Material for regenerating tissue, and method for producing the same
Patent Information
- Application Number
- JP2025011428
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-01-27
- Publication Date
- 2025-10-14
AI Technical Summary
Existing methods for isolating bone marrow-derived mesenchymal stem cells with a sufficient number of leptin receptor-positive cells are either costly, prone to infection, or require extensive processing, making them inefficient for clinical use.
A tissue regeneration material is developed by enzymatically treating bone marrow tissue with a buffer containing 0.5-1.5 mg/mL of collagenase and 0.5-1.5 mg/mL of dispase, allowing for the efficient separation of bone marrow-derived mesenchymal stem cells with a high number of leptin receptor-positive cells in a short period.
This method enables the rapid and cost-effective isolation of bone marrow-derived mesenchymal stem cells with a high number of leptin receptor-positive cells, suitable for regenerative medicine applications, including good cell engraftment and induction of bone and cartilage formation.
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Abstract
Description
[Technical field]
[0001] The present invention relates to a tissue regeneration material containing bone marrow-derived mesenchymal stem cells and a method for producing the same. [Background technology]
[0002] Mesenchymal stem cells exist in bone marrow tissue, adipose tissue, etc., and have the ability to differentiate into mesenchymal cells such as osteoblasts, chondrocytes, and muscle cells, and are therefore expected to be used in the field of regenerative medicine, etc. However, since the amount of mesenchymal stem cells contained in bone marrow fluid is insufficient, when used in clinical treatment, mesenchymal stem cells may be cultured and expanded.
[0003] For example, Patent Document 1 describes a method for isolating bone marrow-derived mesenchymal stem cells, in which cells contained in bone marrow fluid are cultured on a nonwoven fabric containing calcium phosphates at least on the culture surface. According to this method, it is said that specific stem cells can be easily and selectively isolated and cultured with a simple culture procedure. However, since it is necessary to culture the cells on the nonwoven fabric for at least several days to about a week, there is a risk of infection during that time and it is costly to maintain the culture, so further improvements were desired.
[0004] On the other hand, Patent Document 2 describes a method for isolating mesenchymal stem cells using bone, which is a bone tissue portion mainly composed of bone matrix and is tissue distinct from bone marrow. According to this method, it is said that mesenchymal stem cells can be efficiently isolated, and therefore a large amount of mesenchymal stem cells can be easily prepared. However, since the cell population of bone-derived mesenchymal stem cells obtained in Patent Document 2 is different from that of bone marrow-derived mesenchymal stem cells, leptin receptor-positive cells were not necessarily efficiently obtained from bone. In addition, when bone is used as a material, it is time-consuming to crush the bone tissue portion in a mortar or the like, so a simple and efficient method for isolating mesenchymal stem cells has been desired. [Prior art documents] [Patent documents]
[0005] [Patent Document 1] WO2018 / 097198 issue [Patent Document 2] JP 2015-39307 A Summary of the Invention [Problem to be solved by the invention]
[0006] The present invention has been made to solve the above-mentioned problems, and aims to provide a material for tissue regeneration that can obtain bone marrow-derived mesenchymal stem cells having a certain or higher number of leptin receptor-positive cells from bone marrow tissue through simple processing in a short period of time. [Means for solving the problem]
[0007] The above object is to provide a tissue regeneration material comprising bone marrow-derived mesenchymal stem cells isolated from bone marrow tissue, the number of leptin receptor-positive cells being 5.0×10 4 The above-mentioned problems are solved by providing a tissue regeneration material comprising bone marrow-derived mesenchymal stem cells.
[0008] At this time, the number of leptin receptor-positive cells per gram of bone was 96 × 10 4 / g or more, and the bone marrow-derived mesenchymal stem cells are obtained by enzymatically treating bone marrow tissue with a buffer solution containing 0.5 to 1.5 mg / mL collagenase and 0.5 to 1.5 mg / mL dispase.
[0009] The above-mentioned problems are also solved by providing a method for producing a tissue regeneration material comprising bone marrow-derived mesenchymal stem cells separated from bone marrow tissue, the method comprising enzymatically treating the bone marrow tissue with a buffer solution containing 0.5 to 1.5 mg / mL collagenase and 0.5 to 1.5 mg / mL dispase at 36°C or higher and 38°C or lower for 5 minutes or longer and shorter than 30 minutes.
[0010] The above-mentioned object is further to provide a tissue regeneration material kit comprising a powder (X) and a liquid (Y), in which the powder (X) containing collagenase and dispase is mixed with the liquid (Y) containing a buffer solution so that the concentrations of collagenase and dispase are 0.5 to 1.5 mg / mL, respectively, and bone marrow tissue is subjected to an enzyme treatment to thereby increase the number of leptin receptor-positive cells to 5.0 × 10 4 The above-mentioned problems are also solved by providing a tissue regeneration material kit comprising a powder material (X) and a liquid agent (Y), which is characterized by providing a tissue regeneration material containing bone marrow-derived mesenchymal stem cells. Effect of the Invention
[0011] According to the present invention, bone marrow-derived mesenchymal stem cells having a certain number of leptin receptor positive cells can be obtained from bone marrow tissues by a simple process in a short time. The tissue regeneration material containing bone marrow-derived mesenchymal stem cells obtained in this manner has good cell engraftment after transplantation and can induce bone and cartilage formation derived from bone marrow, making it particularly suitable for the field of regenerative medicine. [Brief description of the drawings]
[0012] [Figure 1] FIG. 1 shows the results of measuring the number of leptin receptor-positive cells while varying the concentrations of collagenase and dispase. [Diagram 2] FIG. 1 is a diagram showing the use of the tissue regeneration material of the present invention by intravenous administration to mice. [Diagram 3] FIG. 1 shows the results of HE staining (left) and immunohistochemical staining (right) of tissue sections obtained by implanting the tissue regeneration material of Comparative Example 1 into a mouse and inducing ectopic bone tissue subcutaneously in the back. [Figure 4] FIG. 1 shows the results of HE staining (left) and immunohistochemical staining (right) of tissue sections obtained by implanting the tissue regeneration material of Example 1 into a mouse and inducing ectopic bone tissue subcutaneously in the back. [Diagram 5]The tissue regeneration materials of Comparative Example 1 and Example 1 were each transplanted into mice, and ectopic bone tissue was induced subcutaneously in the back. The tissue sections obtained were subjected to immunohistochemical staining to compare the results (Comparative Example 1: left side, Example 1: right side). [Figure 6] FIG. 1 shows the results of double fluorescent immunostaining using monoclonal antibodies against GFP and RUNX2 for tissue sections obtained by implanting the tissue regeneration material of Example 1 into a mouse and inducing ectopic bone tissue subcutaneously in the back. [Figure 7] FIG. 1 shows the results of double fluorescent immunostaining using monoclonal antibodies against GFP and osteocalcine for tissue sections obtained by implanting the tissue regeneration material of Example 1 into a mouse and inducing ectopic bone tissue subcutaneously in the back. [Figure 8] The tissue regeneration materials of Comparative Example 1 and Example 1 were implanted into mice, respectively, to induce ectopic bone tissue subcutaneously in the back. The tissue sections obtained were then subjected to immunohistochemical staining to compare the results of observing the interstitium around the ectopic bone (Comparative Example 1: left side, Example 1: right side). [Figure 9] The tissue regeneration material of Example 1 was transplanted into a mouse, and ectopic bone tissue was induced subcutaneously in the back of the mouse. The tissue sections obtained were stained immunohistochemically to observe the interstitium around the ectopic bone (left side) and fluorescent double immunostaining to observe the interstitium around the ectopic bone (right side). [Figure 10] FIG. 1 shows the results of observing the interstitium around the ectopic bone in tissue sections obtained by implanting the tissue regeneration material of Example 1 into a mouse and inducing ectopic bone tissue subcutaneously in the back, by double fluorescent immunostaining using monoclonal antibodies against GFP and RUNX2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013] The tissue regeneration material of the present invention is a tissue regeneration material obtained by isolating bone marrow-derived mesenchymal stem cells from bone marrow tissue, and the number of leptin receptor-positive cells is 5.0×10 4 The present invention is characterized by containing bone marrow-derived mesenchymal stem cells as described above.
[0014] Leptin receptor positive cells are considered to be the most useful cells for differentiation into mesenchymal cells such as osteoblasts, chondrocytes, and muscle cells, but since the amount of mesenchymal stem cells contained in bone marrow fluid is insufficient, it has been difficult to obtain bone marrow-derived mesenchymal stem cells with a certain number of leptin receptor positive cells. As can be seen from the comparison between the Examples and Comparative Examples described below, if at least one of collagenase and dispase does not meet a specific concentration range, the number of leptin receptor positive cells does not reach a certain number, whereas the present inventors have clarified through their studies that bone marrow-derived mesenchymal stem cells with a certain number of leptin receptor positive cells can be obtained by using collagenase and dispase in a specific concentration range. Thus, it was first confirmed by the present inventors that the number of leptin receptor positive cells varies greatly depending on the conditions for enzyme treatment of bone marrow tissue.
[0015] The tissue regeneration material of the present invention has a leptin receptor positive cell count of 5.0 × 10 4 The tissue regeneration material of the present invention contains bone marrow-derived mesenchymal stem cells as described above, and can be suitably used for differentiating into cells belonging to the mesenchymal system, such as osteoblasts, chondrocytes, and muscle cells. In particular, since bone marrow-derived mesenchymal stem cells can be obtained from bone marrow tissue by simple processing in a short time, the tissue regeneration material of the present invention can be suitably used as a transplantation material. As is clear from the examples described below, transplantation of the tissue regeneration material of the present invention results in good cell engraftment after transplantation, and bone marrow-derived bone and cartilage formation can be induced, which is particularly suitable for the field of regenerative medicine, and the tissue regeneration material of the present invention can be suitably used as an osteo- and chondrogenesis agent.
[0016] The number of leptin receptor positive cells in the present invention is the number of cells measured by flow cytometry, and represents the number of cells in a tissue regeneration material prepared by measuring the total number of bone marrow cells in advance and preparing a constant number of total cells in 0.2 mL of buffer solution. Flow cytometry is a method of optically detecting a suspension containing fluorescently labeled cells by passing it through a thin tube and irradiating it with a laser light of a certain wavelength, and it is possible to quantitatively measure the number of leptin receptor positive cells. The device used for flow cytometry is a flow cytometer. Measurements can be performed using a flow cytometer sold by various companies and following the instruction manual. In addition, the total number of bone marrow cells can be quantitatively measured using a cell counter sold by various companies. The cell counter may be a flow cytometer used in the above-mentioned flow cytometry, a cell counter that measures the number of cells by measuring the change in electrical resistance when cells pass through an opening, or a cell counter that measures the number of cells by image processing a microscope image by autofocus. Measurements can be performed using these cell counters according to the instruction manual.
[0017] In the present invention, the number of leptin receptor positive cells is 5.0 × 10 4 In the case of bone marrow-derived mesenchymal stem cells showing less than 5.2 × 10, the amount is insufficient for engraftment in a living body and is not suitable for use as a tissue regeneration material. From this perspective, the number of leptin receptor positive cells in the tissue regeneration material of the present invention is 5.2 × 10 4 More than 5.5×10 is preferable. 4 More than 5.8×10 is preferable. 4 In the present invention, the number of leptin receptor positive cells is usually 9.9 × 10 4 The following is the result.
[0018] In addition, the tissue regeneration material of the present invention has a leptin receptor positive cell count of 96 × 10 per gram of bone. 4 It is preferable that the number of leptin receptor positive cells per gram of bone is 96×10 4In the case of bone marrow-derived mesenchymal stem cells with leptin receptor positive cells per gram of bone, the number of leptin receptor positive cells per gram of bone is less than 100 × 10 4 / g or more is more preferable, and 105×10 4 / g or more is more preferable, and 110×10 4 In the present invention, the number of leptin receptor positive cells per gram of bone is usually 190×10 4 The following is the result.
[0019] The tissue regeneration material of the present invention is preferably obtained by enzymatically treating bone marrow tissue with a buffer solution containing 0.5 to 1.5 mg / mL collagenase and 0.5 to 1.5 mg / mL dispase. The inventors' studies have revealed that bone marrow-derived mesenchymal stem cells having a certain or higher number of leptin receptor-positive cells can be obtained by using collagenase and dispase in a specific concentration range. As can be seen from the comparison between the examples and comparative examples described below, in Comparative Examples 1, 3, 4, and 5 in which both the collagenase concentration and the dispase concentration do not satisfy the above concentration range, Comparative Examples 2 and 6 in which the collagenase concentration satisfies the above concentration range but the dispase concentration does not satisfy the above concentration range, and Comparative Example 7 in which the dispase concentration satisfies the above concentration range but the collagenase concentration does not satisfy the above concentration range, the leptin receptor-positive cell count does not reach a certain level or higher, and the amount is insufficient for engraftment in a living body. In contrast, in Example 1, where collagenase and dispase are in the above concentration range, bone marrow-derived mesenchymal stem cells showing a certain or higher number of leptin receptor positive cells can be obtained, and therefore can be suitably used as a transplant material. The lower limit of collagenase concentration is preferably 0.6 mg / mL, more preferably 0.8 mg / mL, and even more preferably 0.9 mg / mL. Meanwhile, the upper limit of collagenase concentration is preferably 1.4 mg / mL, more preferably 1.3 mg / mL, and even more preferably 1.2 mg / mL. Also, the lower limit of dispase concentration is preferably 0.6 mg / mL, more preferably 0.8 mg / mL, and even more preferably 0.9 mg / mL. Meanwhile, the upper limit of dispase concentration is preferably 1.4 mg / mL, more preferably 1.3 mg / mL, and even more preferably 1.2 mg / mL.
[0020] The buffer solution containing the collagenase and the dispase is not particularly limited as long as it has an osmotic pressure equivalent to that of intracellular fluid, etc., and Hanks' balanced salt solution (HBSS), phosphate buffered saline (PBS), physiological saline, Ringer's solution, cell culture medium, etc. can be preferably used. Among them, Hanks' balanced salt solution (HBSS) and cell culture medium are more preferably used.
[0021] A preferred embodiment of the present invention is to enzymatically treat bone marrow tissue with a buffer solution containing collagenase and dispase at 36°C to 38°C for 5 minutes to 30 minutes. In this way, bone marrow-derived mesenchymal stem cells having a certain number of leptin receptor positive cells can be separated from bone marrow tissue by a simple and short treatment. In particular, the short time required for enzymatic treatment is advantageous in that tissue regeneration material can be obtained by enzymatic treatment in a medical setting and used as a transplant material on the spot. From this viewpoint, the time required for enzymatic treatment is more preferably 25 minutes or less, and even more preferably 20 minutes or less.
[0022] Bone marrow tissue can be preferably collected from the inside of a bone. For example, a method of collecting bone marrow tissue by cutting a bone and washing the inside of the bone with a buffer solution or the like can be mentioned. From the viewpoint of simpler operation, a method of collecting bone marrow tissue by washing the inside of the bone with a buffer solution containing the collagenase and dispase is preferably adopted. The type of bone is not particularly limited as long as it contains bone marrow tissue, and includes femur, tibia, fibula, jawbone, humerus, etc., among which femur or tibia is preferably used, and femur is more preferably used.
[0023] As explained above, in the present invention, bone marrow-derived mesenchymal stem cells can be obtained by simple processing in a short time, so that tissue regeneration material can be obtained by enzymatic processing in the medical field, and the tissue regeneration material can be used on the spot as a transplant material. From this viewpoint, a tissue regeneration material kit consisting of a powder material (X) and a liquid agent (Y) is provided, in which the powder material (X) containing collagenase and dispase and the liquid agent (Y) containing a buffer solution are mixed so that the concentrations of collagenase and dispase are 0.5 to 1.5 mg / mL, respectively, and the bone marrow tissue is subjected to enzymatic processing, thereby increasing the number of leptin receptor-positive cells to 5.0 x 10 4 One embodiment of the present invention is a tissue regeneration material kit comprising powder (X) and liquid (Y) characterized in that it provides a tissue regeneration material containing bone marrow-derived mesenchymal stem cells as described above. The tissue regeneration material kit comprises powder (X) and liquid (Y), and is characterized in that the powder (X) containing collagenase is mixed with liquid (Y) containing a buffer solution containing 0.5 to 1.5 mg / mL of dispase so that the collagenase concentration is 0.5 to 1.5 mg / mL, and the bone marrow tissue is subjected to an enzyme treatment, thereby increasing the number of leptin receptor-positive cells to 5.0 x 10 4 Another embodiment of the present invention is a tissue regeneration material kit comprising powder (X) and liquid (Y), characterized in that it provides a tissue regeneration material containing bone marrow-derived mesenchymal stem cells exhibiting the above properties. Furthermore, the tissue regeneration material kit comprising powder (X) and liquid (Y) is characterized in that the powder (X) containing dispase is mixed with liquid (Y) containing a buffer solution containing 0.5 to 1.5 mg / mL of collagenase so that the concentration of dispase is 0.5 to 1.5 mg / mL, and the bone marrow tissue is subjected to an enzyme treatment, thereby increasing the number of leptin receptor-positive cells to 5.0 x 10 4 One embodiment of the present invention is a tissue regeneration material kit comprising a powder material (X) and a liquid agent (Y), characterized in that it provides a tissue regeneration material containing bone marrow-derived mesenchymal stem cells as described above.
[0024] The tissue regeneration material of the present invention can be suitably used not only for humans but also for mammals other than humans. The tissue regeneration material of the present invention may be directly administered to the site where the tissue regeneration of bone, cartilage, blood vessels, fat cells, etc. is to be performed, or may be administered by intravenous injection. In particular, since bone marrow-derived mesenchymal stem cells can be obtained by a simple process in a short time, the tissue regeneration material may be obtained by enzymatic processing in the medical field and the tissue regeneration material may be administered on the spot, and the tissue regeneration material has the advantage that the tissue regeneration material can be administered without sterilization or reoperation. When administering, other medicines may be used in combination. For example, it is a suitable embodiment to administer the tissue regeneration material of the present invention while administering BMP-2 to the bone defect site. It is also a suitable embodiment to culture and administer the tissue regeneration material of the present invention, and it is also a suitable embodiment to differentiate and then administer the tissue regeneration material of the present invention. In the present invention, a tissue regeneration material containing bone marrow-derived mesenchymal stem cells showing a certain or higher number of leptin receptor positive cells can be obtained by a simple process in a short time, and therefore a wide range of applications can be achieved in regenerative medicine, experiments, etc. EXAMPLES
[0025] The present invention will now be described in more detail with reference to examples.
[0026] [reagent] (1)10×PBS(Phosphate Buffer Saline) A solution of 80 g of NaCl (1370 mM), 2 g of KCl (27 mM), 11.5 g of Na2HPO4 (81 mM), and 2 g of KH2PO4 (14.7 mM) was diluted with sterilized water to make 1 L. (2) 1×PBS The above (1) 10x PBS was diluted 10-fold with sterile water.
[0027] [Experimental animals] Seven-week-old female GFP transgenic mice (C57BL / 6-Tg(CAG-EGFP)) and seven-week-old female syngeneic wild-type mice (C57BL / 6J) were used.
[0028] [Total number of bone marrow cells] The number of cells in the collected bone marrow cells was counted using a cell counter (TC20 TM The cells were counted using a fully automated cell counter (BIO RAD). The measurement method is as follows. 10 μL was taken from the Digestion Buffer containing the bone marrow cells poured out in a petri dish, stained with the same amount of Trypan Blue (Trypan Blue #1450021, BIO RAD), and the number of cells in the 10 μL was immediately counted using the cell counter. Based on the information obtained from this, the total number of bone marrow cells was calculated.
[0029] [Number of leptin receptor positive cells] In the Examples and Comparative Examples, the bone marrow cells after the enzyme treatment were subjected to measurement of the number of leptin receptor-positive cells using a flow cytometer ("MACSQuant Analyzer 10" manufactured by Wakenyaku Co., Ltd.).
[0030] [Transplantation of tissue regeneration materials] Wild-type mice (C57BJ / 6) were irradiated with a total dose of 10 Gy of X-rays using an X-ray irradiator (Hitachi Medical Corporation, "MBR-1520R"), and the tissue regeneration material was administered via the tail vein at 0.5 mL per mouse using a 27G syringe.
[0031] [IBM (insoluble bone matrix) transplantation] The tissue regeneration materials of Example 1 and Comparative Example 1 described below were transplanted into mice according to the method described in the above "Transplantation of tissue regeneration materials", and a bone marrow engraftment period of 28 days was allowed. Then, 150 mg of IBM (Insoluble Bone Matrix) and 10 μg of BMP-2 (manufactured by PEPRO TECH) were transplanted subcutaneously into the back of the mouse to forcibly induce ectopic bone tissue in the back subcutaneous region. The IBM used was made by crushing the femur and tibia of a dehydrated rat with a grinder and decalcifying it with 0.5 M HCl. 28 days after the transplantation into the back subcutaneous region, the mouse was sacrificed, and the ectopic bone tissue in the back subcutaneous region was extracted, fixed in formalin, decalcified with EDTA, and then tissue sections were prepared according to the usual method.
[0032] [Hematoxylin (HE) staining] The tissue sections obtained above were deparaffinized with xylene, rehydrated using 100% to 70% ethanol (in the order of 100%, 90%, 80%, 70%) and purified water, and then stained with HE. After dehydration and clearing using 70% to 100% ethanol (in the order of 70%, 80%, 90%, 100%) and xylene, the sections were mounted with Entellan (Millipore Corporation) and histologically observed using a system biological microscope (Olympus "BX53").
[0033] [Immunohistochemical staining] After deparaffinization of the obtained tissue sections, endogenous peroxidase was blocked with 0.3% hydrogen peroxide methanol solution for 30 minutes at room temperature (25°C) and washed with purified water. Monoclonal antibodies against GFP (abcam), RUNX2 (abcam), and Osteocalcine (abcam) were used as antibodies, and observations were performed using a system biological microscope (Olympus "BX53"). Negative controls were performed using only secondary antibodies, and all were negative.
[0034] [Fluorescent immunostaining] Double fluorescent immunostaining was performed using monoclonal antibodies against GFP (Abcam), RUNX2 (Abcam), and Osteocalcine (Abcam). Each antibody was diluted in TBS (Tris Buffered Saline). After the secondary antibody reaction, the cells were counterstained with 1 μg / ml DAPI (4',6-diamidino-2-phenylindole) for 3 minutes. After washing, the cells were mounted in Fluorescence mounting medium (Dako) and observed under an all-in-one fluorescence microscope (Keyence BZ700).
[0035] Example 1 [Digestion Buffer] This was prepared by dissolving 10 mg of Collagenase Type IV (Life Technologies, 17104-019) and 10 mg of Dispase (Life Technologies, 17105-041) in 10 mL of HBSS (Thermo Fisher Science, 14175-095).
[0036] [Materials for tissue regeneration] A GFP transgenic mouse (approximately 18.9 g) was euthanized, and the femur and tibia were collected. Each femur weighed approximately 0.052 g. Both ends of the femur were cut with a blade and placed in a petri dish. 4 mL of Digestion Buffer was poured into both sides of the bone using a 1 mL syringe (21 G needle) to pour the bone marrow tissue onto the petri dish. The total number of bone marrow cells was calculated as described above and found to be 1.0 × 10 7 The cells were collected in a 15 mL tube (A) after the Digestion Buffer was removed from the petri dish. The bone marrow tissue remaining on the petri dish was washed with 2 mL of new Digestion Buffer and collected in tube (A). Tube (A) was left in a 37°C water bath for 10 minutes to carry out enzyme treatment. Tube (A) was mixed by inversion and left on ice for 2 minutes. The remaining bone marrow tissue was precipitated, and the supernatant was placed in a new 15 mL tube (B) and stored on ice. 4 mL of new Digestion Buffer was added to tube (A) and tube (A) was left in a 37°C water bath for 10 minutes to carry out enzyme treatment. Tube (A) was mixed by inversion, and tubes (A) and (B) were centrifuged at 1500 rpm for 5 minutes. The supernatant was removed using an aspirator, and 1 mL of Cell Lysis Buffer (NH4Cl (150 mM), NaHCO3 (10 mM), EDTA2Na (1 mM)) was added to each of tubes (A) and (B). After suspension by pipetting, the contents of tubes (A) and (B) were combined into tube (A). After 1 minute, 5 mL of 2% FBS (Thermo Fisher Science, Fetal Bovine Serum) / PBS was added and suspended, followed by centrifugation at 1500 rpm for 5 minutes. The supernatant was removed using an aspirator, diluted with 0.1 M PBS / HBSS, and approximately 1.0 × 107 The number of leptin receptor positive cells per femur (approximately 0.052 g) was 6.0 × 10 4 That is, the number of leptin receptor positive cells per gram of femur was 115.4 × 10 4 The results are summarized in Table 1. The results of histological analysis of tissue sections obtained by inducing ectopic bone tissue subcutaneously in the back of mice are shown in Figures 4, 5 (right side), 6, 7, 8 (right side), 9, and 10, respectively.
[0037] Comparative Example 1 A tissue regeneration material was prepared in the same manner as in Example 1, except that 10 mL of HBSS (Thermo Fisher Science, 14175-095) was used instead of the Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 0.3 × 10 4 That is, the number of leptin receptor positive cells per gram of femur was 5.8 × 10 4 / g. The results are summarized in Table 1. The results of histological analysis of tissue sections obtained by inducing ectopic bone tissue subcutaneously in the back of mice are shown in Figures 3, 5 (left side), and 8 (left side), respectively.
[0038] Comparative Example 2 A tissue regeneration material was prepared in the same manner as in Example 1, except that only 10 mg of Collagenase Type IV (Life Technologies, 17104-019) was dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 1.0 × 10 4 That is, the number of leptin receptor positive cells per gram of femur was 19.2 × 10 4 The results are summarized in Table 1.
[0039] Comparative Example 3 A tissue regeneration material was prepared in the same manner as in Example 1, except that only 20 mg of Dispase (Life Technologies, 17105-041) was dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 2.4 × 10 4 That is, the number of leptin receptor positive cells per gram of femur was 46.2 × 10 4 The results are summarized in Table 1.
[0040] Comparative Example 4 A tissue regeneration material was prepared in the same manner as in Example 1, except that 1 mg of Collagenase Type IV (Life Technologies, 17104-019) and 2 mg of Dispase (Life Technologies, 17105-041) were dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 4.6 × 10 4 In other words, the number of leptin receptor positive cells per gram of femur was 88.5 × 10 4 The results are summarized in Table 1.
[0041] Comparative Example 5 A tissue regeneration material was prepared in the same manner as in Example 1, except that 2 mg of Collagenase Type IV (Life Technologies, 17104-019) and 1 mg of Dispase (Life Technologies, 17105-041) were dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 1.2 × 10 4 In other words, the number of leptin receptor positive cells per gram of femur was 23.1 × 10 4The results are summarized in Table 1.
[0042] Comparative Example 6 A tissue regeneration material was prepared in the same manner as in Example 1, except that 10 mg of Collagenase Type IV (Life Technologies, 17104-019) and 20 mg of Dispase (Life Technologies, 17105-041) were dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 3.8 × 10 4 That is, the number of leptin receptor positive cells per gram of femur was 73.1 × 10 4 The results are summarized in Table 1.
[0043] Comparative Example 7 A tissue regeneration material was prepared in the same manner as in Example 1, except that 20 mg of Collagenase Type IV (Life Technologies, 17104-019) and 10 mg of Dispase (Life Technologies, 17105-041) were dissolved in 10 mL of HBSS (Thermo Fisher Science, 14175-095) to prepare a Digestion Buffer. The number of leptin receptor-positive cells per femur (approximately 0.052 g) was 3.6 × 10 4 In other words, the number of leptin receptor positive cells per gram of femur was 69.2 × 10 4 The results are summarized in Table 1.
[0044] [Table 1]
[0045] From the results of Figures 3 to 5 comparing Comparative Example 1 and Example 1, it was confirmed that all of the osteoblasts, chondrocytes, and bone cells were GFP negative in Comparative Example 1, and GFP-positive bone marrow-derived osteoblasts, chondrocytes, and bone cells were confirmed in the ectopic bone and ectopic cartilage in Example 1, and the engraftment of the cells after transplantation was good. In addition, GFP-positive inflammatory cells were confirmed in both Comparative Example 1 and Example 1. In addition, from the results of Figure 6, the expression of RUNX2 was confirmed in the GFP-positive bone marrow-derived cells, and from the results of Figure 7, the expression of Osteocalcine was confirmed in the GFP-positive bone marrow-derived cells, and it was confirmed that the GFP-positive cells in Example 1 were bone marrow-derived bone cells and chondrocytes. In addition, from the results of Figure 8 in which the interstitium around the ectopic bone was observed, it was confirmed that the interstitial cells were GFP negative in Comparative Example 1, but that many GFP-positive cells were present in the interstitial cells around the ectopic bone in Example 1. 9 and 10, RUNX2 expression was confirmed in GFP-positive bone marrow-derived cells present in the stroma, and GFP-positive cells in the stroma surrounding the ectopic bone were confirmed to be RUNX2-positive bone precursor cells derived from bone marrow. From the above, it can be seen that transplantation of tissue regeneration material containing bone marrow-derived mesenchymal stem cells with a certain number of leptin receptor-positive cells or more results in good cell engraftment after transplantation and enables induction of bone and cartilage formation derived from bone marrow.
Claims
1. A tissue regeneration material obtained by separating bone marrow-derived mesenchymal stem cells from bone marrow tissue, The number of leptin receptor-positive cells was 5.0 × 10 ４ A tissue regeneration material characterized in that it contains bone marrow-derived mesenchymal stem cells having a cell count of 100 or more, and the tissue regeneration material obtained by enzyme treatment is transplanted in situ as a transplant material and used to allow the cells to engraft after transplantation.
2. The number of leptin receptor-positive cells per gram of bone was 96 x 10 ４ The tissue regeneration material according to claim 1, comprising bone marrow-derived mesenchymal stem cells having a cell density of 1000 cells / g or more.
3. 3. The tissue regeneration material according to claim 1, wherein the bone marrow-derived mesenchymal stem cells are obtained by enzymatically treating bone marrow tissue with a buffer solution containing 0.5 to 1.5 mg / mL of collagenase and 0.5 to 1.5 mg / mL of dispase.
4. A bone / cartilage forming agent comprising the tissue regeneration material described in any one of claims 1 to 3.
5. A method for producing a tissue regeneration material obtained by separating bone marrow-derived mesenchymal stem cells from bone marrow tissue, comprising: The method for producing a tissue regeneration material according to any one of claims 1 to 3, wherein bone marrow tissue is subjected to an enzyme treatment using a buffer solution containing 0.5 to 1.5 mg / mL of collagenase and 0.5 to 1.5 mg / mL of dispase at 36°C or higher and 38°C or lower for 5 minutes or longer and shorter than 30 minutes.