells expressed cyclooxygenase-2 (Cox-2), which is a prostaglandin-producing enzyme related to local inflammation, at the protein level. Expressed (FIG. 5).

7). Examples of use of PDL-tk1 cells 1) Study on proliferation / differentiation ability of periodontal ligament cells The reactivity to various periodontal disease therapeutic agents was evaluated by the proliferation ability of cells and the ability to form hard tissues. The proliferation ability was determined using BrdU uptake ability as an index. In addition, formazan formation ability may be used as an index.

  As a result, by adding growth factors (TGF-β1, bFGF, IGF-I, EGF) to the culture solution, it was possible to induce vigorous BrdU incorporation activity (an index of DNA synthesis activity) (FIG. 6). ).

  Here, when growth inhibition is observed, the induction of apoptosis is examined.

  On the other hand, with regard to hard tissue forming ability (differentiation ability), ALP activity, collagen production ability, calcification formation ability, activity of various related enzymes and expression of transcription factors were used as indices. At least, if cell death such as apoptosis is not confirmed as a result, it can be said that the safety screening of the drug has passed.

2) Examination of the affinity of periodontal ligament cells for biomaterials Seeding cells on solid materials for the purpose of investigating the affinity with various biomaterials used in dental implants (for example, hydroxyapatite and titanium) Then, cell proliferation / differentiation ability was evaluated in the same manner as in (1). Furthermore, a section was prepared and histological examination was added.

  FIG. 7 shows the morphology of the obtained PDL-tk1 cells.

  Moreover, calcification (alizarin red-stained image) in vitro could be induced by culturing with StemXvivo (registered trademark) (FIG. 8). Similar results were obtained by adding a cocktail of dexamethasone, vitamin C and beta glycerophosphate, which are considered to be the main components of Stem X Vivo (registered trademark).

  Furthermore, transplantation was performed subcutaneously on the back of nude mice, and von Kossa staining-positive calcification was formed in one week (FIG. 9).

It is a graph which shows a mode that ALP activity rises according to culture | cultivation days. It is a graph which shows a mode that the quantity of the procollagen type I C terminal peptide (PIP) derived from the cell contained in a culture supernatant rises according to a culture | cultivation day. It is the immuno-staining image and Western blot (Westernblot) data which show the expression of osteopontin. It is an immuno-staining image which shows the expression of osteocalcin. It is the immuno-staining image and Western blot data which show the expression of Cox-2. It is a graph which shows BrdU uptake | capture activity by adding a growth factor (TGF- (beta) 1, bFGF, IGF-I, EGF) in a culture solution. It is a phase-contrast micrograph which shows the spindle-like form of a PDL-tk1 cell (a scale bar represents 50 micrometers). It is a light micrograph which shows calcification (alizarin red dyeing | staining image) in vitro by culturing by a stem X Vivo (trademark). It is an optical microscope photograph which shows a mode that the calcification of positive von Kossa staining is formed in the subcutaneous part of a nude mouse in one week after transplantation.

Claims (13)

A human periodontal ligament cell line characterized by having the ability to differentiate into osteoblasts. The human periodontal ligament cell line according to claim 1, wherein the human periodontal ligament cell line is derived from stem cells of human periodontal ligament tissue. The human periodontal ligament cell line according to claim 1 or 2, wherein the receipt number is FERM AP-21302. The human periodontal ligament cell line according to any one of claims 1 to 3, wherein the cell density increases according to the number of days of culture, but the alkaline phosphatase activity increases significantly accordingly. The human periodontal ligament cell line according to any one of claims 1 to 4, which produces a large amount of type I collagen and / or osteopontin and / or osteocalcin. The human periodontal ligament cell line according to any one of claims 1 to 5, wherein the cell period factor exhibits vigorous responsiveness. The cell growth factor is transforming growth factor-β1 and / or basic fibroblast growth factor and / or insulin-like growth factor-I and / or epidermal growth factor. Human periodontal ligament cell line. The human periodontal ligament cell line according to any one of claims 1 to 7, which expresses cyclooxygenase-2. An osteoblast differentiated from the human periodontal ligament cell line according to any one of claims 1 to 8. An artificial bone produced from the osteoblast according to claim 9. A method for using osteoblasts, wherein the osteoblasts according to claim 9 are embedded in a base material and calcified. A method for using osteoblasts, wherein the osteoblasts according to claim 9 are transplanted and mineralized in bone tissue deficient. A method for using osteoblasts, characterized by using calcified osteoblasts according to claim 9.