JP7797072B2 - Bone substitute material and its manufacturing method - Google Patents

Description

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

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 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 grafts 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.

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. 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 a risk of unknown 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 the body, 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, posing a risk of increased infection and fragility.

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, there is a need for the development of new bone substitute materials that can create a microenvironment that promotes bone remodeling.

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 clusters from human iPSCs (Patent Document 1). While iPSC-derived spheroids (cell clusters) 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.

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 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 substitute materials with excellent osteoconductivity and osteogenic induction capabilities, but this has not yet been put to practical use.

International Publication No. 2018/181960 International Publication No. 2015/064705 International Publication No. 2020/175592

S. Pigeot, etal., Advanced Materials, 2021, 33, 2103737, https://doi.org/10.1002/adma.202103737. H. Egusa,et al., Stem Cells Dev,2014, 23(18),2156-69. H. Egusa, et al., Plos One, 2010,5(9) :e12743. doi: 10.1371/journal.pone.0012743.

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 used to inactivate cells, these methods may denature proteins. Therefore, we considered freeze-drying to be a better method, as it can inactivate cells while maintaining the physiological activity and structure of proteins. Compared to conventional drying methods, freeze-drying dries materials at low temperatures, thereby suppressing thermal denaturation and chemical changes in the material. It also slows the rate of product degradation 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, a mass of iPS cells induced to differentiate into osteoblasts was pre-frozen overnight in a -80°C freezer, then freeze-dried on a cooling stage by fixing the temperature at -10°C and gradually lowering the air pressure to 6-20 Pa overnight, producing a bone prosthesis without secondary drying. However, because a shelf-type freeze dryer, which can accurately 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 production method, it may be possible to produce a bone prosthesis with even greater bone formation induction and inflammation suppression capabilities.

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 objective of the present invention is to provide a bone substitute material that is highly safe and has higher osteoconductivity and osteogenic induction ability. Another objective is to provide a method for producing a bone substitute material that can be supplied stably, has high production efficiency, and is low-cost.

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 including 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.

(2) A method for producing a bone filler according to (1), further comprising a secondary drying step.
Conventionally, a secondary drying step has not been carried out, but by carrying out the secondary drying step to desorb unfrozen water, a high-performance bone prosthetic material can be produced.

FIG. 1 is a diagram showing a schematic diagram of the process of freeze-drying a cell mass produced from iPS cells. FIG. 1 is a diagram showing a schematic configuration of the tray-type freeze dryer used. Study of pre-freezing conditions (1). Study of freezing temperature and time. Study of pre-freezing conditions (2). Study of stabilizers. Study of primary drying conditions (1). A diagram showing the results of a study of drying shelf temperature conditions during freeze-drying. Study on primary drying conditions (2). A diagram showing the results of the study, including the drying shelf temperature condition of -80°C during freeze-drying. Study of primary drying conditions (3). Figure showing the expression of osteogenic differentiation marker genes depending on the drying shelf temperature conditions during freeze-drying. Study of primary drying conditions (4). A diagram showing the results of a study on freeze-drying time. FIG. 10 is a diagram showing the results of a study on secondary drying time. 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 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). The images show hematoxylin and eosin (HE) staining (top row) and immunohistochemical staining with anti-galectin-3 antibody of the femoral defect site 3 weeks after miPS-BGM implantation. The arrows indicate the edges of the defect site. Scale bar: 1.0 mm. The top row is an enlarged image of the area enclosed by the dotted line in the HE-stained image in the top row of Figure 6A. The bottom row is an enlarged image of the area enclosed by the dotted line in the immunohistochemical stained image in the bottom row of Figure 6A. * indicates remaining miPS-BGM, and arrows indicate neutrophils. Scale bar: 100 μm. The insets are enlarged images of the area enclosed by the square in each figure. Scale bar in inset: 10 μm. The area of remaining miPS-BGM is shown (mean ± SD, n = 12-14). This figure shows the effect of miPS-BGM on macrophage activity. The photographs are micrographs of macrophages cultured in growth medium (GM) or growth medium supplemented with miPS-BGM one day later; scale bar: 200 μm. The graph shows the expression of inflammation- and anti-inflammatory-related genes one or three days after culturing macrophages in growth medium or growth medium containing miPS-BGM. 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. 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 position of miPS-BGM. Scale bar: 100 μm. The number of hMSC cells around miPS-BGM (mean ± SD, n = 3). Figure 1 shows the results of a wound healing assay. Phase-contrast microscopic images of hMSCs immediately after scratch formation (0 h) and after 9 h of culture (upper row, scale bar: 200 μm), and a graph showing the number of migrated cells in the scratch area after 9 h (mean ± SD, n = 3) (lower row). 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 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. Macrophotographs of miPS-BGM with different primary drying temperatures. A graph showing the results of compression tests on miPS-BGM with different primary drying temperatures. SEM images of miPS-BGMs with different primary drying temperatures. SEM image of miPS-BGM produced using a cooling stage freeze dryer. A diagram showing the results of elemental composition analysis of miPS-BGM produced using a tray-type freeze dryer. Figure showing the results of elemental composition analysis of miPS-BGM produced using a cooling stage freeze-dryer and non-decalcified freeze-dried human bone. A graph showing the results of measuring the amount of calcium elution from miPS-BGM at different primary drying temperatures. XRD patterns of miPS-BGMs with different primary drying temperatures. TEM/STEM images of miPS-BGMs with different primary drying temperatures. Scale: left panel: 200 nm, right panel: 100 nm. A diagram showing the length of fibrous apatite in miPS-BGM measured from a TEM image. Evaluation of human iPS-BGM using a cytokine antibody array. Human freeze-dried bone (FDBA) was used as a control for comparison. FIG. 1 shows the results of an analysis by ALP staining of the bone differentiation induction ability of human iPS-BGM at different primary drying temperatures. 1 shows the results of a wound healing assay, including a phase-contrast microscope image of hMSCs cultured for 6 hours after scratch formation (top row) and a graph showing the number of migrated cells in the scratch area after 6 hours (bottom row).

Bone filler refers to a material other than autologous bone used for the purpose of bone regeneration, and can be used interchangeably with artificial bone, bone substitute, bone regenerating agent, etc. The bone filler of the present invention is made from stem cells and has osteoconductive and osteogenic inducing 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 it is implanted into natural bone in the body, bonding the material to the bone and forming an integrated unit. Osteogenic inducing properties refer to the ability to stimulate the differentiation of mesenchymal stem cells, such as osteoblasts, into new bone at sites of bone loss or degeneration due to disease or injury.

The stem cells used in the production of the bone substitute material of the present invention are not particularly limited as long as they can be induced to become osteoblasts, but examples 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.

As mentioned above, even freeze-drying can cause chemical changes such as oxidation, deamidation, and hydrolysis, as well as aggregation due to changes in protein higher-order structure, depending on the conditions. Therefore, optimizing the freeze-drying conditions can lead to the production of high-performance bone substitute materials. The production of regenerative medicine products used in treatment 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 electricity, 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.

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 the same way 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.

1. Culturing iPS cells Induction of stem cells into bone cells and production of cell masses by 3D culture can be performed by known methods, including but not limited to the following method. The following experiments were performed using mouse gingival fibroblast-derived iPS cells (miPSCs, Non-Patent Documents 2 and 3) generated by retroviral introduction of Oct3/4, Sox2, and Klf4. miPSCs were cultured on inactivated SNLP76.7-4 feeder cells (provided by Dr. Allan Bradley of the Stanger Institute, UK) 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 x 10 ^-4 M 2-mercaptoethanol (Thermo Fisher Scientific), penicillin (50 U)/streptomycin (50 μg/mL) (Fujifilm Wako Pure Chemical Industries, Ltd.)). miPSCs were passaged every 5–6 days using a 0.25% w/v trypsin-1 mM/L EDTA solution. Embryoid bodies (EMBs) were formed in ES medium on low-adhesion culture dishes for 2 days. Subsequently, the miPSCs were cultured in suspension in ES medium supplemented with 1 μM all-trans retinoic acid for an additional 2 days (Non-Patent Document 2).

SNLP76.7-4 cells used as feeder cells were cultured in a medium containing 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.

2. Cell mass formation The embryoid bodies were cultured in a 70 mL flask 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 flask was placed on a seesaw shaker and cultured with shaking at 0.3 Hz for 30 days. The osteoblast differentiation medium was changed every two days.

3. Freeze-Drying As mentioned above, we believe that the freeze-drying conditions can lead to the production of 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 drying 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 object to be dried, the temperature inside the drying chamber can be precisely controlled, achieving excellent drying efficiency. Because precise temperature control during drying is considered important, freeze-drying was performed using a shelf-type freeze dryer (EYELA DRC-1100, Tokyo Rikakikai) here; 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 and a radiant heating vacuum freeze dryer. Note that the shelf temperature adjustment range of this dryer is -40°C to 30°C, so a liquid nitrogen vacuum freeze dryer (NRL-BC02S, Taiyo Nippon Sanso) was used only for the study in which the primary drying temperature was set to -80°C.

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 investigated. The drying shelf temperature was set to increase 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%.

3.1 Investigation of Initial Freezing Conditions Initial freezing is a process in which a 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, rapid expansion of water as the vacuum level increases during the subsequent primary drying process may lead to boiling of the sample. Because the conditions of the initial freezing 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.

Extracts of miPS-BGM prepared under each condition listed in Table 1A were added to osteoblast differentiation medium. Human bone marrow-derived mesenchymal stem cells (hMSCs, provided by Dr. Mabuchi, Tokyo Medical and Dental University, and Dr. Matsuzaki, Shimane University) were cultured using this medium, and the osteogenic induction ability 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 x ¹⁰ cells/mL at 37°C and 5% _CO2 . The growth medium was changed every 3-4 days. When hMSCs reached 80% confluence, they were harvested with trypsin and seeded into 24-well plates at a cell density of 4 x ¹⁰ cells/mL. After 24 hours, the growth medium was replaced with osteoblast differentiation medium (Lonza).

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 (pore size 0.22 μm, 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 induction, 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.

Alizarin Red S staining was performed as follows. After washing the fixed hMSCs twice with deionized water, 1% ARS solution (pH 6.3, Muto Chemicals) was added and stained for 20 minutes with gentle shaking at room temperature. 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 Chemicals) 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 Chemicals) was added to the supernatant, and the absorbance of the supernatant was measured at 405 nm using a microplate reader (iMark, BIO RAD).

The results are shown in Figure 2A. The top panel shows photographs of stained wells, and the bottom panel shows quantitative results. miPS-BGM pre-frozen at -80°C was found to promote osteogenic differentiation more effectively than miPS-BGM pre-frozen at -30°C. iPS-BGM pre-frozen at -80°C for 6 hours or longer significantly promoted osteogenic differentiation in hMSCs (P<0.05). Pre-freezing at -80°C for even 1 hour was more effective at promoting osteogenic differentiation than 24 hours at -30°C, 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 a bone substitute with high osteogenic differentiation potential. Furthermore, based on these results, it is suggested that pre-freezing at temperatures below -40°C, preferably below -60°C, is recommended.

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 CaCl ₂ (Nacalai Tesque) were used as stabilizers. Each additive was dissolved in PBS to obtain a 10% solution. A 25 cm ² culture flask of miPSC cell mass was incubated in PBS containing 5 ml of each additive at 4°C for 1 hour and then frozen at -80°C for 6 hours. The frozen sample was 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).

3.2 Investigation 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, and osteogenic differentiation induction ability was evaluated. 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. In the figure, GM indicates proliferation medium, and OS indicates osteoblast differentiation medium without miPS-BGM. The lower panel shows quantitative results, with different letters indicating significant differences (P < 0.05, ANOVA followed by Tukey's multiple comparison test; the same applies to the following experiments unless otherwise noted) (mean ± SD, n = 3). It was shown that bone substitutes prepared at a drying shelf temperature of -10°C during primary drying had significantly higher osteogenic differentiation induction ability than bone substitutes prepared at 30°C. Furthermore, preparation at lower temperatures, such as -20°C and -40°C, resulted in higher bone differentiation induction ability. We analyzed whether freeze-drying at temperatures lower than -40°C resulted in higher bone differentiation induction ability (Figure 3B). miPS-BGM was prepared under the primary drying conditions of -80°C, as shown in Table 1B, and bone differentiation induction ability was evaluated by Alizarin Red S staining. No difference in bone differentiation induction ability was observed between primary drying at -80°C and primary drying at -40°C. Although no difference in performance was observed, setting a lower primary drying temperature requires cooling costs, such as the use of liquid nitrogen, making primary drying at -40°C preferable from a cost perspective. Since a temperature of -10°C results in higher bone differentiation induction ability compared to 30°C (room temperature), the primary drying temperature should be set to -10°C or below. Furthermore, since a temperature of -20°C or below results in higher bone differentiation induction ability, primary drying at -20°C or below is more preferable.

Furthermore, quantitative real-time RT-PCR analysis was used to analyze the expression of bone differentiation-related genes (COL1A1, RUNX2, 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 an 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 known methods. 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 quantitatively analyzed using the 2- ^ΔΔCt method and normalized to GAPDH expression. The primer sequences used are shown in Table 2. The primers used were primers for human sequences.

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

Next, the drying shelf temperature was fixed at -40°C, and the primary drying time was optimized. Pre-freezing was performed at -80°C for 24 hours, followed by secondary drying at 30°C for 2 hours (Table 1C). The results revealed that under these conditions, hMSC osteogenic differentiation was best maintained when the miPS-BGM was dried for 0.5 hours (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, 3, and 6 hours.

3.3 Investigation of Secondary Drying Conditions Next, we optimized the secondary drying time. Because the purpose of secondary drying is to remove unfrozen water, the drying temperature can be room temperature; in this case, it was 30°C. Furthermore, the pre-freezing was performed at -80°C for 24 hours, and the primary drying time was fixed 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 osteogenic differentiation of hMSCs (Figure 4). Given that the amount of unfrozen water is likely minimal, a shorter drying time, such as 10 minutes, may be sufficient.

4. Evaluation of iPS-BGM in a rat femur defect model The above experimental results demonstrated that the shelf temperature during primary drying significantly affects the quality of miPS-BGM. The following experiment was 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).

The quality of miPS-BGM, which differs depending on the temperature during primary drying, was analyzed using a rat femoral defect model. The therapeutic effects of miPS-BGM prepared at different primary drying temperatures were evaluated using a rat model with a femoral defect. The rat model used was 11-week-old Sprague-Dawley rats (CLEA) weighing 340-410 g (average 380 g). Anesthesia was induced by inhalation of 2% isoflurane, and combined anesthetics (1 mg/mL medetomidine hydrochloride (Domitor, Meiji Seika Kaisha) and 5 mg/mL midazolam (Dormicum, Astellas Pharma) were 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 (3 mm x 5 mm, critical size) was created under perfusion using a Diamant syringe. Then, 0.2 cc of miPS-BGM was implanted into the femoral defect. The implantation site was wrapped in a 10 mm x 20 mm absorbable collagen membrane (Cocken Tissue Guide, Koken). The femur was splinted using 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 37°C heating mat (Natsume Seisakusho). After 3 weeks, the rats were euthanized and subjected to micro-CT and histological examination.

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 one week. Bone mass, bone mineral density, and bone mineral content at the miPS-BGM implantation site were evaluated 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.

Bone regeneration was promoted in the miPS-BGM implantation group compared to the non-implantation group (Figure 5A). Furthermore, new bone formation was significantly higher in the miPS-BGM implantation group compared to the non-implantation group (Figure 5B).

Pathological analysis of the bone defects was performed. Femoral bone samples were degreased by immersion in 70% ethanol and then 100% ethanol for one day each, for a total of two days, and then decalcified in 0.5 mol/L EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for four 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 five 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 activated overnight at 60°C using 10 mM citrate buffer (pH 6.0, Fujifilm Wako Pure Chemical Industries). The slides were washed with PBS and treated with 3% hydrogen peroxide (Fujifilm Wako Pure Chemical Industries) at room temperature for 10 minutes to inhibit endogenous peroxidase activity. The slides were then washed with PBS and left to stand for 30 minutes in 1% bovine serum albumin (Fujifilm Wako Pure Chemical Industries). The reaction was then stopped by incubation 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.

HE staining of the bone defect area revealed neutrophil infiltration, a marker of acute inflammation, in the miPS-BGM implantation groups manufactured at -10°C and 30°C (hereafter, miPS-BGM will be referred to as miPS-BGM (-10°C) and miPS-BGM (30°C) based on the primary drying temperature at which it was manufactured) (Figures 6A, top and 6B, top). Meanwhile, galectin-3-positive macrophages accumulated around the miPS-BGM (-40°C) (Figures 6A, bottom and 6B, bottom), but were barely observed around the miPS-BGM (-10°C) and miPS-BGM (30°C) groups. Analysis of the total area of miPS-BGM remaining in a granular form revealed that the area of miPS-BGM (30°C) was significantly larger than that of miPS-BGM (-40°C) and miPS-BGM (-10°C) (Fig. 6C). Furthermore, no tumor formation was observed macroscopically or histologically in 17 rat femur samples implanted with miPS-BGM.

5. Evaluation of Inflammatory Responses The effects of miPS-BGM prepared at various primary drying temperatures on inflammatory responses were 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 in sodium pyruvate-free 4.5 g/L glucose-containing DMEM (Nacalai Tesque), 10% FBS (Japan Bioserum), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Industries), penicillin (50 U)/streptomycin (50 μg/mL) at 37°C in a humidified atmosphere _of 5% CO . Cells were passaged twice, refreshing the medium every three days. After culturing to 80% confluence, cells were collected and seeded into 6-well plates at a cell density of 3.0 x ¹⁰ cells/mL. After 24 hours, the medium was replaced with 5 mg/mL miPS-BGM suspension medium, and the cells were cultured for 3 days. The cells were then collected and analyzed for the expression of pro-inflammatory and anti-inflammatory genes by real-time RT-PCR. The primer sequences used are shown in Table 3. The primers used were primers for mouse sequences.

Macrophage J774A.1 cells were cultured in growth medium containing pulverized miPS-BGM, and gene expression of inflammatory (Il-1β, Tnfα, Il-6) and anti-inflammatory (Il-10, Tgfβ1, Il-11) cytokines was analyzed using real-time RT-PCR analysis on days 1 and 3. As shown in the upper images of Figure 7, in the groups to which miPS-BGM produced at either temperature was added, cells aggregated and formed colonies on day 1 compared to the growth medium (GM) group. Gene expression of Tgfβ1 on day 1 after addition of miPS-BGM was significantly higher than that in the GM group. The expression of Tgfβ1, Il-10, and Il-11 genes was higher in the miPS-BGM (-40°C) group than in the GM, miPS-BGM (-10°C), and miPS-BGM (30°C) groups. The expression of Il-1β, Tnfα, and Il-6 genes was significantly reduced in all miPS-BGM groups compared with the GM group. The expression of Il-1β gene was lower in the miPS-BGM (-40°C) group than in the miPS-BGM (-10°C) and miPS-BGM (30°C) groups, although this was not significantly different (Figure 7, bottom graph).

6. Protein Profile of miPS-BGM 6.1 Measurement of Protein Amount The amount of protein released from miPS-BGM prepared at each primary drying temperature and suspended in PBS for two days was measured. 0.09 g of miPS-BGM, which had been primary dried at each temperature, was suspended in 500 μL of PBS and shaken at 37°C and 54 rpm. After two days, the supernatant was collected. The concentration of total protein released from miPS-BGM was measured using a 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 that had been primarily dried at temperatures of -40°C, -10°C, and 30°C. hMSCs and miPS-BGM were co-cultured in growth medium on a dish at a cell density of 5 x ^10. After 7 days, hMSCs were washed with PBS and then 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) at room temperature for 5 minutes. 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 then 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 that the number of cells around miPS-BGM (-40°C) and miPS-BGM (-10°C) was significantly higher than that of miPS-BGM (30°C) (Figure 8C).

6.2 Effect on Migratory Activity The effect of proteins released from miPS-BGM on the cell migration activity of hMSCs was evaluated using a scratch wound healing assay. The wound healing assay was performed as follows: hMSCs were seeded in a 12-well plate at a cell density of 1.0 × ¹⁰ cells/mL. After 24 hours, a scratch was created by linearly scraping the hMSCs with 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) suspended at a concentration of 5 mg/mL. Immediately after scratching and 9 hours later, images of hMSCs were taken and quantified using ImageJ (NIH Image) (Figure 9). The number of cells in the scratch area for each group was counted from the images. The number of migratory cells in the medium containing miPS-BGM (-40°C) was significantly higher than that in the medium containing miPS-BGM (-10°C) or miPS-BGM (30°C) (P<0.05) (Figure 9, lower graph).

6.3 Analysis of Protein Components The above results indicate that cellular behavior around miPS-BGM is primarily controlled by the constituent proteins of miPS-BGM. Therefore, we comprehensively analyzed the protein components 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 substitute, 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). Compared to β-TCP, miPS-BGM showed higher levels of 121 cytokines/cytokine-related proteins. Although details are not shown here, miPS-BGM maintained higher levels of 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.

To clarify the effect of freeze-drying on the proteins that make up miPS-BGM, proteome analysis (mass spectrometry) was used to analyze changes in protein composition before and after freeze-drying. miPS 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 protein composition.

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

7.1 Morphological characteristics by macroscopic observation As shown in Figure 12A, the morphology of miPS-BGM (-40°C) and miPS-BGM (-10°C) was round and granular, with similar diameters, whereas miPS-BGM (30°C) did not maintain its granular morphology and appeared to be finely crushed.

7.2 Compression Test Results To prepare the molded specimens for compression tests, 100 μL of miPSC cell aggregates were 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 at a crosshead speed of 1.0 mm/min using an Instron universal testing machine (Instron 3342) equipped with a 50 N load cell. Bluehill™ 2 software was used for machine operation and data analysis. All tests were repeated six times, and the mean values and standard deviations 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).

7.3 Scanning Electron Microscopy Figure 12C shows the SEM microstructure of miPS-BGM prepared using a tray-type freeze dryer. The miPS-BGM samples, which were 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 contained aggregates of relatively uniformly sized spherical structures with a particle size of approximately 10 μm (average particle size 7 μm). On the other hand, the miPS-BGM (30°C) samples exhibited flake-like structures with an average particle size of 1–2 μm.

Figure 12D shows the SEM microstructure of miPS-BGM (Patent Document 2) fabricated using a conventional cooling-stage freeze dryer (JFD-320 Freeze Drying Device, JEOL) at a primary drying temperature of -10°C. SEM observation revealed aggregates of spherical structures with particle sizes of 5 μm or less, with smaller surface granular structures and less porosity than miPS-BGM fabricated 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.

Furthermore, EDX was performed on the miPS-BGMs produced using the tray freeze dryer to evaluate the elemental composition of each miPS-BGM. SEM-EDX images revealed the elemental distribution of Ca, P, C, and O in the surface layer 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), at 1.34 and 1.41, respectively. In contrast, the Ca/P ratio for miPS-BGM (30°C) was 0.43 (Figure 12E).

EDX was also performed on miPS-BGM (Patent Document 2) and non-decalcified freeze-dried human bone (OraGRAFT Cortical: LifeNet Health) produced using a cooling-stage freeze-dryer with the primary freeze-drying temperature set at -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) produced using the tray-type freeze-dryer described above. Furthermore, the Ca/P ratio of non-decalcified human freeze-dried bone was 1.41, a value similar to the Ca/P ratio of miPS-BGM (-10°C) produced using the tray-type freeze-dryer. Therefore, it was suggested that the miPS-BGM produced using the tray-type freeze dryer has a Ca/P ratio closer to that of natural bone compared to the miPS-BGM described in Patent Document 2, and is more suitable as an artificial bone (bone filler).

Thus, even though the primary drying temperatures appear to be the same, the reason for the different properties of miPS-BGM is thought to be 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 is able to control the temperature inside the drying chamber by controlling the shelf temperature, and the presence of an internal sensor allows for more accurate temperature control. Furthermore, it is thought that this is because the vacuum state is reached quickly, at less than 3 Pa in just 10 minutes, allowing for rapid drying. This shows that temperature control during freeze drying is important for producing high-performance bone substitute materials.

7.4 Calcium Elution Test A calcium elution test was performed on miPS-BGM using different primary drying 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 (30°C) was significantly lower than that of miPS-BGM (-40°C) and (-10°C) (P<0.05) (Figure 12G).

7.5 Analysis by X-ray Diffraction (XRD) Furthermore, the microstructure was analyzed using X-ray diffraction (XRD). The composition of miPS-BGM was analyzed using X-ray diffraction analysis with a SmartLab 9SW (Rigaku Corporation). 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 similar to the pattern of carbonate apatite and were high and sharp, indicating that miPS-BGM has high crystallinity and purity.

7.6 Transmission Electron Microscopy (TEM) Analysis 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. The TEM image of miPS-BGM revealed a cluster structure 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-BGMs dried at different temperatures can be distinguished based on the average length of their 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 IL10 in macrophages, suggesting that the short, needle-like crystal structure obtained under dry conditions at -40°C may have an effect on local immune control.

8. Analysis of Human iPS Cell-Derived Bone Filler 8.1 Analysis of Protein Components of Human iPS Cell-Derived Osteoblast Masses Protein components of human iPS cell-derived osteoblast masses before freeze-drying were analyzed. Human iPS osteoblast masses were prepared according to Patent Documents 1 and 3. Human iPS osteoblast masses were collected in Eppendorf tubes and stored in a freezer at -80°C. For comparison, human iPS cell masses 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).

Mass spectrometry identified 7,678 proteins contained in the human iPS osteoblast cell mass. Excluding proteins constitutively expressed in human iPS cells (proteins expressed before osteoblast differentiation induction), 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 detected. Collagen, COL1A2, COL4A1, COL5A1, COL6A6, COL15A1, COL16A1, COL21A1, and COL26A1 were also detected.
8.2 Analysis of human iPS cell-derived bone filler using antibody arrays The protein components of human iPS cell-derived bone filler (hiPS-BGM) were also analyzed in the same manner as in 6.3. hiPS-BGM was prepared according to the inventors' method (Patent Documents 1 and 3). After ultrasonically pulverizing hiPS-BGM in PBS, the supernatant collected using a centrifuge was stored in a -80°C freezer. For comparison, human freeze-dried bone (FDBA: OraGRAFT (LifeNet Health)) was treated in the same manner, and a supernatant sample was prepared. Comprehensive analysis using antibody arrays was performed using a Human Antibody Array L-1000 (Filgen, Inc.). The proteins contained in hiPS-BGM and FDBA were comparatively analyzed, and were compared with the protein data contained in miPS-BGM to identify common proteins (FIG. 14).

While existing bone substitute materials (FDBA) contain almost no protein, hiPS-BGM contains 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; angiogenesis proteins such as EGFR-VEGF/PK1, angiopoietin-like 1, angiopoietin-like factor, PDGF-AA, angiopoietin-2, VEGF R3, VEGF R1, and VEGF; and beta IG-H3 and IL-17B. It was revealed that the stratum corneum contains many proteins related to bone metabolism, such as IFN-beta, IFN-beta, calcitonin, IL-17R, MMP-10, and TNF-beta. Other hiPS-BGM includes COCO, Cadherin-13, Clusterin, C5/C5a, CD30 Ligand/TNFSF8, Kallikrein 5, IL-4, IGFBP-1, IL-1 F8/FIL1 eta, GRO, IL-8, Glut3, IL-29, ALCAM, CNTF, IL-21 R, CD163, IL-5, CD71, IL-21, EDG-1, Prolactin, IL-26, Growth Hormone (GH), GDF11, CD40 Ligand/TNFSF5/CD154, Cathepsin It was revealed that the hiPS-BGMs were rich in proteins such as 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).

9. Evaluation of the osteogenic induction ability of hiPS-BGM The temperature conditions for primary drying of hiPS-BGM were investigated. As with miPS-BGM, hiPS-BGM was pre-frozen at -80°C, and cell aggregates were primary freeze-dried at different shelf temperatures to produce cells. 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 ability of hiPS-BGM was evaluated based on the degree of differentiation. hMSCs were induced using osteogenic differentiation medium in the same manner as described in 3.1.

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 (pore size 0.22 μm, 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 induction, hMSCs were fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries) and stained for alkaline phosphatase (ALP) to evaluate the degree of osteogenic differentiation.

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 positive region was analyzed using ImageJ image analysis software.

The results are shown in Figure 15. The upper panel shows an ALP staining image on day 14 of bone differentiation induction, and the lower panel shows the quantitative results. In the figure, "No Addition" indicates bone differentiation medium without hiPS-BGM. The lower panel shows the quantitative results (mean ± SD, n = 3). Bone substitutes prepared at a drying shelf temperature of -10°C during primary drying were shown to have significant bone differentiation induction ability compared to bone substitutes prepared at 30°C. Furthermore, preparation at a lower temperature of -40°C has even higher bone differentiation induction ability. Since -10°C has higher bone differentiation induction ability compared to 30°C, the primary drying temperature should be set to -10°C or below. Furthermore, since -40°C or below has higher bone differentiation induction ability, it is more preferable to perform primary drying at -40°C or below.

10. Effect of hiPS-BGM on Migratory Activity The effect of proteins released from hiPS-BGM on the cell migration activity of hMSCs was evaluated using a scratch wound healing assay. The wound healing assay was performed using hMSCs as described in Section 6.2. hMSCs were seeded onto a 12-well plate at a cell density of 4.0 x ¹⁰ 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 with culture medium, and the medium was replaced with growth medium containing hiPS-BGM (-40°C), hiPS-BGM (-10°C), or hiPS-BGM (30°C) suspended at a concentration of 5 mg/mL. Suspension was performed using an ultrasonic grinder as described above. Images of hMSCs were taken immediately after scratching and 6 hours later (Figure 16, upper image shows image 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 in the medium containing hiPS-BGM (-10°C) or miPS-BGM (30°C) (P<0.001) (Figure 16, lower graph).

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.

Here, detailed analysis was performed using mouse iPS cells, but the analysis results for protein components, bone formation 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 for bone substitute materials produced using human iPS cells.

Claims (4)

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. The method for producing a bone substitute material according to claim 1, characterized in that the primary drying process is carried out for a period of 10 minutes or more and 6 hours or less. The method for producing a bone substitute material according to claim 1, characterized in that the pre-freezing step is carried out at a temperature of -40°C or lower and -80°C or higher. 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.