TW201204834A - Combination for tooth regeneration - Google Patents

Abstract

A combination for tooth regeneration used in combination with adipose stem cells is provided, and the combination comprises a growth factor and an artificial and/or natural polymer. Tooth regeneration can be carried out by implanting the combination along with adipose stem cells into a tooth socket.

Description

201204834 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a combination for regenerating teeth, and more particularly to a combination of a species with adipose stem cells and for regenerating teeth.曰[Prior Art] In dental treatment, when the teeth are missing due to factors such as rainbow teeth, age, orthodontic surgery (4), when they are removed, they will be treated with filling, implant or denture reconstruction. . Whether filling, implant or denture reconstruction, artificial materials are currently used to replace the original tooth components, such as titanium, £ or composite resin. However, the human body may be repulsive to artificial materials, and the material of the human body is easily damaged due to lack of wear and tear, and even the entire tooth is lost. Therefore, if the teeth can be naturally regenerated, the problems caused by the treatment or reconstruction of the teeth using artificial materials can be avoided. The technology of regenerative medicine involves the differentiation of stem cells into specific cell types and combined with tissue engineering to grow into new tissues or organs to replace damaged tissues or organs, thereby exerting normal physiological functions. In the case of dental regenerative medicine, dental pulp stem cells, periodontal ligament stem cells and bone marrow mesenchymal stem cells have been used as sources of stem cells, so that although dental pulp stem cells and periodontal ligament stem cells can be used as the most direct regeneration of teeth. The source of cells, because it is rare in the human body, it is difficult to obtain sufficient amount for treatment; while the bone marrow mesenchymal stem cells have the disadvantage of being scarce in availability, they are easy to age due to age, and must Obtained in the form of bone marrow extraction, so there are many limitations in practical applications. On the other hand, in addition to stem cell sources, how to use appropriate growth hormone and scaffold components, 3 201204834 to differentiate stem cells and further grow into well-structured and functional teeth, is also the key to dental regenerative medicine. . Therefore, in order for regenerative medicine to be practically applied to clinical dental treatment, a stable, abundant and readily available source of stem cells is required. The present invention is directed to the above needs, and the inventors of the present invention have found that the use of a combination of an adipose stem cell and a growth hormone and a biomedical material can successfully regenerate the teeth, and the regenerated tooth has a sound structure and function. SUMMARY OF THE INVENTION One object of the present invention is to provide a combination with adipose stem cells for regenerating teeth comprising a growth hormone and an artificial and/or natural polymer. Another object of the present invention is to provide a combination for regenerating teeth comprising adipose stem cells, a growth hormone, and an artificial and/or natural polymer. The detailed description of the present invention and the preferred embodiments thereof will be described in the following description of the present invention. [Embodiment] As mentioned above, the current filling, implant or denture reconstruction is the use of artificial materials as a substitute for natural tooth components, but it has the disadvantage of causing repulsion or repeated repair due to wear, and dental regenerative medicine can solve This problem. However, currently studied dental pulp stem cells, periodontal ligament stem cells or bone marrow mesenchymal stem cells are not a stable and abundant source of stem cells. Adipose-derived stem cells belong to adult stem cells and are characterized by their aging resistance. They can be obtained in large quantities by general liposuction, so they can be used as a source of stem cells that are stable and rich in 201204834. In addition, adipose stem cells have low immunological repellency, so that allogeneic transplantation of adipose stem cells between animals can be performed, and is not limited to autologous transplantation, and therefore, it has high flexibility of application. In view of the above, the present invention applies adipose stem cells to dental regenerative medicine, and provides a combination with adipose stem cells for regenerating teeth, and the combination comprises a growth hormone and an artificial and/or natural polymer. In the combination of the present invention, growth hormone selected from the group consisting of bone morphogenetic protein (BMP), dentin matrix protein (DMP), growth differentiation factor (Growth/differentiation factor, Gdf), and combinations of the foregoing. The growth hormone acts as a message molecule that causes the adipose stem cells to differentiate and form dental tissue. Osteogenic proteins, also known as bone morphogenetic proteins, are currently known to have twenty different osteogenic proteins, which are a transforming growth factor that induces differentiation of undifferentiated mesenchymal stem cells. Chondrogenic cells or osteoblasts promote proliferation, so osteogenic proteins are involved in the growth and repair and reconstruction of cartilage and osteogenesis. In addition to osteogenic protein, it promotes the formation of cartilage and osteogenesis, and also regulates the development and morphogenesis of embryonic cells in tissues or organs. A dentine matrix protein, or dentin matrix protein, is a non-colloidal protein involved in the calcification of bones and teeth, which is secreted by bone cells or dentate mother cells to promote extracellular or dentate formation. Growth differentiation factors are important biomessages for cell development, which promote chondrocyte proliferation and periodontal tissue formation. Preferably, the combination of the invention may comprise a growth hormone selected from the group consisting of osteogenic protein-2 (BMP-2), osteogenic protein-4 (BMP-4), osteogenic protein-7 (BMP-7). , osteogenic protein-11 (BMP-11), dentine matrix protein-1 (DMP-1), growth differentiation factor-11 (Gdf-11), and combinations thereof. Most preferably, the combination comprises osteogenic protein-2. 201204834 Osteogenic protein-2 is a disulfide homodimer protein with a molecular weight of about 30 kilodaltons (kDal), which can be obtained by automated purification in an object or by artificial gene selection. However, due to the low content of osteogenic protein-2 in animals, and the osteogenic protein-2 derived from different individuals has a very different activity in inducing stem cell differentiation, it is usually an osteogenic protein selected by artificial gene-- 2. In one embodiment of the present invention, recombinant human BMP-2 is used. In the combination of the present invention, a polymer is included as a scaffold or a backbone when the adipose stem cells differentiate and further form dental tissues, allowing the cells to form and maintain a three-dimensional structure. A polymer selected from the group consisting of collagen, polyglutamic acid (PGA), polylactic acid-glycolic acid (PLGA), and poly-acid can be used in combination with the present invention. #5 (calcium polyphosphate), hexafluoroisopropanol silk (HFIP silk), and combinations thereof. Preferably, collagen is used in combination in the present invention. Collagen is also known as collagen. Twenty-nine different collagen proteins are known to exist in tissues of animals. Collagen has a special three-strand-wound structure that maintains a entangled structure between the three independent collagen peptide chains by a hydrogen bond formed by glycine, which imparts excellent mechanical strength and elasticity to collagen. In addition, collagen can form a more stable structure via cross-linking. Because collagen has excellent toughness and elasticity and is highly biocompatible, it is often used in tissue engineering. Therefore, in the present invention, it is preferred to use collagen as a cell scaffold; and among all collagens, type I collagen and type II collagen are in the teeth and cartilage tissues. Main ingredients. Therefore, in the present invention, it is more preferred to use collagen of the first type or the second type. 201204834 The combination of the present invention may be in the form of a mixture, or in the form of a separate component of each component, in principle, as long as the growth hormone in the combination does not interact with the artificial or natural polymer, then it may be in the form of a mixture (eg, Dry powder mixture). For example, when :: osteogenic protein and collagen are included in the ::, the two do not interact with each other 'that is, the osteogenic protein and collagen are mixed together. However, it may be different from raw or natural polymers.

The environment (e.g., different pH values, preservatives, water content, etc.) may be used in the form of separate components to maintain the optimum activity or condition of each component. On the other hand, depending on the growth hormone selected and the artificial or natural compound (4), the present invention is in the form of a solution, a powder, a suspension or the like. Since the growth hormone is mainly two egg tarts, in order to prevent growth hormone denaturation, it is generally required to be placed in a buffer solution of 3 salts, or the growth hormone can be powdered and placed in a low-temperature cold green manner. Store at a low temperature below 4°C. In addition, if the selected polymer is protein f (e.g., _ protein silk noodle), the village is in the form of a solution or a powder. Side Modes • The components of the combinations of the present invention may be mixed or separated from each other, and may be in the form of a powder, a solution, a liquid, or the like, respectively, in the form of a powder, a solution, a suspension, or the like, or 'Collection - the ingredients are dissolved in another age, etc., and the special limit material' can be added to the combination towel of the present invention to prevent the (four) or desiccant from extending the shelf life. The present invention is generally known to those skilled in the art, based on the teachings herein, and may be used in the present invention. The amount is adjusted depending on the type of growth hormone or polymer selected. Preferably, the combination of the present invention has a weight ratio of growth hormone to polymer of from about 1:1 _ to about 1.12 〇, _' more preferably from about 1:6,000 to about 1:60,000, most preferably about 201204834 9,000 to approximately 1: 12,000. The combination of the invention, together with adipose stem cells, can be transplanted into a tooth defect or extracted from the alveolus for tooth regeneration. Here, the amount of the adipose stem cells can be adjusted as needed. For example, when the combination is used for repair or regeneration of a part of the teeth, the amount of the adipose stem cells can be low; and when the combination is used for the whole tooth regeneration, Increase the amount of fat 2 stem cells. The amount of Lingancheng is generally from about 1 to 2 to about 5,106' per gram of growth hormone, preferably from about 5χ1〇3 to about 5χ1〇5, more preferably from about 5χΐ〇4 to about 1x10. Further, as described above, since the adipose stem cells have low immunological repellency, allogeneic transplantation of adipose stem cells between animals can be performed. Therefore, in the present invention, in addition to the adipose stem cells from the animal to be transplanted, Use adipose stem cells from other animal species of the same species. The present invention also provides another combination for regenerating teeth comprising a fat stem cell, a growth hormone, and a human and/or natural polymer. Wherein, the form, amount and form of the growth hormone and the artificial and/or natural polymer, and the source and content of the adipose stem cells are as defined above. In addition, #high sensitivity of adipose-derived stem cells, which are based on harsh environmental conditions, can be used to prevent vitality and prevent contamination, and in order to avoid growth hormone or polymer affecting adipose stem cells, therefore, in this combination, Lin Stem cells are usually mixed with growth hormone and polymer before transplantation. Since the key to tooth regeneration is that adipose stem cells must successfully differentiate and form tooth tissue, the vitality of the adipose stem cells is maintained, and the activity or freshness of the growth hormone and the compound are maintained to regenerate the teeth under optimal conditions. Consider the person. To this end, adipose stem cells, growth hormone, and artificial and/or natural polymers can be mixed in an appropriate ratio to provide a combination of the regenerated tooth of the present invention 201204834 before the transplantation operation is performed, so that the components in the combination are highly active. In the fresh state, the teeth are regenerated. In one embodiment of the present invention, a combination comprising adipose stem cells, osteogenic protein-2, and collagen is prepared before the transplantation operation is performed, and the weight ratio of osteogenic protein-2 to collagen is Approximately 1:11,250, and the amount of adipose stem cells is about 5 x 104 per gram of growth hormone, and transplanted into the alveolar of the animal for tooth regeneration. Since the combination of the present invention allows for the regeneration of teeth, it can be used for the repair, replacement, and/or reconstruction of teeth. The invention is further illustrated by the following specific embodiments. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. [Preparation Example] Preparation of a combination for regenerating teeth Referring to Fig. 1, a combination for regenerating teeth of the present invention was prepared in the following steps and ratios, and transplanted. A. Configure 1.35 ml of solution A (on ice) containing: 5.7><DMEM (Dulbecco's modified Eagle's medium) 0.5 ml of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) , 0.1 molar concentration) 0.5 ml 0.25 ml 50 μl 50 μl 2.15 ml 1.5 ml

NaHC03 (2.5 wt%)

CaCl2 (0.17 molar concentration)

NaOH (1 equivalent concentration) B, configuration 3.65 ml of solution B, containing: DMEM (containing 23.3% by weight fetal bovine serum) Type I collagen (3.75 mg / ml) 201204834 C, cultured rabbit adipose tissue (rabbit adipose tissue- The derived stem cell 'rADSCs' or rabbit dental pulp stem cells (rDPSCs), as shown in Fig. 2, showed typical cell morphology of mesenchymal stem cells. After cultured rabbit adipose stem cells or rabbit dental pulp stem cells were trypsinized for 3 minutes, the cell suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and 5 χ 106 cells were collected and placed at 36.5 μl. The solution is mashed to make a cell suspension. D. Add 1 μg/ml of osteogenic protein-2 (R&D Systems Catalog Number 355-BEC) to the cell suspension prepared in step C. E. Mix 13.5 μl of solution A and solution Β (containing osteogenic protein-2 with adipose stem cells or dental pulp stem cells) on ice, and immediately add to a 48-well cell culture dish. F. The cell culture plate was placed in a cell culture chamber containing 5% by volume of CO 2 at 37 ° C for 2 hours, and after the collagen was polymerized and solidified into a gel, the transplantation was carried out.

[Example I A. Transplantation for regenerative teeth first] The New Zealand White Rabbit (purchased from Taiwan, Lin Guoyu Rabbit Factory, No. 115, Siwei Road, Beidou Town, Changhua County) was anesthetized and then removed one of the upper front teeth. And a lower incisor' and waiting to be transplanted. The combination gel prepared in Example 1 (including osteogenic protein-2 and adipose stem cells or dental pulp stem cells) was directly transplanted and placed in a sulcus in which the rabbit was removed, and the gingiva was sutured with a suture. In addition, a collagen gelatin 201204834 gel containing osteogenic protein-2 but no stem cells was implanted into the alveolar detachment of the upper incisor to serve as a control group, and the gingiva was also sutured by suture, that is, the transplantation step was completed. Β·X-ray photography and anatomical analysis After 15 weeks of completion of the transplant operation, the rabbits were removed by X-ray photography to remove the canines of the upper and lower incisors. As a result, as shown in Figure 3A, the lower door can be observed. There was tooth regeneration at the transplant site, however, there was no tooth regeneration in the tooth groove of the upper door as a control group (Fig. 3A, arrow). In addition, observations were performed anatomically, and the results were as shown in Fig. 3B, showing that teeth were newly regenerated in the alveolar cells containing a combination of adipose stem cells or dental pulp stem cells, but no tooth regeneration occurred in the alveolar cells of the control group. (Fig. 3B, white arrow). The results of this test demonstrate that the combination of the present invention can successfully regenerate teeth. C. H&E tissue staining analysis Regenerated tooth tissue and normal staining of rabbits stained with H&E staining reagent (including Mayer's Hemalum Solution (available from MerckTM) and Eosin Y Solution Alcoholic (purchased from Sigma-AldrichTM)) The tooth tissues were analyzed and compared, and the results are shown in Figures 4A to 4C. As shown in Fig. 4A, the tissue structure similar to normal teeth can be regenerated in the alveolus transplanted by a combination of dental pulp stem cells or adipose stem cells. In addition, as shown in Fig. 4B, the detailed structure of the regenerated tooth has the same tooth shape (D: Dentin), gingival mass (C: Cementum), and periodontal ligament (PDL). And under the observation of a high-magnification (ΙΟΟΟχ) microscope, Sharpey's fiber extending from the periodontal strip to the gingival mass (Fig. 4B, white arrow) can be seen. In addition, as shown in Fig. 4C, the regenerated tooth tissue has a structure of the pulp, which represents the ability of the regenerated tooth to self-renew. 201204834 D·Green immunofluorescence staining analysis of regenerative tooth tissue of rabbits stained with green immunofluorescence staining reagent (including Wyperber's factor (v〇n lake coffee (10) factor) or Beta 3-tubulin (abulin) The results were compared with the normal teeth _, and the results are shown in Fig. 5A and Fig. 5B. As shown in Fig. 5A, new blood vessels are formed in the newly grown pulp and periodontal ligament (pDL) which are transplanted by a combination of dental pulp stem cells or adipose stem cells. The material, as shown in Fig. 5B, is dominated by nerve fibers in the pulp and periodontal strips regenerated by the rabbit (Fig. 5B 'white arrow'). The analysis results of the above B to C show that the combination of the present invention can successfully regenerate the teeth, and the regenerated new teeth have the same intact structure as the normal teeth, so that the normal chewing function can be performed. The above-described embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as set forth in the appended claims. [Simple description of the diagram]. 1 is a flow chart of preparation and transplantation of a combination for regenerating teeth of the present invention; FIG. 2 is an optical micrograph of rabbit dental pulp stem cells and rabbit adipose stem cells; FIG. 3A is a tooth of a rabbit upper incisor and a lower incisor The χ 摄影 摄影 第 第 ; ; ; ; 第 第 第 第 第 第 第 第 第 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; And Figures 5A and 5B show green immunofluorescence staining of rabbit new tooth tissue and normal tooth tissue. [Main component symbol description] (none).

Claims (1)

201204834 VII. Patent Application Range: 1. A combination with adipose stem cells and used to regenerate teeth, comprising a growth hormone and an artificial and/or natural polymer. 2. The combination of claim 1, wherein the growth hormone is selected from the group consisting of bone morphogenetic protein (BMP), dentin matrix protein (DMP), growth differentiation factor (Growth/differentiation). Factor ' Gdf), and combinations of the foregoing. 3. The combination of claim 2, wherein the growth hormone is selected from the group consisting of osteogenic protein-2, osteogenic protein-4, osteogenic protein-7, osteogenic protein-11, dentine matrix protein _- 1. Growth Differentiation Factor-11, and combinations thereof. 4. The combination of claim 3, wherein the growth hormone is osteogenic protein-2. 5. The combination of claim 1, wherein the polymer is selected from the group consisting of collagen, polyglutamic acid (PGA), polylactic acid-glycolic acid (PLGA). ), calcium polyphosphate, hexafluoroisopropanol silk 'ΗΠΡ silk, and combinations of the foregoing. 6. The combination of claim 5, wherein the polymer is collagen. The combination of any one of claims 1 to 6, wherein the weight ratio of the growth hormone to the polymer is from about 1:1,200 to about 1:120,000. 8. The combination of claim 7, wherein the weight ratio of the growth hormone to the polymer is from about 1:6 to about 1:60,000. 9. The combination of claim 8, wherein the weight ratio of the growth hormone to the polymer is from about 1:9 to about 1:12,000. 10. A combination for regenerating teeth comprising adipose stem cells, a growth hormone, and a polymer of 201204834 artificial and/or natural. 11. The combination of claim 10, wherein the growth hormone is selected from the group consisting of osteogenic proteins, dentine matrix proteins, growth differentiation factors, and combinations thereof. 12. A combination of 4, wherein the growth hormone is selected from the group consisting of osteogenic protein-2, osteogenic protein-4, osteogenic protein-7, osteogenic protein _u, dentine matrix protein _ 1. Growth differentiation factor _U, and combinations thereof. 13' is a combination of μ and 12 wherein the growth hormone is osteogenic. The combination of claim 10, wherein the polymer is selected from the group consisting of collagen protein polyglycolic acid, polylactide-glycolic acid, calcium polyphosphate, hexafluoroisopropanol filament, and combinations thereof. 15. The combination of claim 14, wherein the polymer is collagen. The combination of any one of the items 1 to 15 wherein the weight ratio of the growth hormone to the polymer is about 丨: 12 〇〇 to about i: (9), and the content of the adipose stem cells is per gram of growth. The hormone is about 5χ1〇2 to about 5乂1〇6. 17. The combination of claim 16, wherein the weight ratio of the growth hormone to the polymer is from about 6,000 to about 〇〇〇〇6 〇〇〇〇, and the content of the adipose stem cells is about 5 x 10 每 3 per gram of growth hormone. It is about 5χ1〇5. 18. The combination of claim 17 wherein the weight ratio of the growth hormone to the polymer is from about 1: 9,000 to about 丨:12, 〇〇〇, and the content of the adipose stem cells is about 5 x 1 per gram of growth hormone. 4 to about 丨... 15