Bone substitute material and its manufacturing method

Abstract

In the present invention, stem cells are induced to differentiate into osteoblasts by three-dimensional culturing and cell masses are formed to produce a bone substitute. By employing lyophilization, it is possible to reduce the risk of tumor formation and, additionally, to control immunogenicity and local inflammation. By optimizing the temperature and time of an initial freezing step, a primary drying step, and a secondary drying step, it is possible to provide a bone substitute achieving good performance and having reduced cost. It is possible to provide a bone substitute having high safety and higher osteoconductivity and osteoinductivity.

Description

[Technical Field]

[0001] The present invention relates to a bone substitute material used in surgically treating damaged bone tissue, and a method for producing the same. [Background technology]

[0002] Bone tissue has multiple important functions, including protecting the brain and internal organs, supporting the skeleton, producing blood, and storing electrolytes such as calcium. Bone tissue can be damaged by trauma, surgical removal of bone tumors, infection, osteomyelitis, and other factors. When the damage is mild, osteoblasts proliferate and differentiate, resulting in bone regeneration and healing. However, when the defect is large or the environment prevents osteoblasts from functioning effectively, treatment using bone transplants may be necessary. As the population ages, the number of elderly people with low bone density is increasing, and the market for bone grafts and artificial bone graft materials is expanding year by year. This has led to the development of bone substitute materials.

[0003] Bone substitute materials can be broadly divided into autogenous bone, allogeneic bone, xenogeneic bone, and artificial bone graft materials. Autogenous bone grafts are considered the gold standard for large bone defects, but they have drawbacks such as donor site invasiveness, limited supply, and infection risk. Freeze-dried bone grafts and demineralized freeze-dried bone grafts have been reported to have relatively good osteoinductivity, but they are limited in supply and may pose unknown risks of viral and bacterial infection. Furthermore, artificial bone substitute materials have also been identified with the following problems. For example, β-tricalcium phosphate (β-TCP) is rapidly absorbed in vivo, preventing sufficient bone mass. In contrast, hydroxyapatite and carbonate apatite are less resorbable and are useful for creating space for bone regeneration, but they remain for long periods of time, increasing the risk of increased infection and fragility.

[0004] In the field of dentistry, these drawbacks have been addressed by combining autologous bone and bone substitutes. However, it has been reported that approximately 25% of patients who underwent maxillary sinus floor augmentation using a combination of autologous and artificial bone experienced bone resorption one year after surgery, and that bone height decreased by 25% six months after autologous bone grafting using the onlay graft method, indicating that bone regeneration is insufficient. Therefore, the development of new bone substitute materials that can create a microenvironment that promotes bone remodeling is awaited.

[0005] Induced pluripotent stem cells (iPSCs) can be generated by introducing specific factors into somatic cells. They exhibit excellent proliferation and pluripotency, making them promising candidates for regenerative medicine. In recent years, iPSCs have been used to artificially generate three-dimensional (3D) tissues (organoids) resembling in vivo organs. Organoids are generated through the self-aggregation and self-organization of stem cells, mimicking the developmental process of in vivo organs. Because the structure and function of organoids resemble those of in vivo organs, they have been applied to basic research in embryology and physiology, as well as pathological elucidation, drug discovery research, and transplantation for regenerative medicine. The inventors have developed a method for 3D culturing iPSCs in a bioreactor, inducing bone differentiation, and generating osteoblastic masses from human iPSCs (Patent Document 1). While iPSC-derived spheroids and organoids are promising biomaterials, transplanting undifferentiated living cells carries the risk of tumorigenesis. Furthermore, since the implanted material is a foreign substance to the body, it is also necessary to control immunogenicity and local inflammation.

[0006] Recently, a new concept in regenerative medicine has been proposed: inactivating cells while preserving the tissue components of tissues created in vitro from stem cells (Non-Patent Document 1). Using this method, tissues can be stored long-term in a "seed" state and then transplanted into patients to induce tissue regeneration, thereby providing the required amount of tissue when needed. Furthermore, inactivating undifferentiated iPSCs may potentially reduce the risk of tumor formation and control inflammation. Inactivating cells induced from stem cells may potentially enable the creation of bone substitutes with excellent osteoconductivity and osteogenic induction, but this has not yet been put to practical use. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 181960 [Patent Document 2] International Publication No. 2015 / 064705 [Patent Document 3] International Publication No. 2020 / 175592 [Non-patent literature]

[0008] [Non-Patent Document 1] S. Pigeot, etal., Advanced Materials, 2021, 33, 2103737, https: / / doi.org / 10.1002 / adma.202103737. [Non-patent document 2] H. Egusa,et al., Stem Cells Dev,2014, 23(18),2156-69. [Non-patent document 3] H. Egusa, et al., Plos One, 2010,5(9) :e12743. doi: 10.1371 / journal.pone.0012743. Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have developed a bone prosthesis material with excellent osteoconductivity and osteogenic induction by inactivating cells while retaining their activity in promoting bone remodeling. While methods such as radiation, heat treatment, and drug treatment are commonly used to inactivate cells, these methods may denature proteins. Therefore, we considered freeze-drying, which can inactivate cells while maintaining the physiological activity and structure of proteins. Compared to conventional drying methods, freeze-drying dries materials at low temperatures, suppressing thermal denaturation and chemical changes in the material. It also reduces the rate of product deterioration during the drying process, allowing for long-term storage at room temperature. However, freeze-drying conditions may potentially cause chemical changes such as oxidation, deamidation, and hydrolysis, as well as aggregation due to changes in the higher-order structure of proteins. These chemical and physical changes during the manufacturing process may reduce the physiological activity of bone prosthesis materials intended for implantation in vivo and may also act as antigens. The present inventors have already produced a bone regenerating agent by freeze-drying iPS cells induced to differentiate into osteoblasts (Patent Document 2). Specifically, iPS cell masses induced to differentiate into osteoblasts were pre-frozen overnight in a -80°C freezer, then freeze-dried on a cooling stage at a fixed temperature of -10°C while gradually reducing the air pressure to 6-20 Pa overnight, producing a bone prosthesis without secondary drying. However, because a shelf-type freeze dryer, which can precisely control the freeze-drying temperature by program and has excellent drying efficiency, was not used, it cannot be said that the temperature and time control of each process and the drying of the sample were sufficient. By improving and controlling the manufacturing method, it may be possible to produce bone prosthesis with even greater bone formation induction and anti-inflammatory properties.

[0010] Bone tissue is a composite material consisting of cells, organic matrix, and inorganic matrix. Various biomaterials have been used to treat bone defects, but these biomaterials have certain limitations in their osteogenic induction ability and production efficiency. The present invention aims to provide a bone substitute material that is highly safe and has higher osteoconductivity and osteogenic induction ability. Another objective of the present invention is to provide a method for producing a bone substitute material that can be supplied stably, has high production efficiency, and is low-cost. [Means for solving the problem]

[0011] The present invention relates to the following bone prosthesis and method for producing the bone prosthesis. (1) A method for producing a bone filler derived from stem cells, characterized by comprising a step of inducing differentiation from stem cells into osteoblasts to form cell masses, a pre-freezing step, and a primary drying step of freeze-drying at a temperature of -80°C or higher and -10°C or lower. It was found that precisely adjusting the temperature during the freeze-drying process to produce bone prosthesis materials leads to their performance. In particular, it was found that controlling the shelf temperature during the primary drying process to freeze the materials at a constant temperature in the drying chamber leads to improved performance of the bone prosthesis materials.

[0012] (2) The method for producing a bone filler according to (1), further comprising a secondary drying step. Conventionally, a secondary drying step was not performed, but by performing the secondary drying step to desorb unfrozen water, a high-performance bone prosthesis can be produced. [Brief explanation of the drawings]

[0013] [Figure 1A] A diagram showing the freeze-drying process of cell aggregates produced from iPS cells. [Figure 1B] FIG. 1 is a diagram showing a schematic configuration of the tray-type freeze dryer used. [Figure 2A] Study of pre-freezing conditions (1). Study of freezing temperature and time. [Figure 2B] Study of pre-freezing conditions (2). Study of stabilizers. [Figure 3A] Study of primary drying conditions (1). A diagram showing the results of a study of drying shelf temperature conditions during freeze-drying. [Figure 3B] Study on primary drying conditions (2). A diagram showing the results of the study, including the drying shelf temperature conditions during freeze-drying, at -80°C. [Figure 3C] Study of primary drying conditions (3). Graph showing the expression of osteogenic differentiation marker genes depending on the drying shelf temperature conditions during freeze-drying. [Figure 3D] Study of primary drying conditions (4). A diagram showing the results of a study on freeze-drying time. [Figure 4] FIG. 10 is a diagram showing the results of a study on secondary drying time. [Figure 5A] Figure showing the evaluation results of implantation into a rat femoral defect model. The evaluation results of miPS-BGM (mouse iPS-bone graft material) after primary drying at various temperatures are shown. 3D micro-CT image (top) and cross-sectional image of the midline of the defect (bottom). The arrow indicates the edge of the bone defect. Scale bar: 1.0 mm. [Figure 5B] Figure 1 shows the results of quantitative analysis of bone parameters using 3D micro-CT images. Bone volume (BV / TV) and bone mineral content (BMC) were analyzed at the defect site (mean ± SD, n = 3–7). [Figure 6A] The images show hematoxylin and eosin (HE) staining (top) and immunohistochemical staining with anti-galectin-3 antibody of the femoral defect site 3 weeks after miPS-BGM implantation. The arrow indicates the edge of the defect. Scale bar: 1.0 mm. [Figure 6B] The upper row shows an enlargement of the dotted-framed area in the HE-stained image in the upper row of Figure 6A. The lower row shows an enlargement of the dotted-framed area in the immunohistochemical stained image in the lower row of Figure 6A. * indicates remaining miPS-BGM, and arrows indicate neutrophils. Scale bar: 100 μm. The insets are enlargements of the boxed areas in each panel. Scale bar in inset: 10 μm. [Figure 6C] The area of ​​remaining miPS-BGM is shown (mean ± SD, n = 12–14). [Figure 7] Figure showing the effect of miPS-BGM on macrophage activity. Photographs show microscopic images of macrophages cultured in growth medium (GM) or growth medium supplemented with miPS-BGM after 1 day; scale bar: 200 μm. Graph shows the expression of inflammation- and anti-inflammatory-related genes after 1 or 3 days of culture of macrophages in growth medium or growth medium containing miPS-BGM. [Figure 8A] A graph showing the total amount of protein released from miPS-BGM prepared at primary drying temperatures of -40°C, -10°C, and 30°C. [Figure 8B]Fluorescence image of F-actin in human bone marrow-derived mesenchymal stem cells (hMSCs) cultured in the presence of miPS-BGM. The dotted circle indicates the location of miPS-BGM. Scale bar: 100 μm. [Figure 8C] The number of hMSC cells surrounding miPS-BGM (mean ± SD, n = 3). [Figure 9] Figure 1 shows the results of the wound healing assay. Phase-contrast microscopic images of hMSCs immediately after scratch formation (0 h) and after 9 h of culture (top row, scale bar: 200 μm), and a graph showing the number of migrated cells within the scratch area after 9 h (mean ± SD, n = 3) (bottom row). [Figure 10] Evaluation of miPS-BGM using a cytokine antibody array. miPS-BGM produced at a primary drying temperature of -40°C was compared with β-TCP as a control. The x-axis shows the fluorescent signal intensity of the 308 target proteins, and the y-axis shows the fluorescent signal intensity ratio (miPS-BGM / β-TCP intensity). [Figure 11] Figure showing the results of proteome analysis of miPS-BGM. Venn diagram showing the number of proteins identified as common or specific between the miPS cell clumps before freeze-drying and miPS-BGM. [Figure 12A] Macrophotographs of miPS-BGM with different primary drying temperatures. [Figure 12B] A diagram showing the results of compression tests on miPS-BGM with different primary drying temperatures. [Figure 12C] SEM images of miPS-BGMs dried at different temperatures. [Figure 12D] SEM image of miPS-BGM produced using a cooling stage freeze dryer. [Figure 12E] Figure showing the results of elemental composition analysis of miPS-BGM produced using a tray-type freeze dryer. [Figure 12F] Figure 1 shows the results of elemental composition analysis of miPS-BGM prepared using a cooling stage freeze-dryer and non-decalcified freeze-dried human bone. [Figure 12G] FIG. 1 shows the results of measuring the amount of calcium elution from miPS-BGM at different primary drying temperatures. [Figure 13A]XRD patterns of miPS-BGMs dried at different primary drying temperatures. [Figure 13B] TEM / STEM images of miPS-BGM with different primary drying temperatures. Scale: left panel: 200 nm, right panel: 100 nm. [Figure 13C] A diagram showing the length of fibrous apatite in miPS-BGM measured from a TEM image. [Figure 14] Evaluation of human iPS-BGM using a cytokine antibody array. Human freeze-dried bone marrow (FDBA) was used as a control for comparison. [Figure 15] Figure 1 shows the results of ALP staining analysis of the bone differentiation induction ability of human iPS-BGM at different primary drying temperatures. [Figure 16] Figure 1 shows the results of a wound healing assay. The upper panel shows phase-contrast microscopic images of hMSCs cultured for 6 hours after scratch formation, and the lower panel shows a graph showing the number of migrated cells in the scratch area after 6 hours. DETAILED DESCRIPTION OF THE INVENTION

[0014] Bone fillers refer to materials other than autologous bone that are used for the purpose of bone regeneration, and can be used interchangeably with artificial bone, bone substitutes, bone regenerating agents, etc. The bone filler of the present invention is made from stem cells and has osteoconductive and osteogenic inductive properties. Osteoconductive properties refer to the ability of a medical material, such as an implant, to form bone along the surface of the material when embedded in natural bone in a living body, bonding the material to the bone and forming an integrated unit. Osteogenic inductive properties refer to the ability to promote the differentiation of mesenchymal stem cells, such as mesenchymal stem cells, into osteoblasts and other cells to form new bone at sites of bone loss or degeneration due to disease or injury.

[0015] The stem cells used in producing the bone filler of the present invention are not particularly limited as long as they can be induced to become osteoblasts, and examples thereof include pluripotent stem cells and somatic stem cells. Examples of pluripotent stem cells include iPS cells (induced pluripotent stem cells), EG cells (embryonic germ cells), and ES cells (embryonic stem cells). Examples of somatic stem cells include mesenchymal stem cells, skeletal stem cells, hematopoietic stem cells, and neural stem cells, but mesenchymal stem cells and skeletal stem cells are preferred because they differentiate into osteoblasts.

[0016] As mentioned above, even freeze-drying can cause chemical changes such as oxidation, deamidation, and hydrolysis, as well as aggregation due to changes in the higher-order structure of proteins, depending on the conditions. Therefore, optimizing the freeze-drying conditions can lead to the production of high-performance bone substitutes. The production of regenerative medicine products for therapeutic use requires ensuring product quality, and optimizing the freeze-drying process to ensure consistent quality is essential. Furthermore, because the freeze-drying process requires a long drying time and consumes a large amount of power, optimizing the freeze-drying cycle is crucial to maintaining high quality while increasing production efficiency. As shown in the following examples, the inventors optimized the freeze-drying process, particularly the primary drying conditions, and completed the present invention. By optimizing the primary drying conditions, we were able to improve the bone replacement properties of bone graft materials derived from miPS cells (mouse iPS cells) and hiPS cells (human iPSCs) in vivo.

[0017] The present invention will be explained below with reference to data. In the following experiments, iPS cells derived from mouse gingival fibroblasts and osteoblasts derived from human iPS cells are used, but any stem cells may be used. It goes without saying that bone filler can be produced in a similar manner regardless of animal species. Furthermore, iPS cells are not limited to gingival fibroblasts; cells established from various cells, such as blood cells, skin fibroblasts, and dental pulp cells, can also be used.

[0018] 1. iPS cell culture Stem cells can be induced to become bone cells and cell masses can be produced by 3D culture using known methods, including but not limited to the following methods. The following experiments were performed using mouse gingival fibroblast-derived iPS cells (miPSCs; Non-Patent Documents 2 and 3) generated by retroviral transduction of Oct3 / 4, Sox2, and Klf4. miPSCs were grown on inactivated SNLP76.7-4 (provided by Dr. Allan Bradley of the Stanger Institute, UK) feeder cells in ES medium (Dulbecco's Modified Eagle's Medium (DMEM, Nacalai Tesque), 15% FBS (Biosera), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 x 10 -4 M non-essential amino acids (Thermo Fisher Scientific), 1 × 10 -4 The miPSCs were cultured in a 0.25 w / v% trypsin-1 mM EDTA solution containing 2-mercaptoethanol (Thermo Fisher Scientific), penicillin (50 U), and streptomycin (50 μg / mL) (Fujifilm Wako Pure Chemical Industries). The miPSCs were passaged every 5–6 days using a 0.25 w / v% trypsin-1 mM EDTA solution. Embryoid bodies (EMBs) were formed in ES medium using low-adhesion culture dishes for 2 days. The miPSCs were then cultured in suspension in ES medium supplemented with 1 μM all-trans retinoic acid for an additional 2 days (Non-Patent Document 2).

[0019] SNLP76.7-4 cells used as feeder cells were cultured in Dulbecco's modified Eagle's medium (DMEM, glucose 4.5 g / L), 7% fetal bovine serum (Japan Bioserum), 2 mM L-glutamine, and penicillin (50 U) / streptomycin (50 μg / mL). When the cells reached 90% confluence, they were treated with 12 μg / mL mitomycin C (Fujifilm Wako Pure Chemical Industries) at 37°C for 2.5 hours to inhibit DNA replication and inactivate the cells. They were then washed twice with physiological saline (PBS) and used as feeder cells.

[0020] 2.Cell cluster formation The embryoid bodies were cultured in 70 mL flasks in osteoblast differentiation medium (α-modified medium (Nacalai Tesque), 15% FBS (Gibco), 0.01 μM dexamethasone (Merck), 10 mM β-glycerophosphate (Merck), 50 μg / mL ascorbic acid-2-phosphate (Merck), 1% antibiotic-antimycotic solution (Thermo Fisher Scientific)) to induce osteoblast differentiation. The flasks were placed on a seesaw shaker and cultured at 0.3 Hz for 30 days. The osteoblast differentiation medium was changed every 2 days.

[0021] 3.Lyophilization As mentioned above, we believe that the freeze-drying conditions are crucial for producing high-performance bone prosthesis materials. Therefore, we investigated the freeze-drying conditions in detail. Freeze-drying consists of three stages: initial freezing (formation of ice crystals), primary drying (sublimation of ice crystals), and secondary drying (desorption of unfrozen water) (Figure 1A). A typical shelf-type freeze dryer consists of a drying chamber, a condenser (cold trap), a pressure-reducing device (vacuum pump), and a temperature control device (Figure 1B). A shelf-type freeze dryer has temperature-controllable shelves inside the chamber to cool and heat the product during the drying process. By controlling the temperature of the drying shelves that come into contact with the material to be dried, the temperature inside the chamber can be precisely controlled, achieving excellent drying efficiency. Because precise temperature control during drying is considered important, we used a shelf-type freeze dryer (EYELA DRC-1100, Tokyo Rikakikai) for freeze-drying. However, any freeze dryer can be used as long as it can accurately control the sample temperature during drying. Examples of such dryers include a closed tube freeze-drying system, a radiant heating vacuum freeze-drying device, etc. Note that the shelf temperature adjustment range of this dryer is from -40°C to 30°C, so a liquid nitrogen vacuum freeze-drying device (NRL-BC02S, Taiyo Nippon Sanso) was used only in the study where the primary drying temperature was set to -80°C.

[0022] After 30 days of osteoblast differentiation induction, the miPS cell aggregates were washed with PBS and transferred to glass vials (19 mm diameter, 48 mm length, Nichiden Rika Glass Co., Ltd.). They were stored in a freezer at -80°C or -30°C until freeze-drying, and then rapidly transferred to a freeze-dryer for freeze-drying. Table 1 summarizes the freeze-drying conditions tested. The shelf temperature was increased at a rate of 1.5°C / min between the primary and secondary drying steps. After secondary drying, the chamber was purged with nitrogen gas to prevent deterioration of the freeze-dried miPS-BGM (miPS-bone graft material; hereafter, freeze-dried miPS will be referred to as miPS-BGM). The miPS-BGM was stored in an auto-dry desiccator (AS ONE Corporation) at room temperature (23°C) and humidity below 15%.

[0023] [Table 1]

[0024] 3.1 Consideration of initial freezing conditions Prefreezing is a process in which the sample is cooled below the eutectic point, which is the temperature at which the entire sample freezes. This process is crucial for ensuring the porosity of the dried sample by forming and growing ice crystals. If freezing is insufficient, the water may rapidly expand as the vacuum level increases during the subsequent primary drying process, leading to sample boiling. Because the conditions of the prefreezing process affect the efficiency of the subsequent drying process, we investigated the conditions. Furthermore, we investigated whether the use of various stabilizers, such as sugars and sugar alcohols, in the initial freezing process could reduce damage to proteins and maintain their stability during freezing and primary drying.

[0025] Extracts of miPS-BGM prepared under each condition in Table 1A were added to osteoblast differentiation medium, and human bone marrow-derived mesenchymal stem cells (hMSCs, provided by Dr. Mabuchi, Tokyo Medical and Dental University, and Dr. Matsuzaki, Shimane University) were cultured in this medium. The osteogenic induction potential of miPS-BGM was evaluated based on the degree of differentiation. hMSCs were cultured in MSC growth medium (DMEM (pyruvate-free, 4.5 g / L glucose, Nacalai Tesque), 20% FBS (GE Healthcare), 1% penicillin (50 U) / streptomycin (50 μg / mL), 10 mM HEPES (Dojindo Laboratories), and 10 ng / mL recombinant b-FGF (Fujifilm Wako Pure Chemical Industries). hMSCs were cultured in 10 cm culture dishes at a cell density of 3 × 10 4 The cells were cultured at 4 × 10 cells / mL at 37°C and 5% CO2. The growth medium was changed every 3–4 days. When the hMSCs reached 80% confluence, they were harvested by trypsinization and cultured at a cell density of 4 × 10 cells / mL. 5 The cells were seeded at 1000 cells / mL onto a 24-well plate, and after 24 hours, the proliferation medium was replaced with osteoblast differentiation medium (Lonza) and cultured.

[0026] miPS-BGM was suspended in osteoblast differentiation medium at a concentration of 5 mg / mL and pulverized on ice using an ultrasonicator (BRANSON Digital Sonifier SFX150, EMERSON) at 20% ultrasonic amplitude for 2 minutes. After sonication, the suspension was filtered through a syringe filter (0.22 μm pore size, Merck) and added to osteoblast differentiation medium for hMSCs. The osteoblast differentiation medium was changed every 3 days. Seven days after the start of osteogenic differentiation, hMSCs were fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries), and calcium deposits were stained with Alizarin Red S (ARS) to assess the degree of osteogenic differentiation.

[0027] Alizarin Red S staining was performed as follows. After washing the fixed hMSCs twice with deionized water, 1% ARS solution (pH 6.3, Muto Chemical Co., Ltd.) was added and stained for 20 minutes at room temperature with gentle shaking. After washing four times with deionized water, the hMSCs were observed under an optical microscope (BX51, Olympus). To quantify the staining, 10% acetic acid (Fujifilm Wako Pure Chemical) was added to each well. The plate was then left at room temperature for 30 minutes, after which the solution was collected in a microtube and vortexed for 30 seconds. The sample was heated at 85°C for 10 minutes and then cooled on ice for 5 minutes. After centrifugation at 20,000 × g for 15 minutes, 10% ammonium hydroxide (Fujifilm Wako Pure Chemical) was added to the supernatant, and the absorbance of the supernatant was measured at 405 nm using a microplate reader (iMark, BIO RAD).

[0028] The results are shown in Figure 2A. The upper panel shows photographs of stained wells, and the lower panel shows quantitative results. miPS-BGM pre-frozen at -80°C promoted osteogenic differentiation more effectively than miPS-BGM pre-frozen at -30°C. iPS-BGM pre-frozen at -80°C for 6 hours or longer promoted significant osteogenic differentiation in hMSCs (P<0.05). Pre-freezing at -80°C for even 1 hour was more effective than pre-freezing at -30°C for 24 hours, and no difference was observed after 6 hours or longer. Therefore, freezing for 1 hour or longer, preferably 2 hours or longer, more preferably 4 hours or longer, and even more preferably 6 hours or longer, can produce bone substitutes with high osteogenic differentiation potential. Furthermore, these results suggest that pre-freezing at temperatures below -40°C, preferably below -60°C, is recommended.

[0029] Stabilizers such as sugars and sugar alcohols are known to protect proteins during freezing, so various stabilizers were investigated. Sucrose (Merck), trehalose (Nacalai Tesque), maltose (Nacalai Tesque), maltitol (Nacalai Tesque), lactose (Nacalai Tesque), and CaCl2 (Nacalai Tesque) were used as stabilizers. Each additive was dissolved in PBS to obtain a 10% solution. 25 cm 2A single culture flask's worth of miPSC cell aggregates was incubated in 5 ml of PBS containing each additive at 4°C for 1 hour, then frozen at -80°C for 6 hours. The frozen samples were then lyophilized at room temperature (23°C) for 6 hours. The promotion of osteogenic differentiation of hMSCs was evaluated as described above. The results are shown in Figure 2B. None of the stabilizers altered the osteogenic differentiation potential of miPS-BGM (Figure 2B).

[0030] 3.2 Consideration of primary drying conditions The temperature conditions for primary drying were investigated. As shown in Table 1B, pre-freezing was performed at -80°C, and the cell masses were primary freeze-dried at different temperatures. Alizarin Red S staining was performed as described above to evaluate osteogenic differentiation. The results are shown in Figure 3A. The upper panel shows images of hMSCs stained with Alizarin Red S on day 7 of osteogenic differentiation induction. GM (proliferation medium) and OS (osteoblast differentiation medium without miPS-BGM) are shown in the figure. The lower panel shows quantitative results. Different letters indicate significant differences (P < 0.05, Tukey's multiple comparison test after ANOVA; the same applies to the following experiments unless otherwise noted) (mean ± SD, n = 3). Bone substitutes prepared at a drying shelf temperature of -10°C during primary drying were shown to have significantly higher osteogenic differentiation potential than those prepared at 30°C. Furthermore, bone substitutes prepared at lower temperatures (-20°C and -40°C) had higher osteogenic differentiation potential. We analyzed whether freeze-drying at temperatures lower than -40°C resulted in higher osteogenic differentiation (Figure 3B). miPS-BGM was prepared at a primary drying temperature of -80°C (as listed in Table 1B), and osteogenic differentiation was assessed by Alizarin Red S staining. No difference in osteogenic differentiation was observed between primary drying at -80°C and primary drying at -40°C. Although no difference in performance was observed, primary drying at -40°C is preferable from a cost perspective because lower primary drying temperatures require the use of liquid nitrogen and other cooling costs. A primary drying temperature of -10°C results in higher osteogenic differentiation compared to 30°C (room temperature), so primary drying at -10°C or below is sufficient. Furthermore, primary drying at -20°C or below results in higher osteogenic differentiation, so primary drying at -20°C or below is preferred.

[0031] Furthermore, quantitative real-time RT-PCR analysis was performed to analyze the expression of bone differentiation-related genes (COL1A1, RUNX2, and OCN) under each temperature condition. After 7 days of culturing hMSCs in osteogenic differentiation medium supplemented with miPS-BGM, RNA was extracted from the cells using TRIzol reagent (Thermo Fisher Scientific). RNA was isolated and purified using the RNeasy Mini Kit (Qiagen). After DNA removal using a DNA-free DNA Removal Kit (Thermo Fisher Scientific), cDNA was synthesized using 1 μg of total RNA using a known method. Quantitative real-time RT-PCR analysis was performed using Thunderbird SYBR qPCR Mix (Toyobo) and a StepOnePlus real-time PCR system (Thermo Fisher Scientific). Target gene expression was measured using a 2- △△Ct The results were analyzed quantitatively using the qPCR method and normalized by GAPDH expression. The primer sequences used are shown in Table 2. The primers used were primers for human sequences.

[0032] [Table 2]

[0033] Freeze-dried miPS-BGM at -40°C significantly increased the expression of osteogenic differentiation-related genes COL1A1, RUNX2, and OCN in hMSCs (P<0.05) (Figure 3C).

[0034] Next, we optimized the primary drying time by fixing the drying shelf temperature at -40°C. Pre-freezing was performed at -80°C for 24 hours, followed by secondary drying at 30°C for 2 hours (Table 1C). Under these conditions, we found that a primary drying time of 0.5 hours for miPS-BGM maintained hMSC osteogenic differentiation at its highest level (Figure 3D). The time required for complete drying varies depending on factors such as the sample volume, so a shorter drying time is acceptable as long as complete drying is achieved. For example, under these experimental conditions, a drying time of 10 minutes or more, preferably 15 minutes or more, is considered sufficient. There was little difference observed between primary drying times of 1 hour, 3 hours, and 6 hours.

[0035] 3.3 Consideration of secondary drying conditions Next, we optimized the secondary drying time. Because the purpose of secondary drying is to remove unfrozen water, room temperature is sufficient; here, it was performed at 30°C. Furthermore, we performed pre-freezing at -80°C for 24 hours and the primary drying time at -40°C for 1 hour (Table 1D). The results demonstrated that a secondary drying time of 0.5 hours or longer for miPS-BGM maintained hMSC osteogenic differentiation (Figure 4). Given the likely minimal amount of unfrozen water, a shorter drying time, such as 10 minutes, may be sufficient.

[0036] 4. Evaluation of iPS-BGM in a rat femoral defect model The results of the above experiments clearly showed that the shelf temperature during primary drying significantly affects the quality of miPS-BGM. The following experiments were conducted by setting the pre-freezing and secondary drying conditions constant (pre-freezing: -80°C / 6 hours, secondary drying: 30°C / 0.5 hours) and varying the primary drying conditions (primary drying: -40°C, -10°C, or 30°C / 0.5 hours).

[0037] The quality of miPS-BGM was analyzed using a rat femoral defect model to assess the effect of different drying temperatures on the quality of miPS-BGM. The therapeutic effects of miPS-BGM prepared at different drying temperatures were evaluated using a rat model with a femoral defect. The rat model consisted of 11-week-old Sprague-Dawley rats (CLEA) weighing 340-410 g (average 380 g). Anesthesia was induced by inhalation of 2% isoflurane, and a combination of anesthetics (1 mg / mL medetomidine hydrochloride (Domitor, Meiji Seika Kaisha) and 5 mg / mL midazolam (Dormicum, Astellas Pharma)) was used. Continuous anesthesia was achieved by adding 5 mg / mL butorphanol (Betorfar, Meiji Seika Kaisha) to 0.9% w / v sodium chloride (physiological saline, Otsuka Pharmaceutical). After shaving the left leg, the rat was disinfected with povidone-iodine (Kaneichi Chemical) and locally anesthetized with xylocaine. The skin and muscle were incised to expose the femur, and a round steel burr (Drendel + Zweiling) was inserted into the center of the femur. A cortical bone defect (3mm x 5mm, critical size) was created under perfusion using Diamant. Then, 0.2cc of miPS-BGM was implanted into the femoral defect. The implantation site was wrapped in a 10mm x 20mm absorbable collagen membrane (Cocken Tissue Guide, Koken). The femur was splinted with a prefabricated titanium stent, and then the midsection of the femur was wrapped and ligated with hydraulic polyurethane resin-reinforced glass fiber (Castlite α, Alcare) and stainless steel wire. Muscle and skin were sutured with 5-0 polypropylene sutures (Prolene, Ethicon). The rats were operated on on a heating mat (Natsume Seisakusho) at 37°C. After 3 weeks, the rats were euthanized and subjected to micro-CT and histological examination.

[0038] Three weeks after miPS-BGM implantation, femoral defects were scanned using micro-CT to quantify bone mass, bone mineral density, and bone mineral content. Femoral samples were fixed in 10% neutral buffered formalin at 4°C for 1 week. Bone mass, bone mineral density, and bone mineral content at the miPS-BGM implantation site were assessed using a 3D micro X-ray CT scanner (Scan Xmater-E090, Comscan Techno) and bone structure analysis software (TRI / 3D-BON, Ratoc Systems Engineering). X-ray scans of the specimens were performed at an energy level of 80 kV / 60 μA through a 1 mm-thick brass filter. 3D images were reconstructed based on a bone mineral content calibration curve obtained by scanning a hydroxyapatite phantom under the same X-ray conditions. A separate analysis area was defined for each specimen. The cortical region was defined as the area connecting the outer and inner basic lamellae on both sides of the defect. The specific threshold for bone tissue was determined by overlaying the segmented image onto the original grayscale X-ray image.

[0039] Bone regeneration was enhanced in the miPS-BGM implant group compared to the non-implant group (Fig. 5A). Furthermore, new bone formation was significantly higher in the miPS-BGM implant group compared to the non-implant group (Fig. 5B).

[0040] Pathological analysis of the bone defects was performed. Femoral bone samples were degreased by immersion in 70% ethanol and then 100% ethanol for 1 day each, for a total of 2 days, and then decalcified in 0.5 mol / L EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for 4 weeks. After dehydration with ethanol, acetone, and xylene, the samples were embedded in paraffin. 3.5 μm-thick paraffin sections were stained with hematoxylin and eosin (HE) for morphological observation. Galectin-3, a macrophage marker molecule, was visualized by immunohistochemical staining. For immunohistochemical staining, paraffin sections were deparaffinized by washing twice for 5 minutes in 100% xylene (Fujifilm Wako Pure Chemical Industries, Ltd.) and xylene / ethanol (50 / 50), and then rehydrated in graded ethanol (100%, 95%, 80%, 70%) and distilled water. The antigen was then retrieved overnight at 60°C using 10 mM citrate buffer (pH 6.0, Fujifilm Wako Pure Chemical). The slides were washed with PBS and treated with 3% hydrogen peroxide (Fujifilm Wako Pure Chemical) for 10 minutes at room temperature to inhibit endogenous peroxidase activity. The slides were then washed with PBS and incubated with 1% bovine serum albumin (Fujifilm Wako Pure Chemical) for 30 minutes to stop the reaction. The slides were then incubated overnight at 4°C with anti-galectin-3 antibody (Galectin-3 / LGALS3 Rabbit mAb, Cell Signaling Technology). The slides were then washed with PBS containing Tween 20 (TBST) and incubated with a secondary antibody (mouse anti-rabbit IgG-HRP, Santa Cruz Biotechnology) for 1 hour at room temperature. The samples were washed with PBST and visualized by incubation with DAB substrate (Merck) for 15 minutes, followed by counterstaining with hematoxylin.

[0041] HE staining of the bone defects revealed neutrophil infiltration, a marker of acute inflammation, in the miPS-BGM implanted groups prepared at -10°C and 30°C (hereafter, miPS-BGM is referred to as miPS-BGM (-10°C) and miPS-BGM (30°C) based on the primary drying temperature at which it was prepared) (Figures 6A, upper and 6B, upper). Galectin-3-positive macrophages, on the other hand, accumulated around the miPS-BGM (-40°C) implant (Figures 6A, lower and 6B, lower). Galectin-3-positive macrophages were rarely observed around the miPS-BGM (-10°C) and miPS-BGM (30°C) implants. Analysis of the total area of ​​remaining granular miPS-BGM revealed that the area of ​​miPS-BGM (30°C) implants was significantly larger than that of miPS-BGM (-40°C) and miPS-BGM (-10°C) (Figure 6C). Furthermore, no tumor formation was observed macroscopically or histologically in 17 rat femur samples implanted with miPS-BGM.

[0042] 5. Evaluation of inflammatory response The effect of miPS-BGM prepared at various primary drying temperatures on inflammatory responses was evaluated. The mouse macrophage-like cell line J774A.1 (JCRB9108) was obtained from the Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition. Cells were cultured at 37°C in a humidified atmosphere with 5% CO2 in DMEM (Nacalai Tesque) containing 4.5 g / L glucose without sodium pyruvate, 10% FBS (Japan Bioserum), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.). The medium was refreshed every 3 days and the cells were passaged twice. After culturing until 80% confluent, the cells were collected and plated at 3.0 × 10 cells per well in a 6-well plate. 5 Cells were seeded at a cell density of 1000 cells / mL. After 24 hours, the medium was replaced with 5 mg / mL miPS-BGM suspension medium and cultured for 3 days. The cells were collected and analyzed for expression of pro- and anti-inflammatory genes by real-time RT-PCR. The primer sequences used are shown in Table 3. The primers used were for mouse sequences.

[0043] [Table 3]

[0044] Macrophage J774A.1 cells were cultured in growth medium containing pulverized miPS-BGM, and gene expression of pro-inflammatory (Il-1β, TNFα, Il-6) and anti-inflammatory (Il-10, Tgfβ1, Il-11) cytokines was analyzed by real-time RT-PCR analysis on days 1 and 3. As shown in the upper panel of Figure 7, cells in the miPS-BGM-treated groups at either temperature showed greater cell aggregation and colony formation than those in the growth medium (GM) group on day 1. Tgfβ1 gene expression was significantly higher on day 1 in the miPS-BGM-treated group than in the GM group. Expression of Tgfβ1, Il-10, and Il-11 genes was higher in the miPS-BGM (-40°C)-treated group than in the GM, miPS-BGM (-10°C), and miPS-BGM (30°C)-treated groups. Gene expression of Il-1β, Tnfα, and Il-6 was significantly reduced in all miPS-BGM-treated groups compared with the GM group. Il-1β gene expression was lower in the miPS-BGM (-40°C)-treated group than in the miPS-BGM (-10°C)-treated and miPS-BGM (30°C)-treated groups, although not significantly different (Fig. 7, bottom graph).

[0045] 6. Protein profile of miPS-BGM 6.1 Protein measurement The amount of protein released from miPS-BGM prepared at each primary drying temperature and suspended in PBS for 2 days was measured. 0.09 g of miPS-BGM prepared at each temperature was suspended in 500 μL of PBS and shaken at 37°C and 54 rpm. After 2 days, the supernatant was collected. The concentration of total protein released from miPS-BGM was measured using Pierce protein assay (Thermo Fisher Scientific). miPS-BGM (-40°C) released significantly more protein than miPS-BGM (-10°C) or miPS-BGM (30°C) (Figure 8A). To confirm the effect of proteins released from miPS-BGM on hMSC proliferation and migration, hMSCs were cultured in the presence of miPS-BGM prepared at -40°C, -10°C, or 30°C. hMSCs and miPS-BGM were cultured in growth medium on dishes at a cell density of 5 × 10 5 hMSCs were co-cultured at 100°C / 100°F. After 7 days, hMSCs were washed with PBS and fixed with 3.7% paraformaldehyde at room temperature. The fixed cells were then washed with PBS and permeabilized with 0.5% Triton-X (Fujifilm Wako Pure Chemical Industries, Ltd.) for 5 minutes at room temperature. The cells were then stained with Rhodamine Phalloidin (Thermo Fisher Scientific) for 40 minutes. After washing twice with PBS, the cells were incubated with DAPI-containing mounting medium (VECTASHIELD) for 20 minutes and observed under a confocal laser scanning microscope (LSM780, Zeiss) (Figure 8B). Immunofluorescence images of miPS-BGM and hMSCs showed that hMSCs accumulated around the miPS-BGM. Quantification of hMSC cell number revealed significantly higher cell numbers around miPS-BGM (-40°C) and miPS-BGM (-10°C) than those in miPS-BGM (30°C) (Figure 8C).

[0046] 6.2 Effect on migration The effect of proteins released from miPS-BGM on the migration ability of hMSCs was evaluated using a scratch wound healing assay. hMSCs were cultured at a cell density of 1.0 × 10 5hMSCs were seeded into 12-well plates at 1000 cells / mL. After 24 hours, a scratch was created by scraping the hMSCs linearly using a 200 μL pipette tip. Cell debris was gently washed away with medium, and the medium was replaced with growth medium containing miPS-BGM (-40°C), miPS-BGM (-10°C), or miPS-BGM (30°C) at 5 mg / mL. Immediately after scratching and 9 hours later, images of the hMSCs were taken and quantified using ImageJ (NIH Image) (Figure 9). The number of cells within the scratch area in each group was counted from the images. The number of migrating cells in the medium containing miPS-BGM (-40°C) was significantly higher than in the medium containing miPS-BGM (-10°C) or miPS-BGM (30°C) (P < 0.05) (Figure 9, lower graph).

[0047] 6.3 Analysis of protein components These results suggest that cellular behavior around miPS-BGM is primarily controlled by the miPS-BGM's constituent proteins. Therefore, we comprehensively analyzed the protein composition of miPS-BGM (-40°C) using a protein array (Figure 10). After lyophilization, 0.05 g of miPS-BGM was added to 2 mL of PBS and crushed in an ultrasonic grinder for 2 minutes. After centrifugation (1000 × g, 10 minutes), 1 mL of the supernatant was collected in a microtube and stored in a -80°C freezer. β-TCP (Kyocera), a widely used bone filler, was used as a control. A total of 308 proteins in miPS-BGM were evaluated using the RayBio™ Label-Based Mouse Antibody Array L-308 (RayBiotech). The abundance of 121 cytokines / cytokine-related proteins was found to be higher in miPS-BGM than in β-TCP. Although details are not shown here, miPS-BGM retained more bone metabolism-related proteins such as M-CSF, IL-27, adiponectin / Acrp30, and IL-6, osteoblast-related proteins such as SPARC, IL-17E, Decorin, TGF-β1, osteoprotegerin, IGF-1, osteopontin, osteoactivin / GPNMB, and VCAM-1, and angiogenesis-related proteins such as PDGF C, endostatin, VEGF, and VEGFC.

[0048] To clarify the effects of freeze-drying on the proteins that make up miPS-BGM, we used proteome analysis (mass spectrometry) to analyze changes in protein composition before and after freeze-drying. miPS cells were induced to differentiate into bone for 30 days, and 0.2 g of cell mass was collected in a microtube and stored in a -80°C freezer for 24 hours to prepare the cell mass before freeze-drying. The cell mass before freeze-drying and miPS-BGM were evaluated using DIA proteome analysis (LC-MS / MS (DIA), Promega) (Figure 11). 5,812 proteins were identified, of which 98.7% (5,749 proteins) were common between before and after freeze-drying. This revealed that freeze-drying had little effect on the protein composition.

[0049] 7. Morphological characteristics of miPS-BGM The morphological differences of miPS-BGM prepared at different primary drying temperatures were evaluated using appearance, compression test, scanning electron microscope (SEM), energy dispersive X-ray analysis (EDX), Ca concentration measurement, X-ray diffraction (XRD), and transmission electron microscope (TEM).

[0050] 7.1 Morphological characteristics by macroscopic observation As shown in Figure 12A, miPS-BGM (-40°C) and miPS-BGM (-10°C) were round and granular, with similar diameters, whereas miPS-BGM (30°C) did not maintain its granular shape and appeared finely pulverized.

[0051] 7.2 Compression test results To prepare the molded specimens for compression testing, 100 μL of miPSC cell mass was mixed with 1 mg of gelatin hydrogel, filled into a polypropylene cylinder (4 mm diameter, 4.5 mm height), and frozen at -80°C for 24 hours. Freeze-drying was performed under the temperature conditions listed in Table 1E. Cylindrical specimens were subjected to compression failure using an Instron universal testing machine (Instron 3342) equipped with a 50 N load cell at a crosshead speed of 1.0 mm / min. Bluehill™ 2 software was used for testing machine operation and data analysis. All tests were repeated six times, and the mean and standard deviation were calculated. The compression test results showed that miPS-BGM (-40°C) and miPS-BGM (-10°C) exhibited statistically significant increases in compressive load compared to miPS-BGM (30°C) (Figure 12B, P<0.05).

[0052] 7.3 Scanning electron microscope observation Figure 12C shows the SEM microstructure of miPS-BGM prepared using a tray-type freeze dryer. The miPS-BGM samples dried at each primary drying temperature and then secondary dried were observed using a scanning electron microscope (SU8000, Hitachi High-Technologies) at an accelerating voltage of 5 kV. SEM observation revealed that the miPS-BGM (-40°C) and miPS-BGM (-10°C) samples consisted of aggregates of relatively uniformly sized spherical structures with a particle size of approximately 10 μm (average particle size 7 μm). In contrast, the miPS-BGM (30°C) samples exhibited flake-like structures with an average particle size of 1–2 μm.

[0053] Figure 12D shows the SEM microstructure of miPS-BGM (Patent Document 2) prepared using a conventional cooling-stage freeze dryer (JFD-320 Freeze Drying Device, JEOL) at a primary drying temperature of -10°C. SEM observation revealed spherical aggregates with particle sizes of 5 μm or less. The surface granular structure was smaller and less porous than miPS-BGM prepared at the same primary drying temperature using a tray-type freeze dryer with precisely programmable freeze-drying temperature control (Figure 12C, top). The morphology observed by macroscopic observation and the granular structure observed by SEM are thought to correlate with the primary drying temperature. Therefore, the moisture content, porosity, and true density, which are closely related to these morphologies, may correlate with the primary drying temperature.

[0054] Furthermore, we performed EDX analysis on miPS-BGM prepared using a tray-type freeze dryer to evaluate the elemental composition of each miPS-BGM. SEM-EDX images revealed the elemental distribution of Ca, P, C, and O on the surface of the miPS-BGM, suggesting the presence of carbonate apatite. Furthermore, the Ca / P ratio (atomic composition%) was similar between miPS-BGM (-40°C) and miPS-BGM (-10°C), being 1.34 and 1.41, respectively. In contrast, the Ca / P ratio for miPS-BGM (30°C) was 0.43 (Figure 12E).

[0055] In addition, EDX was performed on miPS-BGM (Patent Document 2) and undecalcified freeze-dried human bone (OraGRAFT Cortical, LifeNet Health) prepared using a cooling-stage freeze-dryer at a primary freeze-drying temperature of −10°C, and the Ca / P ratio (atomic composition %) was calculated (Figure 12F). The Ca / P ratio of miPS-BGM (Patent Document 2) was 1.20, a lower value than the Ca / P ratio (1.41) of miPS-BGM (−10°C) prepared using the tray-type freeze-dryer described above. Furthermore, the Ca / P ratio of undecalcified human freeze-dried bone was 1.41, a value similar to that of miPS-BGM (−10°C) prepared using the tray-type freeze-dryer. Therefore, miPS-BGM prepared using the tray-type freeze-dryer has a Ca / P ratio closer to that of natural bone than the miPS-BGM described in Patent Document 2, suggesting its suitability as an artificial bone (bone substitute).

[0056] Thus, even though the primary drying temperatures appear to be the same, the properties of miPS-BGM differ. This is likely due to the fact that, while a cooling stage freeze dryer allows samples to be placed on a cooled stage for freeze-drying, it is not possible to control the temperature of the drying chamber itself. The shelf freeze dryer used in this study allows for temperature control within the drying chamber by controlling the shelf temperature, and the presence of an internal sensor allows for more accurate temperature control. Furthermore, it quickly reaches a vacuum of less than 3 Pa in 10 minutes, allowing for rapid drying. This indicates that temperature control during freeze-drying is important for producing high-performance bone substitutes.

[0057] 7.4 Calcium elution test A calcium elution test was performed on miPS-BGM dried at different temperatures. For the calcium elution test, miPS-BGM was decalcified with 1.5 mL of 0.5 M acetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and stored overnight at room temperature (23°C). Insoluble materials were removed by centrifugation at 2300 × g for 10 minutes. The calcium concentration of the supernatant was measured by the MXB method (OD595) using a Calcium E-Test Kit (Fujifilm Wako Pure Chemical Industries, Ltd.). The calcium concentration of miPS-BGM dried at 30°C was significantly lower than that of miPS-BGM dried at -40°C and -10°C (P < 0.05) (Figure 12G).

[0058] 7.5 Analysis by X-ray diffraction (XRD) Furthermore, the microstructure was analyzed using X-ray diffraction (XRD). XRD analysis was performed using a SmartLab 9SW (Rigaku Corporation) to analyze the composition of miPS-BGM. The XRD pattern is shown in Figure 13A. The XRD pattern of miPS-BGM was not affected by the temperature during primary drying. The characteristic peaks of hydroxyapatite at 2θ = 26.0 (002) and 2θ = 33.0 (112) were high and sharp, similar to the pattern of carbonate apatite, indicating that miPS-BGM has high crystallinity and purity.

[0059] 7.6 Analysis by Transmission Electron Microscope (TEM) For TEM observations, miPS-BGM was embedded in resin, and ultrathin sections (0.05 μm thick) were prepared from the resin blocks. These sections were then observed using a scanning TEM (JEM-ARM200F, JEOL Ltd.) at an accelerating voltage of 80 kV. TEM images of miPS-BGM revealed clusters of needle-like particles (Figure 13B). The average length of the needle-like structures in miPS-BGM (-40°C) was 35 nm, which was significantly shorter than the lengths of the needle-like structures in miPS-BGM (-10°C) and miPS-BGM (30°C) (48 nm and 64 nm, respectively) (P < 0.05). The length of the needle-like structures correlated with the temperature during primary drying (Figure 13C). Therefore, iPS-BGM samples dried at different temperatures can be distinguished based on the average length of the needle-like structures. There are reports that short, needle-like hydroxyapatite crystals have shown anti-inflammatory effects by increasing the expression of anti-inflammatory factors such as IL-10 in macrophages, suggesting that the short, needle-like crystal structure obtained under -40°C dry conditions may have an effect on local immune control.

[0060] 8. Analysis of human iPS cell-derived bone substitute materials 8.1 Analysis of protein components of human iPS cell-derived osteoblastic clusters The protein components of human iPS cell-derived osteoblastic cell clusters before freeze-drying were analyzed. Human iPS osteoblastic cell clusters were prepared according to Patent Documents 1 and 3. Human iPS osteoblastic cell clusters were collected in Eppendorf tubes and stored in a -80°C freezer. For comparison, human iPS cell clusters before the start of osteoblast differentiation induction were used. Protein expression and relative quantitative analysis were performed at Kazusa Genome Technologies, Inc. using DIA proteome analysis (Promega Corporation).

[0061] Mass spectrometry identified 7,678 proteins contained in the human iPS osteoblastic cell mass. Excluding proteins constitutively expressed in human iPS cells (proteins expressed before osteoblast differentiation), proteins with high expression levels were extracted. Proteins involved in bone metabolism, including OGN, IGFBP5, CD34, TIMP1, FBLN5, LPAR1, IGFBP7, MGP, DCN, PDGFRB, IGF1, TAB2, FGFR2, and SPP1, were also found to be abundant. Collagen, COL1A2, COL4A1, COL5A1, COL6A6, COL15A1, COL16A1, COL21A1, and COL26A1 were also detected. 8.2 Analysis of human iPS cell-derived bone substitutes using antibody arrays The protein composition of human iPS cell-derived bone filler (hiPS-BGM) was also analyzed in the same manner as in Section 6.3. hiPS-BGM was prepared according to our method (Patent Documents 1 and 3). After ultrasonically pulverizing hiPS-BGM in PBS, the supernatant was collected using a centrifuge and stored in a -80°C freezer. For comparison, human freeze-dried bone (FDBA:OraGRAFT (LifeNet Health)) was treated in the same manner to prepare a supernatant sample. Comprehensive analysis by antibody array was performed using the Human Antibody Array L-1000 (Filgen, Inc.). The proteins contained in hiPS-BGM and FDBA were compared and compared with the protein content data of miPS-BGM to identify common proteins (Figure 14).

[0062] While existing bone substitute materials (FDBA) contain almost no protein, hiPS-BGM contains many proteins related to bone tissue regeneration, such as bFGF, FGF-23, BMP-2, BMP-3, DMP-1, ALPP, GDF1, BMP-5, IGF-II, IGFBP-2, IGF-I, BMP-15, BMP-8, BMP-7, osteoprotegerin / TNFRSF11B, and BMP-9; proteins related to angiogenesis, such as EGFR-VEGF / PK1, Angiopoietin-like 1, Angiopoietin-like Factor, PDGF-AA, Angiopoietin-2, VEGF-R3, VEGF-R1, and VEGF; and proteins related to bone metabolism, such as beta-IG-H3, IL-17B-R, IFN-beta, calcitonin, IL-17R, MMP-10, and TNF-beta. Other proteins found in hiPS-BGM include COCO, Cadherin-13, Clusterin, C5 / C5a, CD30 Ligand / TNFSF8, Kallikrein 5, IL-4, IGFBP-1, IL-1F8 / FIL1 eta, GRO, IL-8, Glut3, IL-29, ALCAM, CNTF, IL-21R, CD163, IL-5, CD71, IL-21, EDG-1, Prolactin, IL-26, Growth Hormone (GH), GDF11, CD40 Ligand / TNFSF5 / CD154, Cathepsin B, GDF8, GM-CSF, IL-7, CCR4, BNP, IL-2, IL-6, C-peptide, IL-17, GREMLIN, and BTC. By comparing the data from hiPS-BGM (vs. FDBA) with the data from miPS-BGM (vs. FDBA), 68 common proteins were identified (Table 4).

[0063] [Table 4]

[0064] 9. Evaluation of osteogenic potential of hiPS-BGM The temperature conditions for primary drying of hiPS-BGM were investigated. Similar to miPS-BGM, cell aggregates were prepared by pre-freezing at -80°C and primary freeze-drying at different shelf temperatures. An extract of hiPS-BGM was added to osteogenic differentiation-inducing medium, which was then used to culture human bone marrow-derived mesenchymal stem cells (hMSCs). The osteogenic induction potential of hiPS-BGM was evaluated based on the degree of differentiation. hMSCs were induced in osteogenic differentiation medium as described in Section 3.1.

[0065] hiPS-BGM was suspended in osteogenic differentiation medium at a concentration of 5 mg / mL and pulverized on ice using an ultrasonicator at 20% ultrasonic amplitude for 2 minutes. After sonication, the suspension was filtered through a syringe filter (0.22 μm pore size, Merck) and added to osteogenic differentiation medium for hMSCs. The osteogenic differentiation medium was changed every 3 days. 14 days after the start of osteogenic differentiation, hMSCs were fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries) and stained for alkaline phosphatase (ALP) to assess the degree of osteogenic differentiation.

[0066] ALP staining was performed as follows. After fixing, hMSCs were washed three times with purified water, and then a chromogenic substrate (ALP staining kit, Cosmo Bio) was added and stained for 20 minutes at 37°C. After washing three times with purified water, hMSCs were observed under an optical microscope (BX51, Olympus). To quantify the staining, the area of ​​the ALP-stained region was analyzed using ImageJ image analysis software.

[0067] The results are shown in Figure 15. The upper panel shows an ALP staining image on day 14 of osteogenic differentiation induction, and the lower panel shows the quantitative results. In the figure, "No Addition" indicates osteogenic differentiation medium containing no hiPS-BGM. The lower panel shows the quantitative results (mean ± SD, n = 3). It was shown that bone substitutes prepared at a drying shelf temperature of -10°C during primary drying have significantly higher osteogenic differentiation induction ability than bone substitutes prepared at 30°C. Furthermore, preparation at a lower temperature of -40°C has higher osteogenic differentiation induction ability. Because -10°C has higher osteogenic differentiation induction ability than 30°C, the primary drying temperature should be set to -10°C or below. Furthermore, because higher osteogenic differentiation induction ability is obtained at -40°C or below, primary drying at -40°C or below is preferable.

[0068] 10. Effect of hiPS-BGM on migration The effect of proteins released from hiPS-BGM on the migration ability of hMSCs was evaluated using a scratch wound healing assay. The wound healing assay was performed using hMSCs as described in 6.2. hMSCs were cultured at a cell density of 4.0 × 10 5 hMSCs were seeded at 1000 cells / mL into a 12-well plate. After 24 hours, hMSCs were scraped linearly with a 200 μL pipette tip to create a scratch.

[0069] Cell debris was gently washed away with medium, and the medium was replaced with growth medium containing hiPS-BGM (-40°C), hiPS-BGM (-10°C), or hiPS-BGM (30°C) at a concentration of 5 mg / mL. Suspension was performed using an ultrasonic homogenizer as described above. Images of hMSCs were taken immediately after scratching and 6 hours later (Figure 16, upper panel shows images taken 6 hours later), and the number of cells that migrated into the scratch area was analyzed. The number of cells in the scratch area for each group was counted from the images. The number of migrated cells in the medium containing hiPS-BGM (-40°C) was significantly higher than that in the medium containing hiPS-BGM (-10°C) or miPS-BGM (30°C) (P < 0.001) (Figure 16, lower panel).

[0070] As shown above, freeze-drying inactivates cells, eliminating the risk of tumor formation, and by optimizing the temperature and time, it is possible to produce bone substitutes with excellent osteoconductivity and osteogenic induction. Optimal conditions were investigated for all three freeze-drying steps: pre-freezing, primary drying, and secondary drying. While the quality of each step varies depending on the processing temperature and time, the conditions for primary drying are particularly important, and the sample temperature must be maintained accurately. Furthermore, from a cost perspective, it is preferable to complete the three steps in a shorter time and at a higher cooling temperature.

[0071] Although detailed analysis was performed using mouse iPS cells here, the analysis results for protein components, osteogenic induction ability, and migration ability of human iPS cells were equivalent to those obtained with mice. Therefore, the results obtained using mouse iPS cells can be directly applied to the manufacturing method of bone substitute materials produced using human iPS cells.

Claims

1. A method for producing a stem cell-derived bone substitute, comprising: a step of inducing differentiation of stem cells into osteoblasts and forming cell masses; Pre-freezing process, a primary drying step of freeze-drying at a temperature of −80° C. or higher and −10° C. or lower; A method for producing a bone filler, comprising a secondary drying step.

2. 2. The method for producing a bone prosthetic material according to claim 1, wherein the primary drying step is carried out for a period of 10 minutes to 6 hours.

3. 2. The method for producing a bone substitute according to claim 1, wherein the pre-freezing step is carried out at a temperature of -40°C or lower and -80°C or higher.

4. 4. The method for producing a bone substitute according to claim 3, wherein the pre-freezing step is carried out for at least one hour.

Images