KR20160025705A - A composition for Enhancing Attachment of Stem Cell to Substrate - Google Patents
A composition for Enhancing Attachment of Stem Cell to Substrate Download PDFInfo
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- KR20160025705A KR20160025705A KR1020140112558A KR20140112558A KR20160025705A KR 20160025705 A KR20160025705 A KR 20160025705A KR 1020140112558 A KR1020140112558 A KR 1020140112558A KR 20140112558 A KR20140112558 A KR 20140112558A KR 20160025705 A KR20160025705 A KR 20160025705A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
- C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/10—Mineral substrates
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Abstract
Description
The present invention relates to a method for enhancing substrate adhesion of stem cells by complexing fibronectin and calcium phosphate to a substrate.
Over the past several decades, mesenchymal stem cells (MSCs) have been of interest both scientifically and clinically (3, 4, 17) due to their ability to differentiate into different tissues and regenerate themselves. Although there is some controversy about the pluripotency of MSCs, it has been clearly demonstrated that tissue stem cells can differentiate into specific cell types and produce the same tissue as their origin (1, 9). Therefore, various studies on stem cells isolated from various tissues have been conducted, and studies on dental stem cells isolated from dental pulp, dental papilla, and periodontal ligament (PDL) have been actively conducted (1, 9, 10). These tooth stem cells are expected to help regenerate size and periodontal ligament tissue. In the field of regenerative medicine, clinical studies based on regenerative capacity of periodontal ligament cells, known as guided tissue regeneration, have been ongoing for several decades to achieve periodontal regeneration (6, 11, 19). However, in most clinical cases, clinical success has not been achieved due to two important limitations: (i) a problem of minimal regeneration of specific tissues such as ligaments and cementum, It is a problem that occurs because there is a limit to obtaining undamaged periodontal ligaments (12); (Ii) there is a problem that the adhesion between the regenerated specific tissues (ligament and cementum) and the dental root surface is severely unstable (2, 12).
The periodontal ligament is a unique tissue that connects and attaches the gum bone to the teeth through periodontal attachment. It is made up of densely organized fibers and root dentin inserted into the cementum. The adhesion between these periodontal structures is not only a feature of each structure, but also plays an important role in the function of the periodontal ligament. However, in the regenerated periodontal ligament tissue, there are fewer periodontal ligament fibers that are functionally differentiated and inserted into the root surface as compared with the natural periodontal ligament tissue (12). In order to improve the adhesion of functionally differentiated periodontal ligament fibers, the interactions between cells and tooth root surfaces during regeneration are very important. These interactions include cell attachment, proliferation, differentiation, functional arrangement, There is the mineralization of the periphery.
Recent studies have examined various methods of regenerating periodontal tissue using periodontal ligament stem cells (10, 14, 15, 16). (16, 22), and applying collagen / gelatin containing stem cells to the injured root surface (15, 39). In addition, recent studies have reintroduced natural or artificial stem cells surrounding the tooth surface at the site of extraction (7, 20). However, these findings do not provide any evidence that cells attach to the root surface as the first step of periodontal attachment. It is not yet clear whether the transplanted stem cells survive and then become involved in various cell activities or disappear immediately in vivo.
Cells are bound to other cell or tissue surfaces through binding between cell adhesion molecules (CAM) and extracellular matrix (ECM) (5, 13). Accordingly, the present inventors tried to make periodontal ligament stem cells more efficiently adhere to the root surface by simultaneously administering fibronectin and calcium phosphate (CaP) to the root surface.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made extensive efforts to develop an optimal culture or transplantation environment in which stem cells for regenerative therapy are efficiently transferred to tissues requiring regeneration and specifically differentiated into desired cells. As a result, the addition of fibronectin and calcium phosphate during cultivation or transplantation of mesenchymal stem cells, more specifically periodontal ligament stem cells, It is possible to obtain a better regenerated tissue ultimately by improving the differentiation rate and the survival rate, thereby completing the present invention.
Accordingly, an object of the present invention is to provide a composition for improving substrate adhesion of periodontal ligament stem cells.
Another object of the present invention is to provide a composition for tooth implantation.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a composition for improving adhesion of a periodontal ligament stem cell comprising fibronectin and calcium phosphate as an active ingredient.
The present inventors have made extensive efforts to develop an optimal culture or transplantation environment in which stem cells for regenerative therapy are efficiently transferred to tissues requiring regeneration and specifically differentiated into desired cells. As a result, when the mesenchymal stem cells, more specifically, fibronectin and calcium phosphate were administered during cultivation or transplantation of periodontal ligament stem cells, the matrix attachment efficiency of the stem cells was greatly improved to improve the differentiation rate and survival rate Can ultimately lead to a better regenerated tissue. ≪ Desc / Clms Page number 2 >
In the present specification, the term " stem cell " refers to undifferentiated cells at a stage prior to differentiation into each cell constituting the tissue, and has the ability to differentiate into specific cells by a specific differentiation stimulus (environment) . Stem cells can produce self-renewal cells by cell division, unlike cells that have stopped cell division, and when differentiation stimulus is applied, they are differentiated into specific cells, It is characterized by plasticity of differentiation that can be differentiated into various cells by differentiation stimulation. The stem cells used in the present invention are mesenchymal stem cells. Mesenchymal stem cells (MSCs) refer to nonhematopoietic stromal cells capable of differentiating and regenerating into mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon and fat. Although not immortalized, MSCs can have the ability to grow and multiply by a factor of ten while maintaining the ability to grow and multiply. More specifically, the mesenchymal stem cells of the present invention are periodontal ligament stem cells.
As used herein, the term " substrate " refers to a culture container, culture substrate or any other culture support material that physically and chemically interacts with stem cells to be cultured while simultaneously providing a surface for adsorption. Thus, the substrate of the present invention may be an artifact such as a culture container or a scaffold, or may be a part of a living body such as an alveolar bone or a root surface. The substrate need not necessarily have a planar surface, and may have spherical or other various three-dimensional structures to the extent that it can provide a significant surface for cell adsorption. Therefore, in the case of an artificial substrate, a three-dimensional network nanofiber having a nano unit fiber shape similar to an extracellular matrix (ECM) as well as a general culture container such as a culture plate, Including, but not limited to, a variety of synthetic polymers commonly used as artificial substrates for microstructures, micropatterned solid substrates, or porous substrates.
According to a specific embodiment of the present invention, the substrate of the present invention is a root surface.
As used herein, the term " dental root surface " refers to the surface of the root of a tooth inserted into the alveolar bone of the gum.
According to a specific embodiment of the present invention, fibronectin and calcium phosphate used in the present invention are coated on the substrate.
As used herein, the term " coating " means bonding to the surface of the material without changing the basic properties of the material to be coated (modified). For example, the fact that fibronectin and calcium phosphate are coated on the root surface implies that fibronectin and calcium phosphate bind directly or indirectly to the delocalized area of the root surface. Therefore, it is obvious that the term " coating " in this specification does not refer only to the case of forming a layer which completely closes the surface of a coating material. More specifically, the term " coating " of the present invention means to bind and occupy a surface sufficient to obtain the desired stem cell attachment effect on the root surface. According to the present invention, since the fibronectin and calcium phosphate, which are effective ingredients of the present invention, are coated on the root surface, the adhesion of the stem cells is markedly increased and the differentiation is promoted, and the teeth coated with fibronectin and calcium phosphate on the root surface are treated with the periodontal ligament stem When transplanted with cells, efficient tissue assimilation was achieved.
According to a specific embodiment of the present invention, the fibronectin of the present invention is contained at a concentration of 5-100 占 퐂 / ml. More specifically 10 to 60 占 퐂 / ml, most specifically 15 to 30 占 퐂 / ml.
According to a specific embodiment of the present invention, the calcium phosphate of the present invention is contained at a concentration of 20-300 mg / ml. More specifically 50-200 mg / ml, more specifically 70-150 mg / ml, most specifically 80-120 mg / ml.
According to another aspect of the present invention, there is provided a composition for tooth implant comprising fibronectin and calcium phosphate as an active ingredient.
As used herein, the term " tooth implant " refers to the insertion of the same, similar biotissue or artificial tissue to replace missing tooth tissue. The implanted tooth may be an artificial tooth, an autologous tooth, or a tagged tooth taken from a heterologous and homologous species.
As used herein, the term " composition for tooth implantation " means any active ingredient added to the implantation site together with the tooth or artificial substitute to be implanted for efficient tooth implantation, and the same as " tooth implant material " or " It is used as a meaning. As described above, the composition of the present invention is coated on the root surface (or a corresponding site of a prosthetic implant replacing a tooth) of a tooth to be implanted, thereby allowing the periodontal ligament stem cells to be well adsorbed and differentiated, and ultimately, So that the adhesion of the liver and the tissue assimilation can be efficiently performed.
According to a specific embodiment of the present invention, the composition of the present invention further comprises a periodontal ligament stem cell.
The composition of the present invention coated on the root surface or the like can achieve the object of the present invention by adsorbing endogenous autologous periodontal ligament stem cells near the graft site without the addition of periodontal ligament stem cells, Stem cells can be added together. When periodontal ligament stem cells are added, fibronectin and calcium phosphate can be implanted into the coated root surface with periodontal ligament stem cells attached.
According to a specific embodiment of the present invention, the fibronectin and calcium phosphate of the present invention are coated on the tooth root surface of the implanted tooth.
According to a specific embodiment of the present invention, the fibronectin of the present invention is contained at a concentration of 5-100 占 퐂 / ml.
According to a specific embodiment of the present invention, the calcium phosphate of the present invention is contained at a concentration of 20-300 mg / ml.
The features and advantages of the present invention are summarized as follows:
(a) The present invention provides a composition for improving adhesion of periodontal ligament stem cells and a composition for tooth implantation.
(b) The present invention significantly improves root attachment efficiency of periodontal ligament stem cells, thereby significantly improving the survival rate and differentiation rate of periodontal ligament stem cells.
(c) The present invention enables more efficient attachment and tissue assimilation in implanting lost teeth tissue, artificial teeth, autologous teeth, or other teeth taken from different species and the like, and is useful as an efficient tooth implant composition Can be used.
1, Fig. 9 is a schematic view and a photograph of a surgical operation method in In vivo and xvivo .
FIG. 2 is a graph showing the results of analyzing the characteristics of periodontal ligament stem cells as mesenchymal stem cells. Figures 2a and 2b are photographs showing the results of CFU (colony-forming-units) analysis and colony formation per 1000 cells. Figures 2c and 2d are photographs showing differentiation into adipocytes and bone cells, respectively. FIG. 2E is a chart showing FACS analysis results of markers of mesenchymal stem cells. FIG. FIG. 2f is a photograph showing tissue analysis after transplanting BCP (biphasic CaP) with or without periodontal ligament stem cells (PDLSC). BCP alone is implanted on the left side and PDLSC is implanted on the right side with BCP. FIG. 2g shows the result of hematoxylin and eosin (H & E) staining after transplanting BCP (biphasic CaP) with or without periodontal ligament stem cells (PDLSC).
FIG. 3 is a SEM (scanning electron microscope) image of root surfaces of different treatments (FIG. 3a: control and CaP coating, FIG. 3b: fibronectin coating and CaP / fibronectin coating).
Fig. 4 is a photograph of a micro-CT image after re-implantation of root roots (A: adjacent natural teeth, B: a control group transplanted with root alone without PDLSC, and C: root root implanted with PDLSC).
FIG. 5 is a photograph showing the histological analysis of root roots (C, F) transplanted with the control teeth (B, E) and PDLSCs implanted with root alone without natural teeth (A, D), PDLSC.
FIG. 6 is a photograph of a root tooth coated with natural teeth and CaP / fibronectin, which was subjected to histological analysis after transplantation with PDLSC. A is a photograph showing a periodontal ligament tissue of a natural tooth adjacent to a transplantation site, B is a photograph showing a periodontal ligament-like tissue at a transplantation site, C is a photograph showing that a periodontal ligament-like tissue is kept in contact with a bone marrow tissue, D is a photograph showing the resorption of the root and the ossification of the root between the bone tissues, E is a photograph showing that the periodontal ligament-like tissue is inserted into the newly formed cementum, F is a photograph showing the periodontal ligament stem cells in the periodontal ligament- G is a photograph showing periodontal ligament fibers inserted into the root as an immunohistochemical staining image for collagen III, and H is an immunohistochemical staining photograph of periostin showing the presence of a specific tissue layer on the root surface.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Materials and Experiments
Experimental animal
Five beagle dogs (about 15 months old, average weight 9 kg) were used. Experimental animals had natural tooth condition and healthy periodontal. Experimental animal selection, management, surgical procedure and preparation were done by approved methods from experimental animal management and use committee of Yonsei University Medical Center.
Culture of periodontal ligament stem cells derived from autologous beagle dogs
Previously reported human periodontal ligament stem cell cultures (18) were slightly modified to isolate and cultivate the autologous dog PDLSC (aD-PDLSC). After maxillary molar extraction, the teeth were cut in half and divided into two or four individual roots. The periodontal ligament tissue was separated from the surface of the scalp and cut into as small pieces as possible. Then, 100 U / ml of type I collagenase (Wako, Tokyo, Japan) and 2.5 U / ml of dispase, Gibco ) In α-MEM (α-minimum essential medium, Gibco, Grand Island, NY, USA). Single-cell suspensions were obtained, and then 5 × 10 5 cells were added to T75 flasks at 37 ° C. and 5% CO 2 . (Gibco), 100 U of L-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, Mo., USA) as a medium for cell culture, (Gibco) containing 100 μg / ml penicillin and 100 μg / ml streptomycin (Gibco) was used. After 3-7 days of culture, a single cell colony was formed, which was designated as aD-PDLSC passage 0 (P0). In the present invention, P3 or P4 was used.
Characterization of isolated aD-PDLSC as mesenchymal stem cells
The basic characteristics of mesenchymal stem cells were analyzed using the following experimental methods.
CFU (colony-forming-units) analysis
The cells were cultured in a 100-mm cell culture dish at a density of 1 × 10 3 cells / 10 ml, fixed with 4% formaldehyde, and stained with crystal violet (Sigma-Aldrich). After 14 days, the cells were observed using a microscope.
Differentiation into bone and fat
The cells were divided into 6-well cell culture dishes at 1 × 10 5 cells / well, and the cells were cultured until the cells were filled in wells, followed by further culture with the bone differentiation medium. (Gibco), 2 mM L-glutamine (Gibco), 100 mM L-ascorbic acid 2-phosphate, 10 M dexamethasone (Sigma-Aldrich), 2 mM beta -glycerophosphate -Aldrich), 55 mM 2- mercaptoethanol (AMRESCO, Solon, OH, USA ), 1.8 mM KH 2 PO 4 (Sigma-Aldrich), 100 U / ml penicillin (Gibco) and 100 ㎍ / ㎖ streptomycin (Gibco ) Was used, and the medium was changed every 3 days. The cells were stained with oil-red O and alizarin red, respectively, two weeks after induction of differentiation into adipocytes, and four weeks after induction of differentiation into bone cells.
Fluorescence-activated cell sorting analysis (FACS)
The aD-PDLSC cultured in the T75 flask was treated with trypsin-EDTA and then transferred to a 1.7 ml tube (Oxygen, Union City, CA, USA) and 4% paraformaldehyde was added for 15 minutes to fix the cells . The immobilized cells were incubated with 3% bovine serum albumin (BSA) for 1 hour, then incubated with primary antibody against CD14, CD34, CD44 or CD90 for 1 hour, A secondary antibody with fluorescein isothiocyanate was added. After incubation at room temperature for 45 min, cells were washed 3 times and FACS was performed using a flow cytometer (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA).
Ectopic transplantation
In vivo transplantation experiments were performed to evaluate the regenerative capacity of aD-PDLSC. AD-PDLSC was implanted into the subcutaneous pocket of the dorsal area of immunodeficient mice. 6 × 10 6 aD-PDLSC (P3) was mixed with 80 mg of BCP (biphasic CaP, Biomatlante, Vigneux, France) and incubated for 1.5 hours and then transplanted into four mice. BCP alone was transplanted into another four mice as a control. After 8 weeks, the mice were sacrificed and the tissue sections were fixed in 4% neutralized formalin, and the calcification was removed with 5% EDTA (pH 7.2) and immersed in paraffin. The sections were cut at 50 μm intervals and 5 μm thick and stained with H & E (hematoxylin-eosin) and Masson's trichrome.
Preparation of dental root with cell removed
The maxillary premolar was extracted and the cells were removed from the separated root and used as the scaffold of the present invention (Fig. 1). The soft tissue was removed from the cervical area of the extracted root and root canal treatment was performed. The dental pulp was removed using a barbed broach (MANI, Tokyo, Japan) and a Ni-Ti spinning device (Pathfile, Dentsply Maillefer, Ballaigues, Switzerland) and the root was left empty . All roots used in the experiment were sterilized with 12% ethylene oxide gas at 54 ° C for 6 hours and then degassed for 12 hours. The root was removed and the organic matrix was exposed to 5% EDTA solution (pH 7.4) and washed with PBS buffer for 5 minutes.
Evaluation of effect of coating of root surface on adhesion of aD-PDLSC
36 roots were divided into 4 groups and treated differently on the surface. The control group, the fibronectin coating group, the CaP coating group and the CaP / fibronectin group (Fig. 1). The roots removed from the cells for fibronectin coating were immersed in 20 μg / ml of fibronectin solution obtained from bovine plasma (F1141, Sigma-Aldrich) at 4 ° C overnight, and then washed with PBS. For CaP coating, BCP particles were prepared by dissolving roots of roots removed CaP solution in 100 ㎎ / ㎖ of distilled water and immersed in CaP solution for 12 hours at room temperature and then washed with PBS. For CaP / fibronectin coating, CaP coating was first applied followed by fibronectin coating.
In order to evaluate the effect of the coating of the root surface on adhesion of aD-PDLSC, aD-PDLSC, which was suspension-cultured at a concentration of 1.2 × 10 7 cells / ml, was added to a 6- well plate containing each coated root, And cultured for up to 3 days. The culture conditions were an α-MEM / FBS culture medium at 37 ° C. and 5% CO 2 . Samples were prepared by immobilizing the cells on days 1, 2 and 3 after initiation of culture. To evaluate the growth and adhesion of aD-PDLSC on the root surfaces coated with each of fibronectin, CaP and CaP / fibronectin, SEM (scanning electron microscopy, S-300N, Hitachi, Japan).
Re-implantation of teeth using aD-PDLSC cultured on CaP / fibronectin coated root surfaces
The unilateral mandibular premolar was extracted from five dogs and the experiment was carried out according to the following procedure. On the first day of reimplantation, the second and fourth premolars of both mandibles were extracted after being cut in half with two separate roots. Disinfection and treatment of the root canal was performed according to the method described above, and the crown of the tooth was removed. On the second day, the root surface was coated with CaP / fibronectin for 24 hours and then incubated with aD-PDLSC for 60 hours. To ensure an even distribution of aD-PDLSC on the entire surface of the root, the cells were incubated with constant rolling for the first 6 hours. On the fourth day, a blood clot was removed from the extraction socket, the extraction window wall was gently removed, and the prepared root was re-implanted (FIG. 1). The contralateral mandibular premolar was used as a control and reimplanted via the same procedure except that the roots were incubated with the cells. The experimental animals were given intramuscular injection of antibiotics (20 mg / kg cefazoline sodium, Yuhan, Seoul, Korea) and 0.2% chlorhexidine solution was given daily for 14 days. Seven days after surgery, the surgical suture material was removed and the experimental animals were sacrificed by an overdose of pentobarbital sodium (90120 mg / kg, iv) 8 weeks after surgery. The block section containing the experimental site was fixed in 10% formalin for 10 days and the micro-computed tomography (CT) images of the block sample at 35 μm resolution (100 kV and 100 uA) (Micro-CT; Skyscan 1072, Skyscan, Aartselaar, Belgium). The scanned micro-CT images were processed in DICOM format and reconstructed in three dimensions using PC-based software (On-Demand3D, Cybermed, Seoul, Korea). For each experimental group, 8-12 consecutive tomographic sections were selected at 300 ㎛ intervals, and the percentage of periodontal ligament sites relative to the entire length of the root surface and the extent of osteosynthesis were analyzed. The process of treating the tissue by the method described in the ectopic transplantation analysis was performed. From the oral cavity of the experimental root to the surface of the tongue, twelve coronal cut sections were obtained. Ten of these slides were stained with H & E and Masson trichrome, and the remaining two were used for immunohistochemical staining to identify periodontal ligament markers (collagen Ⅲ and periostin). Five HE - stained slides were used to analyze the percentage of periodontal ligament - like tissue. Periodontal ligament - like tissue had or did not have root resorption or osseointegration relative to the entire length of the root surface. (Ab23445, Abcam, Cambridge, MA, USA) and anti-ferriastin antibody (ab14041, Abcam) and an assay kit (Zymed / Invitrogen, Carlsbad, CA, USA) ) Was used.
Statistical analysis
For statistical analysis, SPSS 15.0 (SPSS, Chicago, IL, USA) was used. Paired t-test was used for the analysis. P <0.05 was considered statistically significant.
Experiment result
Characteristic Analysis of aD-PDLSC
A single cell from a periodontal ligament was cultured for 7 to 14 days to form a cell colony similar to that of fibroblasts. The cultured cells had potential stem cell potential in the periodontal ligament Respectively. After 4-7 days of cell division, prolonged spindle fibroblasts were observed and 76114 independent cell clusters were formed from 1 × 10 3 monolayers (FIGS. 2a and 2b). Although the number of independent cell clusters tended to decrease as the cell lineage increased, a high density of cell clusters formed on the culture plate in the fourth passage (P4).
In order to analyze the degree of differentiation, cells were cultured in a culture medium inducing differentiation into bone and adipocytes, respectively. As a result, it was possible to observe accumulation of minerals stained with alizarin-red and cells stained with oil-red-O . These results show that aD-PDLSC has the ability to differentiate into various cells (FIGS. 2c and 2d). FACS analysis showed that aD-PDLSC did not have hematopoietic stem cell markers CD14 and CD34 but had CD44 and CD90 markers of mesenchymal stem cells (FIG. 2e).
The ability of aD-PDLSC to be regenerated into various tissues was confirmed in an in vivo transplantation model (Fig. 2f-2g). It has been observed that unidirectional periodontal ligament-like tissue and mineralized tissue similar to cementum are formed on the surface of the biomaterial. Adhesive cementoblast dispersed between the implanted Sharpey fibers and the inserted fibers into the cemento-pseudo-tissue at the implant site was also observed. In contrast, no mineralized tissue was formed in the control group, confirming that aD-PDLSC is likely to differentiate into various cells in vivo models.
The effect of surface treatment on the degree of adhesion of aD-PDLSC to the root surface of cells removed
Observations for 3 days showed limited cell adhesion in the uncoated control group. In other words, most of the surfaces were exposed without cells attached, and cells attached to only a limited extent were observed, and their shapes were spherical rather than spindle-like. After 3 days, adherence to the surface of the root surface was progressed in some cells, but no more adherent cells were observed compared to the first day of culture (Fig. 3a).
In the group coated with CaP, most of the root surfaces were covered with small CaP crystal particles, and several spherical cells were observed on the first day of observation. On the second day, some of the CaP crystals disappeared and some cells were observed over the remainder of the period, but the number of cells did not increase (Fig. 3a).
In the case of the group coated with fibronectin, adhesion of cells in a sheet-like state was remarkably increased from the first day (Fig. 3B). As a result of observing at high magnification, cells were spreading in various directions in several folded cell layers, which seemed to be adhered to cells in the same cell layer or other cell layers. On the second day, we observed that the process of these cell changes spread more widely, and we observed that the network of sheet-like cell masses was getting bigger. On the third day, it was observed that a sheet-like cell mass extended in one direction and formed a structure similar to a cloth having a uniform pattern. On the first day, gaps on the surface of the root were observed, but over time, these gaps gradually decreased. Most root surfaces were covered with sheet-like cell masses, but these gaps remained partially until the third day.
In the case of the group coated with CaP and fibronectin, adhesion of cells was remarkably increased as compared with other groups. On the first day of observation, a sheet-shaped cell mass was observed, and no gaps or cells remained from the surface were observed, and a cloth-like structure extending in one direction was evident. On the 2nd and 3rd day, some layers of such sheet-like cell mass were observed to be separated at a restricted area, but the surface of the root was not exposed because only very partial layers were separated (FIG. 3b). Based on the results of this in vitro test, root surfaces coated with CaP and fibronectin were used for in vivo reimplantation experiments.
Of aD-PDLSC in in-vivo re-transplantation
Micro CT images showed that most root surfaces (66.32 ± 10.05%) were in direct contact with the alveolar bone at the graft site in the root group only transplantation. In the root implanted with aD-PDLSC, a portion (39.07 ± 5.15%) was in direct contact with the bone, while a wider region (60.93 ± 5.15%) showed periodontal ligament region [Table 1 and Figure 4)]. In the control group, bony ankylosis was observed more frequently and wider than that of implanted roots with aD-PDLSC.
5.15
5.15
13.07
9.10
12.93
10.05
10.05
0.00
Histologically, only the aD-PDLSC cultured on CaP / fibronectin coated roots observed functionally differentiated periodontal ligament-like tissue between the alveolar bone and the implanted root surface (FIGS. 5 and 6). In the control group transplanted only with root, only the irregularly absorbed root surface directly contacting the alveolar bone was observed without mediating the periodontal ligament (Fig. 5). Observations at high magnification showed that the periodontal ligament-like structure, the basic structure of the periodontal ligament, obliquely or vertically arranged collagen fibers, alveolar bone and Sharpey fibers and cells embedded in newly formed cementum (Fig. 6) Of the periodontal ligament. Most root surfaces in contact with the periodontal ligament-like tissue maintained their original shape without reabsorption (panel B in Figure 6). Periodontal ligament collagen fibers arranged at an angle or vertically were inserted into newly formed cementum over the existing cementum. In contrast, other parts of the periodontal ligament-like tissue remained intact with the periodontal ligament-like tissue expanded (Fig. 6, panel D), despite being surrounded by reattachment of the contacting root and newly formed bones.
In another type of periodontal ligament-like tissue, a purple layer containing collagen fibers and cells between mineralized alveolar bone sides was mineralized and maintained. In the case of periodontal ligament-like tissue in contact with bone marrow instead of mineralized tissue, several polynuclear cells appeared outside the surface but retained the original structure (panel C in FIG. 6).
Immunohistochemical staining with periodontal ligament-specific markers (collagen III and ferriostin) revealed dark dyed specific layers adjacent to the root surface and inserted collagen fibers (panel C And D). However, the periodontal ligament-like tissue was narrower than the natural periodontal ligament tissue, and the number of inserted Sharpey fibers was smaller.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
references
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defect model. J. Periodontal Res .; 2013.
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13. Li, L. et al., Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells. Cell Adh. Migr. 6 (1): 59-70; 2012.
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