Anagenesis cell sheet, production method and use method thereof

Abstract

To provide a periodontal ligament anagenesis cell sheet comprising preferable adhesive property to a surface of a dental root.SOLUTION: Cells are cultured in a specific condition, on a cell culture substrate whose surface is covered with a temperature responsive polymer whose upper limit or lower limit critical solution temperature to water is 0-80°C, (1) a culture liquid temperature is set to equal to or greater than the upper limit critical solution temperature or equal to or lower than the lower limit critical solution temperature, (2) a cultured periodontal ligament cell sheet which is cultured is brought into tight contact with a carrier, (3) then the periodontal ligament cell sheet is peeled with the carrier as it is, for forming a periodontal ligament anagenesis cell sheet. The periodontal ligament anagenesis cell sheet has extremely high transplant survival property to a surface of a dental root, can transfer a target cell at high density, and can achieve reconstruction of a periodontal tissue actively. In addition, by performing lamination of cell sheets to be transplanted and providing a three-dimensional polarity, the periodontal tissue can be more efficiently re-constructed, therefore the periodontal ligament anagenesis cell sheet can be expected for clinical applications such as, therapy of periodontal disease, and periodontal ligament regeneration therapy and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a periodontal ligament tissue regeneration cell sheet, a method for manufacturing the same, and a method for using the same, which are useful in fields such as medicine, biology, drug discovery, and pharmacy. [Background technology]

[0002] Japan is an aging society with the world's highest average life expectancy. People's hopes are increasingly focused on quality of life (QOL) rather than simply extending life. Speaking and eating are important functions that contribute to a sense of purpose, especially for the elderly, and in that sense, maintaining the health of the masticatory organs, including tooth preservation, is a major factor influencing QOL. Mastication is an essential function for food intake, and recent research on the masticatory system is revealing that mastication stimulates brain cells, promoting mental and nervous system development and activation, enhancing immune function, and even suppressing obesity, among other systemic functions. Therefore, the decline in masticatory function due to tooth loss can accelerate the path to dementia and potentially lead to lifestyle-related diseases. Currently, the proportion of the population suffering from periodontal disease exceeds 80% among middle-aged and elderly people aged 35-65, and people's demands for periodontal treatment are high and diverse. Furthermore, many related factors are intricately involved in the onset and progression of periodontal disease, and preventive measures centered on oral hygiene, as has been done until now, cannot solve the problem. Unfortunately, when people contract periodontal disease and seek treatment, they often seek functional restoration and aesthetic improvement. At present, conventional treatments, which mainly consist of non-regenerative therapies, are insufficient to meet these needs, and there is a need for innovative treatments that can regenerate lost periodontal tissue.

[0003] Teeth are exposed to the oral cavity by breaking through the epithelium, and the continuity of the epithelium is lost at the boundary between the tooth and the gum, creating an extremely unique environment within the body. The tooth and gum are composed of "epithelial attachment" and "connective tissue attachment." In the former, epithelium called junctional epithelium adheres to the tooth surface (enamel) via hemidesmosomes and a basement plate. In the latter, the periodontal ligament is composed of collagen fibers that calcify and become embedded in the cementum of the root surface, and these fibers also calcify and become embedded in the alveolar bone, transitioning into gingival fibers, thereby firmly bonding the tooth to the alveolar bone and gum.

[0004] Periodontal disease is an inflammatory disease of the periodontal tissues caused by plaque bacteria, and is classified into "gingivitis" and "periodontitis." Gingivitis is when inflammation is limited to the gums, while periodontitis is when the inflammation extends to the periodontal ligament and alveolar bone, and the attachment by the periodontal ligament is destroyed. Normally, it progresses from gingivitis to periodontitis, forming pockets (grooves) around the teeth. As the pockets deepen, plaque bacteria in the pockets proliferate, and the inflammation progresses to deeper areas. In addition to systemic modifying factors (medications, blood disorders, immune disorders, nutritional status, stress, fatigue, smoking, etc.), local corrective factors such as ambiguous trauma, which occurs when mechanical stress on the teeth is excessive, such as teeth grinding, can worsen inflammation. In cases of severe periodontitis with advanced tissue destruction, even if tooth extraction is avoided, the lost periodontal tissue never recovers its original form and function. After healing, significant functional and aesthetic impairments remain, which is a major factor in reducing the patient's quality of life.

[0005] Alveolar bone resorption is one of the characteristic pathological conditions of periodontitis, but the loss of root cementum and periodontal ligament (periodontal ligament) that occurs simultaneously is the essence of periodontitis. As its name suggests, the periodontal ligament has a ligament-like structure, suspending the tooth within the socket of the alveolar bone via the cementum on the root surface, acting as a cushion against strong occlusal pressure, and is also rich in vascular components and has high metabolic activity, playing an important role in maintaining the homeostasis of periodontal tissue. Despite numerous studies aimed at periodontal tissue regeneration over nearly half a century, a suitable treatment method has yet to be established because it is extremely difficult to regenerate not only the alveolar bone but also this periodontal ligament tissue simultaneously. It has been shown that when periodontal ligament regeneration does not occur, the oral epithelium invades the defect and the root becomes bony, resulting in a biologically unstable state of healing, which can lead to the recurrence of periodontitis and tooth loss.

[0006] Currently, scaling and root planing (S-RP) is performed to treat periodontitis. This is a fundamental procedure for treating periodontitis, in which infected tissue is mechanically removed from the affected area. The cementum, periodontal ligament, and gingival tissue on the root surface, mainly contaminated by bacterial infection, are removed using special instruments, and the root surface is given a morphology that facilitates the attachment of surrounding tissues, thus promoting natural healing. The healing morphology mainly consists of epithelial attachment, where epithelium progresses and proliferates on the treated root surface. With this method, surgical intervention is not necessary, and the procedure can be performed with no anesthesia or only local infiltration anesthesia. It is a necessary and sufficient treatment method for areas that do not pose aesthetic or functional problems. However, in cases of advanced periodontitis where alveolar bone has been destroyed, this treatment method alone is insufficient if reinfection is expected after treatment. Also, the procedure tends to be difficult in areas with complex tooth morphology, in which case surgical intervention is indicated. For areas with complex morphologies that cannot be treated solely with S&RP, surgical procedures are performed, such as surgically creating a gingival flap and treating it under direct visualization. The expected healing pattern is basically the same as with S&RP, with epithelial attachment.

[0007] Amidst these circumstances, regenerative medicine technologies aimed at actively reconstructing periodontal tissue have begun to be actively researched. Several technologies are already being used in treatment, including bone resection, bone grafting, and mucosal grafting. Alveolar bone grafting is classified into autogenous, allogeneic, xenogeneic, and artificial bone grafting. Autogenous bone grafting is mainly performed, and it is a method of transplanting bone fragments harvested from another part of the oral cavity to the bone defect site. In allogeneic bone grafting, demineralized freeze-dried allogeneic bone graft (DFDBA) is used in Europe and the United States and has shown good clinical results. Hydroxyapatite (HA) is a representative example of artificial bone. Compared to S and RP alone, the healing morphology of allogeneic bone grafting is thought to be effective in retaining the blood clot at the defect site, inhibiting epithelial invasion while simultaneously providing conditions that facilitate the migration of periodontal ligament cells. However, animal experiments have reported that the main healing morphology is still epithelial attachment that does not cause regeneration of new cementum and periodontal ligament. Autogenous bone grafting has the lowest antigenicity and infection problems and is naturally easily accepted by the recipient site. Furthermore, bone graft materials have the advantage of potentially possessing osteoconductive and osteoinductive properties. However, securing the necessary amount of bone for autogenous bone grafting is difficult. In addition, allogeneic and xenogeneic bone grafting still have antigenic and infectious problems, and regardless of the type of graft material, if the bone defect area is large and the contact area between the root surface and the grafted bone is large, there is a problem of ankylosis (ossified adhesion of the tooth) occurring.

[0008] In the late 1970s and 1980s, a Scandinavian research group confirmed that epithelial tissue and gingival connective tissue lack the ability to regenerate the periodontal ligament, and that bone tissue causes ossicular adhesion to the root surface. They found that for periodontal ligament tissue regeneration to occur, cells derived from periodontal ligament tissue must be present in the defect, especially on the root surface. Based on this concept, guided tissue regeneration (GTR) was devised. This method suppresses epithelial cells, which proliferate rapidly during the healing process and enter the defect early, using a biocompatible barrier membrane, while simultaneously creating space in the defect, thereby enabling periodontal ligament tissue regeneration. This method is currently the most promising method for periodontal tissue regeneration and has achieved good results in areas with bone defect morphologies where space creation is easy. However, this method has drawbacks: the clinical procedure is complex, it is difficult to accurately place the biocompatible membrane on the root surface in areas with complex defect morphologies, and during the healing period, the membrane must be completely covered with a gingival flap due to infection issues, making the prognosis highly dependent on the surgical procedure and environment. Furthermore, these biocompatible membranes come in absorbable and non-absorbable forms. Using the former requires re-operation to remove the membrane, while the latter does not require re-operation but leaves structural problems in terms of space creation. In addition, because it relies on cell migration from the remaining periodontal ligament, the amount of regeneration is limited, and from the perspective of membrane retention, it is only applicable to some cases of vertical bone defects. In extensive chronic periodontitis, which is widely seen in middle-aged and elderly people, horizontal bone resorption is the main pathological condition, and this GTR is often ineffective.

[0009] Thus, even though it is known that periodontal ligament-derived cells are an essential factor in periodontal tissue regeneration, conventional treatments, including the GTR method, although utilizing growth factors, only rely on cell migration from remaining tissue. As a result, the amount of tissue regeneration is greatly influenced by the defect morphology or the amount of remaining periodontal ligament tissue, and the indications for these treatments are currently limited. In severely damaged periodontal tissue, it is becoming clear that relying solely on cell migration from the remaining surrounding tissue is insufficient, and that an exogenous supply of cells is necessary. The goal of regenerative therapy in periodontal tissue is to suppress healing by epithelial attachment and to acquire connective tissue attachment by the periodontal ligament.

[0010] In recent years, several studies have reported on the aim of tissue regeneration through cell transplantation. Many of these involve seeding single cells into a three-dimensional matrix and injecting them into tissue defects, but none have yet achieved the desired tissue reconstruction. Possible reasons for this include the difficulty in selecting the cell source and controlling the localization of cell differentiation within the tissue defect. Periodontal tissue regeneration requires the formation of new cementum on the root surface, and it is necessary to simultaneously regenerate not only the soft tissue of the periodontal ligament but also this cementum and the hard tissue of the alveolar bone, and to functionally connect them. If these tissues are formed with a time lag due to the action of different tissue-specific progenitor cells and growth factors, then cell transplantation methods in periodontal tissue regeneration must be more delicate. In other words, it is not enough to simply inject a single cell into a space-made defect and leave it to tissue differentiation in vivo; it is necessary to specify the placement of cells and place the appropriate cells in each area.

[0011] The cells required for this purpose have traditionally been cultured on glass surfaces or on the surfaces of synthetic polymers that have undergone various treatments. For example, various containers made of polystyrene with surface treatments such as gamma irradiation or silicone coating are widely used as cell culture vessels. Cells cultured and proliferated using such cell culture vessels are detached and recovered from the container surface by treatment with proteolytic enzymes such as trypsin or chemicals. However, when recovering cells that have been proliferated by the aforementioned chemical treatments, the treatment process becomes complicated, the possibility of contamination with impurities increases, and there have been cases where the proliferated cells are denatured or damaged by the chemical treatment, impairing their original function.

[0012] To overcome these drawbacks, several technologies have been proposed. Among them, Okano et al. devised a novel concept called cell sheet engineering, and succeeded in regenerating tissue by culturing cells taken from the patient's own tissue, forming them into sheets, and transplanting them to the affected area. To date, they have completed human clinical applications in the cornea, myocardium, esophagus, periodontal ligament, etc. (Non-patent documents 1-5, Patent documents 1-5). In particular, Patent document 1 describes culturing anterior segment-related cells on a cell culture support coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature of 0-80°C in water, and, if necessary, layering the cultured cell layers using conventional methods. By simply changing the temperature of the support, it became possible to produce cell sheets with sufficient strength by detaching the cultured cell sheets. Furthermore, these cell sheets also retain basement membrane-like proteins, and their engraftment into tissue is clearly improved compared to those treated with the aforementioned dispase. Furthermore, Patent Document 2 describes culturing myocardial tissue cells on a cell culture support coated with a temperature-responsive polymer to obtain a myocardial-like cell sheet. Subsequently, the culture medium temperature is set to above the upper critical dissolution temperature or below the lower critical dissolution temperature, and the cultured, multilayered cell sheet is attached to a polymer membrane. The cell sheet is then peeled off together with the polymer membrane, and a three-dimensional structure is formed using a predetermined method. This method was found to construct a cell sheet and a three-dimensional structure with few structural defects and possessing several functions as myocardial-like tissue in vitro. However, neither method has investigated whether it can regenerate the periodontal ligament or achieve connective tissue attachment by the periodontal ligament, which is intended for regenerative therapy in periodontal tissue. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Patent No. 4716479 [Patent Document 2] Patent No. 4475847 [Patent Document 3] Patent No. 4679795 [Patent Document 4] Patent No. 4921353 [Patent Document 5] Japanese Unexamined Patent Application Publication No. 2011-115604 [Non-Patent Documents]

[0014] [Non-Patent Document 1] Nakamura K. A "super-aged" society and the "locomotive syndrome". J Orthop Sci. 13(1), 1-2, (2008) [Non-Patent Document 2] Hisashi Kurosawa, Rehabilitation Medicine, "Rehabilitation as a Treatment for Knee Osteoarthritis", 42(2), 124-130 (2005) [Non-Patent Document 3] Naomasa Yokota. et al. Clinical results following intra-articular injection of adipose-derived stromal vascular fraction cells in patients with osteoarthritis of the knee. Regenerative Therapy,( 6),108-112(2017) [Summary of the Invention] [Problems to be Solved by the Invention]

[0015] The present invention has been made to solve the problems associated with the defect of the periodontal ligament tissue as described above. That is, the present invention provides a novel periodontal ligament tissue regeneration cell sheet, a manufacturing method, and a method of using the same, based on an idea completely different from the prior art. [Means for Solving the Problems]

[0016] In order to solve the above problems, the inventors have conducted research and development by considering various aspects. As a result, it has been found that a periodontal ligament tissue regeneration cell sheet obtained by culturing cells from periodontal ligament tissue under specific conditions and reconstructing them into a sheet form can attach to the surface of the periodontal ligament tissue and promote the regeneration of the surface layer of the periodontal ligament tissue. And it has been found that the periodontal ligament cells of the present invention have a strong proliferation ability and are useful as a cell source in the field of regenerative medicine. The present invention has been completed based on such findings.

[0017] That is, the present invention provides a periodontal ligament tissue regeneration cell sheet mainly composed of cells obtained by inducing osteogenic differentiation of undifferentiated cells derived from periodontal ligament tissue. In addition, the present invention provides a method for collecting cells from a specific periodontal ligament tissue under specific culture conditions and finally producing a sheet-like periodontal ligament tissue regeneration cell sheet on the surface of a specific substrate coated with a temperature-responsive polymer. Furthermore, the present invention provides a method for using the obtained periodontal ligament tissue regeneration cell sheet. The present invention is considered to be an extremely important invention that can be realized only by using a cell structure based on a novel idea that is unparalleled in the world of cell sheets.

[0018] According to the present invention, there is provided a periodontal ligament tissue regeneration cell sheet mainly composed of cells obtained by inducing osteogenic differentiation of undifferentiated cells derived from periodontal ligament tissue, the periodontal ligament tissue regeneration cell sheet being peeled from a temperature-responsive culture substrate. The periodontal ligament tissue regeneration cell sheet of the present invention includes, but is not limited to, the following preferred embodiments.

[0019] In one embodiment of the present invention, the periodontal ligament tissue regeneration cell sheet of the present invention uses undifferentiated cells derived from periodontal ligament tissue, and more than 85% of the total number of cells constituting the sheet are cells induced to osteogenic differentiation, has no ligament-like microstructure, and has a strength that can be picked up from the culture substrate after culturing. In one embodiment of the present invention, the periodontal ligament cells used in the periodontal ligament tissue regeneration cell sheet of the present invention are characterized in that undifferentiated cells derived from periodontal ligament tissue account for 60% or more of the total number of cells. In one aspect of the present invention, the periodontal ligament tissue regeneration cells used in the present invention are periodontal ligament-derived mesenchymal stem cells. In one aspect of the present invention, the periodontal ligament tissue regeneration cell sheet of the present invention is spontaneously layered.

[0020] Furthermore, the present invention provides a method for producing a periodontal ligament tissue regeneration cell sheet, characterized by culturing periodontal ligament cells on a cell culture substrate coated with a polymer whose hydration power changes in the temperature range of 0 to 80°C at a temperature range where the polymer's hydration power is weak, and then changing the temperature of the culture medium to a state where the polymer's hydration power is strong, thereby peeling off the cultured periodontal ligament cells in a sheet-like manner. The method for producing a periodontal ligament tissue regeneration cell sheet of the present invention is not limited, but preferred embodiments include the following embodiments. In one aspect of the present invention, the present invention provides a method for collecting periodontal ligament tissue using scissors, wherein the tissue collected under sterile conditions is subdivided using scissors, then treated with enzymes, and cultured on a specific substrate. In one aspect of the present invention, the detached periodontal ligament tissue regenerating cell sheet spontaneously layers. The detachment method is a method of detaching the cell culture support without treatment with proteolytic enzymes. Alternatively, the detachment method may involve adhering a carrier to the cultured cells at the end of the culture and detaching the cell sheet together with the carrier. In one aspect of the present invention, periodontal ligament cells may be collected from living tissue. The number of cells seeded during culture is 2 × 10⁶. 3 ~2×10 5 pieces / cm 2 It is possible. In one aspect of the present invention, the polymer used in the method for producing the periodontal ligament tissue regeneration cell sheet of the present invention is poly(N-isopropylacrylamide), which is a polymer whose hydration capacity changes in the temperature range of 0 to 80°C. In one aspect of the present invention, when a temperature-responsive cell culture substrate coated with a temperature-responsive polymer having a specific structure is used, the periodontal ligament tissue regeneration cell sheet of the present invention does not peel off during culture and can be quickly peeled off when used for treatment such as transplantation.

[0021] The present invention provides a method for utilizing a periodontal ligament tissue regeneration cell sheet, characterized by transplanting the obtained periodontal ligament tissue regeneration cell sheet into periodontal ligament tissue or the like. In one embodiment of the present invention, the purpose may be periodontal disease treatment, periodontal ligament regeneration treatment, or reconstruction of other periodontal ligament tissue. [Effects of the Invention]

[0022] The periodontal ligament tissue regeneration cell sheet shown in this invention will engraft on the surface of the periodontal ligament tissue and promote the regeneration of the periodontal ligament tissue. [Brief explanation of the drawing]

[0023] [Figure 1] This figure shows an overview of the temperature-responsive cell culture polymer synthesis process in Example 1. [Figure 2] This figure shows the cell culture state (microscope magnification 4x) of Example 2 and Comparative Example 1. [Figure 3] This figure shows the appearance (visually) of the cell sheet obtained from the temperature-responsive cell culture substrate obtained in Example 1, as shown in Example 2. [Figure 4] This figure shows the results of flow cytometry analysis of cultured cells from Example 2 and Comparative Example 1. [Modes for carrying out the invention]

[0024] The cells used in the periodontal ligament tissue regeneration cell sheet of the present invention are obtained from the periodontal ligament tissue of a living organism. The periodontal ligament tissue can be any periodontal ligament tissue, but as described later, it is preferable that the tissue contains periodontal ligament-derived mesenchymal stem cells that have undifferentiated cells. This is because mesenchymal stem cells have the characteristic of being highly proliferative. As a result, many periodontal ligament tissue regeneration cell sheets can be obtained from the periodontal ligament tissue cells of one individual, meaning that allogeneic transplantation becomes possible, which is preferable as it reduces the burden on the patient and the ethical issues for the patient. Initially, the inventors thought that a cell sheet having ligament-like microstructures was preferable, but later they found that by using periodontal ligament tissue-derived cells containing a high proportion of undifferentiated cells, inducing osteogenic differentiation during culture, and ensuring that more than 85% of the total number of cells constituting the cell sheet are osteogenic cells, a cell sheet without ligament-like microstructures and with sufficient strength to be picked up from the culture substrate after culture is preferable for the present invention, leading to the present invention. It is believed that most of the cells obtained from periodontal ligament tissue used in this invention are not fully differentiated. In this invention, the types of cells contained in the periodontal ligament tissue are currently being analyzed, and specifically, it is thought to be periodontal ligament mesenchymal stem cells, periodontal ligament blasts, bone marrow mesenchymal stem cells, etc., but is not limited to these. In this invention, undifferentiated cells collected from periodontal ligament tissue are induced to differentiate into bone during culture. However, as mentioned above, even if the collected undifferentiated cells are induced to differentiate into bone in this invention, most of them remain undifferentiated cells, and it is believed that many cells are only directed toward osteogenic differentiation. It is believed that by forming cells in this state into a sheet, periodontal ligament tissue can be efficiently regenerated after transplantation. Against this background, in this invention, it is preferable that 85% or more of the total number of cells constituting the sheet consist of cells that have been induced to differentiate into bone, preferably 90% or more, more preferably 95% or more, and most preferably 97% or more. In this case, if it is less than 85%, the tissue will not regenerate efficiently even if the periodontal ligament tissue regeneration cell sheet of this invention is transplanted.The cell sheet in this invention may contain cells other than periodontal ligament cells, and there are no restrictions on the type of cells used. Examples include one or more of the following cells: epithelial cells, epithelial stem cells, fibroblasts, vascular endothelial cells, vascular endothelial progenitor cells, bone marrow-derived cells, adipocytes, and other mesenchymal stem cells. Furthermore, there are no particular restrictions on the proportion of each of these cells contained within the sheet.

[0025] In the present invention, the undifferentiated cells constituting the periodontal ligament tissue regeneration cell sheet should constitute 1% or more, preferably 5% or more, more preferably 10% or more, even more preferably 30% or more, and most preferably 60% or more, of the total number of cells in the cell sheet. If the undifferentiated cells constitute less than 1% of the total number of cells in the cell sheet, the cell proliferation will be slow and the effects of the present invention cannot be fully exhibited, which is undesirable.

[0026] In the present invention, the method for measuring the number of cells that have undergone osteogenic differentiation is not particularly limited, but one example is a method using an ALP marker as an indicator. Examples of such measurements include flow cytometry and spectroscopic instruments, but are not particularly limited.

[0027] The cells used in this invention may be cells directly collected from living tissue, cells directly collected and differentiated in a culture system, or cell lines, but there are no restrictions on their type. The origin of these cells is not particularly restricted, but examples include humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or immunodeficient animals thereof. However, when using the periodontal ligament tissue regeneration cell sheet of this invention for human treatment, it is preferable to use cells derived from humans, pigs, monkeys, or chimpanzees. The culture medium for cell culture in this invention is not particularly restricted as long as it is one that is normally used for the cells being cultured.

[0028] In this invention, it is necessary to separate cells from periodontal ligament tissue into individual cells through a specific process. Methods for this process include physically peeling and subdividing the superficial periodontal ligament tissue layer from tissue collected from the tooth root using scissors or the like, then enzymatically treating the collected tissue to form individual cells for culture; the Explore culture method, which involves placing the periodontal ligament tissue layer on a culture substrate; or a combination of these methods. However, in this invention, the method of enzymatically treating the collected tissue to form individual cells before culturing is most preferable. The culture medium used is not particularly limited and can be appropriately selected according to conventional methods depending on the cells being used. For example, in the case of periodontal ligament cells, MEM medium, F-12 medium, DMEM medium, KCM medium, or mixtures thereof are suitable. Additives commonly used in cell culture, such as serum, may be added to the culture medium. The inventors have found that when culturing periodontal ligament cells, regardless of the form of the culture substrate described later, adding human serum promotes the proliferation of the cultured periodontal ligament cells, resulting in a human periodontal ligament cell sheet with physical strength and flexibility. Here, the concentration of serum used in the culture medium is preferably 0.5 to 35%, more preferably 1 to 20%, more preferably 5 to 15%, and most preferably 8 to 12%. If the serum concentration is lower than 0.5% or higher than 30%, the strength of the cultured cells may be weakened, which is undesirable according to the present invention. Furthermore, to increase the adhesion of cells to the culture substrate surface, collagen, laminin, fibronectin, Matrigel, and mixtures thereof may be pre-coated as needed, and the present invention is not particularly limited by these conditions.

[0029] In the present invention, the above-mentioned cells are cultured on a cell culture support coated with a polymer whose surface changes in hydration capacity in the temperature range of 0 to 80°C, at a temperature range where the polymer's hydration capacity is weak. This temperature is preferably 37°C, which is the temperature at which cells are typically cultured. The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such polymers include the polymer described in Japanese Patent Application Publication No. 2-211865. Specifically, for example, it can be obtained by the homopolymerization or copolymerization of the following monomers. Examples of monomers that can be used include (meth)acrylamide compounds, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, or vinyl ether derivatives, and in the case of copolymers, any two or more of these can be used. Furthermore, copolymerization with monomers other than the above-mentioned monomers, grafting or copolymerization of polymers, or mixtures of polymers and copolymers may also be used. It is also possible to crosslink the polymer within a range that does not impair its inherent properties. In this process, since cells are cultured and detached, the separation is performed in the range of 5°C to 50°C. Examples of temperature-responsive polymers include poly-n-propylacrylamide (lower critical dissolution temperature of the homopolymer is 21°C), poly-Nn-propyl methacrylamide (27°C), poly-N-isopropylacrylamide (32°C), poly-N-isopropyl methacrylamide (43°C), poly-N-cyclopropylacrylamide (45°C), poly-N-ethoxyethylacrylamide (approximately 35°C), poly-N-ethoxyethyl methacrylamide (approximately 45°C), poly-N-tetrahydrofurfurylacrylamide (approximately 28°C), poly-N-tetrahydrofurfuryl methacrylamide (approximately 35°C), poly-N,N-ethylmethylacrylamide (56°C), and poly-N,N-diethylacrylamide (32°C).Examples of monomers used for copolymerization in the present invention include, but are not particularly limited to, hydrated polymers such as polyacrylamide, poly-N,N-diethylacrylamide, poly-N,N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and its salts, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose, and carboxymethylcellulose.

[0030] The method for coating the substrate surface with the above-mentioned polymers used in the present invention is not particularly limited, but for example, the substrate and the monomer or polymer can be coated by electron beam irradiation (EB), gamma ray irradiation, ultraviolet irradiation, plasma treatment, powder treatment, organic polymerization reaction, or by physical adsorption such as coating or kneading. As an example, a block polymer or hyperbranched polymer in which a temperature-responsive polymer is bonded to a hydrophobic polymer may be used, and these may be physically adsorbed to the substrate surface by coating or kneading, and the present invention is not particularly limited. In the present invention, a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded is coated on the substrate surface at a density of 0.8 to 3.0 μg / cm³. 2 This is a temperature-responsive substrate for cell culture coated in a specific ratio. The water-insoluble polymer portion of the block copolymer coated on the substrate surface has the effect of not being released from the substrate surface during cell culture, and even when detaching cultured cells or cell sheets based on temperature changes.

[0031] This invention relates to a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded together, and is applied to the substrate surface at a rate of 0.8 to 3.0 μg / cm³. 2 This is a temperature-responsive substrate for cell culture coated in a specific ratio. The water-insoluble polymer portion of the block copolymer coated on the substrate surface has the effect of not being released from the substrate surface during cell culture, and even when detaching cultured cells or cell sheets based on temperature changes.

[0032] Generally, "block copolymer" means a polymer having at least two compositionally distinct segments, and as understood by those skilled in the art, examples include diblock copolymers, triblock copolymers, random block copolymers, star-branched block copolymers, or hyperbranched block copolymers. The block copolymer used in the present invention typically has a water-insoluble polymer segment (A) and a temperature-responsive polymer segment (B), but the following structure: AB structure containing two block segments Structure; ABA or BAB containing three block segments; and (AB) containing multiple blocks. n - (where n is an integer of 2 or more) may be any of the above. In light of the above structure, the AB structure is preferred for the block copolymer that can be used in the temperature-responsive substrate for cell culture of the present invention. In this specification, if the block copolymer having an AB structure consists of, for example, a segment of poly-n-butyl methacrylate (PBMA) as a water-insoluble polymer segment (A) and a segment of poly-N-isopropylacrylamide (PIPAAm) as a temperature-responsive polymer segment (B), the block copolymer may be written as "B79-IP120" or the like using the number of monomer units of each polymer (for example, 79 units of BMA and 120 units of IPAAm) (see Example 1).

[0033] The term "water-insoluble polymer" as used herein is not particularly limited as long as it is insoluble in water, and examples include polyalkyl acrylates such as poly-n-butyl acrylate and poly-t-butyl acrylate, polyalkyl methacrylates such as poly-n-butyl methacrylate, poly-t-butyl methacrylate and polymethyl methacrylate, and polystyrene.

[0034] As used herein, the "temperature-responsive polymer" means a polymer having a lower critical solution temperature (LCST) and / or an upper critical solution temperature (UCST), which may be any of their homopolymers, copolymers, or mixtures. Examples of such polymers include those described in Japanese Patent Publication No. 06-104061. Specifically, for example, they can be obtained by homopolymerization or copolymerization of the following monomers. Examples of monomers that can be used include (meth)acrylamide compounds, N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, vinyl ether derivatives, and partially acetylated polyvinyl alcohol. In the case of copolymers, any two or more of these monomers can be used. Furthermore, copolymers with monomers other than the above monomers, graft or copolymers of polymers with each other, or mixtures of polymers and / or copolymers may also be used. Also, if necessary, crosslinking can be carried out within a range that does not impair the original properties of the polymer. The coating amount of the temperature-responsive polymer on the surface of the culture substrate is preferably in the range of 0.3 to 6.0 μg / cm 2 , more preferably 0.5 to 4.5 μg / cm 2 , even more preferably 1.0 to 3.5 μg / cm 2 , most preferably 1.5 to 2.0 μg / cm 2When the coating amount is less than 0.3 μg / cm2, cells on the polymer are difficult to remove even when stimulated, resulting in significantly reduced work efficiency, which is undesirable. Conversely, when it is more than 6.0 μg / cm2, cells are difficult to adhere to that area, making it difficult to ensure sufficient cell adhesion. In such cases, if a cell adhesion protein is further coated on top of the temperature-responsive polymer coating layer, the amount of temperature-responsive polymer coating on the substrate surface may be 6.0 μg / cm2 or more, and in this case, the amount of temperature-responsive polymer coating should be 9.0 μg / cm2 or less, preferably 8.0 μg / cm2 or less, and 7.0 μg / cm2 or less is advantageous. If the amount of temperature-responsive polymer coating is 9.0 μg / cm2 or more, cells will not adhere easily even if a cell adhesion protein is further coated on top of the temperature-responsive polymer coating layer, which is undesirable. The type of such cell adhesion protein is not limited in any way, but examples include collagen, laminin, laminin 5, fibronectin, Matrigel, etc., either individually or in mixtures of two or more. Furthermore, the coating method for these cell adhesion proteins can follow conventional methods. Typically, an aqueous solution of the cell adhesion protein is applied to the substrate surface, and then the aqueous solution is removed and the substrate is rinsed. The present invention is a technology that aims to utilize the cell sheet itself using a temperature-responsive culture dish. Therefore, it is undesirable for the amount of cell adhesion protein coating on the temperature-responsive polymer layer to be excessively large. The amount of coating by the temperature-responsive polymer and the amount of coating by the cell adhesion protein can be measured by conventional methods. For example, methods include directly measuring the cell attachment area using FT-IR-ATR, or immobilizing a pre-labeled polymer in a similar manner and estimating the amount from the amount of labeled polymer immobilized on the cell attachment area. Any of these methods may be used. Moreover, the inventors have found that if a substrate coated with the above-mentioned temperature-responsive block polymer is used as a substrate for temperature-responsive cell culture, the substrate surface can be designed so that cells do not peel off during culture, but can be easily peeled off by cooling when desired. With this culture substrate, valuable cells used for treatment can be reliably detached immediately before treatment without wasting any, making it extremely useful for therapeutic procedures.

[0035] In the method of the present invention, cultured cell sheets can be detached and recovered from a temperature-responsive substrate by raising the temperature of the culture substrate to which the cultured cells are attached above the upper critical dissolution temperature or below the lower critical dissolution temperature of the coating polymer on the culture substrate. This can be done in the culture medium or in other isotonic solutions, and the method can be selected according to the purpose. To detach and recover cells more quickly and efficiently, methods such as lightly tapping or shaking the substrate, or stirring the culture medium with a pipette, may be used alone or in combination. Culture conditions other than temperature can follow conventional methods and are not particularly limited. For example, the culture medium used may be a medium to which serum such as known fetal bovine serum (FCS) has been added, or it may be a serum-free medium without such serum added.

[0036] The above will be explained using poly(N-isopropylacrylamide) as an example of a temperature-responsive polymer. Poly(N-isopropylacrylamide) is known as a polymer with a lower critical dissolution temperature of 31°C. In its free state, it undergoes dehydration in water at temperatures above 31°C, causing the polymer chains to aggregate and the solution to become cloudy. Conversely, at temperatures below 31°C, the polymer chains hydrate and become dissolved in water. In this invention, this polymer is coated and fixed to the surface of a substrate such as a petri dish. Therefore, at temperatures above 31°C, the polymer on the substrate surface also undergoes dehydration, but because the polymer chains are coated and fixed to the substrate surface, the substrate surface becomes hydrophobic. Conversely, at temperatures below 31°C, the polymer on the substrate surface hydrates, but because the polymer chains are coated and fixed to the substrate surface, the substrate surface becomes hydrophilic. The hydrophobic surface at this time is a suitable surface for cells to attach and proliferate, while the hydrophilic surface is a surface that cells cannot attach to, and cultured cells or cell sheets can be detached simply by cooling. The inventors refer to this temperature-responsive cell culture substrate, which retains intact membrane proteins and yields cells that are extremely useful in the medical and biological fields and can engraft in tissues and organs, as "SSCW".

[0037] As substrates to be coated, any material that can be shaped, such as glass, modified glass, polystyrene, polymethyl methacrylate, and other compounds commonly used in cell culture, can be used.

[0038] The shape of the culture substrate in this invention is not particularly limited, but examples include dishes, multiplates, flasks, cell inserts via porous membranes, or flat membranes. When cells are cultured using such a culture substrate, periodontal ligament cells are promoted by the presence of cells other than periodontal ligament cells, and the resulting cell sheet of this invention is advantageous because it is physically strong and highly flexible.

[0039] The periodontal ligament tissue regeneration cell sheet in this invention is not damaged by proteolytic enzymes such as dispase and trypsin during culture. Therefore, the periodontal ligament tissue regeneration cell sheet detached from the substrate possesses adhesive proteins, and when mucosal cells are detached in a sheet form, the desmosome structure between cells is retained to a certain extent. This allows for good adhesion to the affected tissue during transplantation, enabling efficient transplantation. Generally, dispase, a proteolytic enzyme, is known to retain 10-40% of the desmosome structure between cells during detachment, but it destroys most of the basement membrane-like proteins between cells and the substrate, resulting in a cell sheet with low strength. In contrast, the periodontal ligament tissue regeneration cell sheet of this invention retains 60% or more of both the desmosome structure and basement membrane-like proteins, thus providing the various effects described above.

[0040] Furthermore, in the method of the present invention, when peeling and recovering the periodontal ligament tissue regeneration cell sheet from the culture substrate, a carrier may be attached to the cultured cells at the end of the culture period, and the periodontal ligament tissue regeneration cell sheet may be recovered together with the carrier, if necessary. Typically, when peeling and recovering a cultured periodontal ligament tissue regeneration cell sheet from a temperature-responsive substrate, the periodontal ligament tissue regeneration cell sheet can be peeled off together with the carrier by attaching a carrier to the cultured cells at the end of the culture period and then raising the temperature above the upper critical dissolution temperature or below the lower critical dissolution temperature of the coating polymer of the culture substrate. The carrier used as needed may be, for example, hydrophilized polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and its derivatives, PVLA, chitin, chitosan, collagen, paper such as Japanese paper, urethane, spandex, or other net-like or stockinette-like polymer materials. In this case, if a net-like or stockinette-like polymer material is used, the degree of freedom of the periodontal ligament tissue regeneration cell sheet is increased, and the periodontal ligament tissue regeneration cell sheet can be provided with a shape suitable for the site of the root tissue to which it is transplanted.

[0041] In the present invention, two or more periodontal ligament tissue regeneration cell sheets may be laminated. The number of laminated sheets is not particularly limited, but the number of laminates is preferably 10 or less, more preferably 5 or less, and more preferably 2 or less. Laminating periodontal ligament tissue regeneration cell sheets improves the cell density per sheet area, and the function of the periodontal ligament tissue regeneration cell sheet is also improved, which is preferable. Furthermore, in the periodontal ligament tissue regeneration cell sheet of the present invention, insofar as the function of the cell sheet of the present invention as described above is not impaired, one or more of the following cells may be mixed in addition to mucosal cells: vascular endothelial cells, vascular endothelial progenitor cells, adipose-derived cells, and mesenchymal stem cells, or these cell sheets may be laminated on the periodontal ligament tissue regeneration cell sheet.

[0042] The method for producing the cell sheet laminate in the present invention is not particularly limited, but for example, it can be obtained by detaching cultured cells in sheet form and, if necessary, stacking the cultured cell sheets using a cell transfer jig. In this case, the temperature of the culture medium is not particularly limited, as long as it is below the upper critical dissolution temperature of the polymer coated on the surface of the culture substrate if the polymer has an upper critical dissolution temperature, and above the lower critical dissolution temperature if the polymer has a lower critical dissolution temperature. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate, or in a high temperature range where cultured cells die, is inappropriate. Culture conditions other than temperature can be carried out according to conventional methods and are not particularly limited. For example, the culture medium used may be a medium to which a known serum such as fetal bovine serum (FCS) has been added, or it may be a serum-free medium without such serum added. Also, if necessary, a jig for moving the cell sheets may be used. Such jigs are not limited in material or shape as long as they can capture the detached cell sheet. However, the materials typically used are polyvinylidene difluoride (PVDF), silicone, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibrin glue, etc., which are brought into contact with the periodontal ligament tissue regeneration cell sheet in the form of a film, porous film, nonwoven fabric, or woven fabric.

[0043] The periodontal ligament tissue regeneration cell sheet obtained by this invention can be transplanted to a predetermined site in the body. Examples of transplantation sites include periodontal ligament defects in the tooth root tissue. It is not particularly limited whether or not intercellular adhesive components such as fibrin glue or collagen have been applied to the transplantation site beforehand.

[0044] The periodontal ligament tissue regeneration cell sheet transplanted in this invention retains basement membrane-like proteins and exhibits excellent engraftment properties. When transplanting, the number of cells can be adjusted according to the purpose, and when the transplanted cells are in sheet form, the overall activity of the cell sheet can be altered by changing its size and shape. The mechanism is currently under investigation, but as a result, transplanting the periodontal ligament tissue regeneration cell sheet of this invention can regenerate the surface of the periodontal ligament tissue.

[0045] By using the periodontal ligament tissue regeneration cell sheet described in this invention, it becomes possible to perform periodontal disease treatment and periodontal ligament regeneration treatment on humans, but the purpose is not particularly limited to these. [Examples]

[0046] The present invention will be described in more detail below based on examples, but these examples are not intended to limit the present invention in any way. [Examples]

[0047] A temperature-responsive block copolymer was prepared from the temperature-responsive polymer poly-N-isopropylacrylamide (PIPAAm) and the hydrophobic polymer poly-n-butyl methacrylate (PBMA), and the copolymer was spin-coated onto the surface of a commercially available 3.5 cmφ cell culture dish (Corning 430165). Specifically, a hydrophobic polymer PBMA synthesized by reversible addition-cleavage chain transfer radical (RAFT) polymerization was used as a macro-RAFT agent to prepare the PBMA-b-PIPAAm block copolymer (Figure 1). The obtained block copolymer was evaluated by nuclear magnetic resonance spectroscopy (1H-NMR) and gel permeation chromatography (GPC), and it was found that the desired product was obtained. The amount of polymer introduced onto the surface of the obtained temperature-responsive block copolymer coating was found to be 1.6 μg / cm³. 2 This material was then sterilized with ethylene oxide gas to create a temperature-responsive culture substrate. [Examples]

[0048] Human periodontal ligament tissue was obtained and harvested using scissors. The tissue was then subdivided using scissors and treated in collagenase solution for 3 hours to obtain periodontal ligament cells according to the present invention. As the temperature-responsive cell culture substrate, commercially available UpCell (manufactured by CellSeed) and the substrate from Example 1 were used. The obtained cells were seeded (5 × 10) onto the aforementioned temperature-responsive cell culture substrate. 4 Cells were cultured in a 3.5cmφ dish and cultured using MEM medium supplemented with 10% bovine serum. Cells on the substrate proliferated normally, and after 2 days of culture, the cells reached a subconfluent state. From the second day of culture, the culture was changed to a bone differentiation induction medium, which was the MEM medium supplemented with 82.1 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone. Subsequently, the culture medium was changed to the differentiation induction medium after 5, 9, and 12 days. After 10 days of culture, some cells on the UpCell were peeling off from the periphery of the substrate surface (near the Petri dish wall), but this did not occur with the substrate coated with the temperature-responsive block copolymer in Example 1. Figure 2 shows the results of microscopic observation of cultured cells 14 days after the start of culture (magnification ×4). After 14 days of culture, the culture medium was cooled to 20°C, and a sheet of cultured periodontal ligament cells was obtained by picking it up with tweezers (Figure 3 shows the appearance of cells before and after detachment from the temperature-responsive cell culture substrate in Example 1). <Comparative Example 1>

[0049] The same experiment as in Example 2 was performed, except that a conventional culture substrate, not a temperature-responsive substrate, was used as the cell culture substrate (Figure 2). After the culture was completed, cooling was attempted, but the periodontal ligament cell sheet of the present invention could not be peeled off the substrate surface. [Examples]

[0050] When cultured cells from Example 2 and Comparative Example 1 were analyzed by flow cytometry six days after the start of culture (Figure 4), the expression level of ALP, a marker for osteogenic differentiation, was the same in all three groups. Furthermore, the expression level of CD105, an undifferentiated cell marker, was also similar among the three groups. This indicates that periodontal ligament cells on a temperature-responsive cell culture substrate can be cultured and induced to differentiate into bone in the same way as cells cultured on a normal culture substrate (Comparative Example 1). It is considered that cells cultured on a temperature-responsive culture substrate do not undergo mutations as a result of being cultured on that substrate.

Claims

1. A periodontal ligament tissue regeneration cell sheet is a sheet-like culture made by culturing undifferentiated cells derived from periodontal ligament tissue, in which 85% or more of the total number of cells constituting the sheet are bone differentiation-inducing cells, the sheet does not have a ligament-like microstructure, and the sheet is strong enough to be picked up from the culture substrate after culturing.

2. The periodontal ligament tissue regeneration cell sheet according to claim 1, wherein undifferentiated cells derived from periodontal ligament tissue account for 60% or more of the total number of cells.

3. The periodontal ligament tissue regeneration cell sheet according to claim 1 or 2, wherein the undifferentiated cells are periodontal ligament-derived mesenchymal stem cells.

4. The periodontal ligament tissue regeneration cell sheet according to any one of claims 1 to 3, wherein the bone differentiation induction marker is an ALP expression level.

5. The periodontal ligament tissue regeneration cell sheet according to any one of claims 1 to 4, wherein the periodontal ligament tissue regeneration cell sheet is spontaneously layered.

6. The periodontal ligament tissue regeneration cell sheet according to any one of claims 1 to 5, wherein the periodontal ligament tissue regeneration cell sheet is artificially laminated.

7. The cell sheet for regenerating periodontal ligament tissue according to any one of claims 1 to 6, wherein the periodontal ligament tissue is from any of humans, rabbits, dogs, cats, pigs, horses, monkeys, chimpanzees, rats, mice, goats, and sheep.

8. The periodontal ligament tissue regeneration cell sheet according to any one of claims 1 to 7, which is used for dental treatment.

9. The cultured periodontal ligament cell sheet according to claim 8 , wherein the dental treatment is treatment of periodontal disease or periodontal ligament regeneration treatment.

10. The periodontal ligament tissue regeneration cell sheet according to any one of claims 8 to 9, wherein the treatment is allogeneic transplantation.

11. A process for harvesting periodontal ligament tissue-derived cells using scissors from periodontal ligament tissue, comprising the steps of: mincing the tissue harvested under clean conditions using scissors; then enzymatically treating the tissue; and culturing the cells on a specific substrate. Culturing the obtained cells on a temperature-responsive cell culture substrate having a surface coated with a polymer whose hydration strength changes in a temperature range of 0 to 80° C. in a temperature range where the hydration strength of the polymer is weak; A method for producing a cell sheet for regenerating periodontal ligament tissue, comprising a step of detaching the cultured cells in sheet form by changing the temperature of the culture medium to a level at which the hydration power of the polymer is strong.

12. The method for producing a cell sheet for regenerating periodontal ligament tissue according to claim 11, wherein the temperature-responsive cell culture substrate has a surface coated with a block polymer consisting of a temperature-responsive polymer and a hydrophobic polymer.

13. The coating amount of the temperature-responsive polymer on the surface of the temperature-responsive cell culture substrate is 0.3 to 8.0 μg / cm ２ The method for producing a periodontal ligament tissue regeneration cell sheet according to claim 11 or 12,

14. The method for producing a cell sheet for regenerating periodontal ligament tissue according to any one of claims 11 to 13, wherein the temperature responsive polymer is poly(N-isopropylacrylamide).

15. The method for producing a cell sheet for regenerating periodontal ligament tissue according to any one of claims 11 to 14, wherein the cell sheet is detached by adhering a carrier to the cultured cells at the end of the culture, and then detaching the cell sheet together with the carrier.

16. The method for producing a cell sheet for regenerating periodontal ligament tissue according to any one of claims 11 to 15, wherein the cell sheet for regenerating periodontal ligament tissue does not detach from the temperature-responsive substrate during the culture process.

17. The method for producing a cell sheet for periodontal ligament tissue regeneration according to any one of claims 11 to 16, wherein the cell sheet contains transfected cells.

18. The method for producing a cell sheet for regenerating periodontal ligament tissue according to any one of claims 11 to 17, wherein the cell sheet for regenerating periodontal ligament tissue is self-stratified when the cells constituting the cell sheet proliferate.

19. A method for producing a periodontal ligament tissue regeneration cell sheet described in any one of claims 11 to 17, further comprising a step of layering the cell sheet on another cell sheet, or layering the cell sheets by repeating that operation.

20. The method for producing a cell sheet for regenerating periodontal ligament tissue according to any one of claims 11 to 19, wherein the cell sheet is detached from the temperature-responsive cell culture substrate without treatment with a proteolytic enzyme.

21. The method for producing a periodontal ligament tissue regeneration cell sheet according to any one of claims 11 to 20, wherein the medium used for culture does not contain human serum.

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