Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• the present invention relates to a bone substitute material used in surgically treating damaged bone tissue, and a method for producing the same.
• the inventors have developed a method to three-dimensionally culture iPSCs in a bioreactor, induce bone differentiation, and produce osteoblastic masses of human iPSCs (Patent Document 1). Although spheroids (cell masses) and organoids derived from iPSCs are promising biomaterials, transplanting undifferentiated living cells carries the risk of tumor development. In addition, because the transplanted material is foreign to the body, it is also necessary to control immunogenicity and local inflammation.
• Non-Patent Document 1 a new concept of regenerative medicine in which cells created in vitro from stem cells are inactivated while retaining their tissue components.
• tissue can be stored for a long period of time in a "seed” state and then transplanted into a patient to induce tissue regeneration, making it possible to supply the required amount of tissue when needed.
• inactivating undifferentiated iPSCs may reduce the risk of potential tumor formation and control inflammation.
• the present inventors have developed a bone filling material that inactivates cells while retaining the activity of promoting bone remodeling, and has excellent osteoconductivity and bone formation induction.
• radiation exposure, heat treatment, drug treatment, and other methods are used to inactivate cells, they may denature proteins, so they thought that freeze-drying, which can inactivate cells while maintaining the physiological activity and structure of proteins, would be better.
• freeze-drying dries materials under low-temperature conditions, suppressing thermal denaturation and chemical changes in materials, and also reducing the rate of product deterioration during the drying process, allowing long-term storage at room temperature.
• freeze-drying conditions may cause chemical changes such as oxidation, deamidation, and hydrolysis, and aggregation due to changes in the higher-order structure of proteins.
• chemical and physical changes during the manufacturing process may reduce the physiological activity of bone filling materials intended for implantation in vivo, and may also act as antigens.
• the present inventors have already produced a bone regenerator by freeze-drying iPS cells that have been induced to differentiate into osteoblasts (Patent Document 2).
• a mass of iPS cells induced to differentiate into osteoblasts is 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.
• a shelf-type freeze-dryer that 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 samples were sufficient.
• 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 bone formation induction ability and production efficiency.
• the objective of the present invention is to provide a bone filler that is highly safe and has higher osteoconductive ability and bone formation induction ability.
• Another objective of the present invention is to provide a method for producing a bone filler that allows for a stable supply, has high production efficiency, and is low cost.
• the present invention relates to the following bone prosthesis and a method for producing the bone prosthesis.
• a method for producing a bone filler derived from stem cells comprising the steps of inducing differentiation of stem cells into osteoblasts to form cell masses, a preliminary freezing step, and a primary drying step of freeze-drying at a temperature between -80°C and -10°C. It was found that the performance of bone prosthesis can be improved by precisely adjusting the temperature during the freeze-drying process. In particular, it was found that controlling the shelf temperature during the primary drying process and freezing at a constant temperature in the drying chamber leads to improved performance of the bone prosthesis.
• the secondary drying step was not carried out, but by carrying out the secondary drying step to desorb the unfrozen water, it is possible to produce a bone prosthesis with good performance.
• FIG. 1 is a diagram showing a schematic diagram of a process for freeze-drying a cell mass produced from iPS cells.
• FIG. 2 is a diagram showing a schematic configuration of the tray-type freeze dryer used.
• FIG. 13 is a diagram showing the results of a study on secondary drying time.
• the evaluation results of miPS-BGM (mouse iPS-bone graft material) that was subjected to primary drying at various temperatures are shown.
• the arrow indicates the edge of the bone defect.
• Scale bar 1.0 mm
• the images show hematoxylin-eosin (HE) staining (upper row) and immunohistochemical staining with anti-galectin-3 antibody of the femoral defect site 3 weeks after miPS-BGM implantation.
• the arrows indicate the edge of the defect site.
• Scale bar 1.0 mm.
• the top row is an enlarged image of the part enclosed by the dotted line in the HE stained image in the top row of Fig. 6A.
• the bottom row is an enlarged image of the part enclosed by the dotted line in the immunohistochemical stained image in the bottom row of Fig. 6A.
• * indicates remaining miPS-BGM, and the arrow indicates a neutrophil.
• Scale bar 100 ⁇ m.
• FIG. 1 shows the results of elemental composition analysis of miPS-BGM produced using a tray-type freeze dryer.
• FIG. 1 shows the results of elemental composition analysis of miPS-BGM produced using a cooling stage freeze-dryer and non-demineralized freeze-dried human bone.
• a bone filler with high bone formation and differentiation ability can be obtained by freezing for 1 hour or more, preferably 2 hours or more, more preferably 4 hours or more, and even more preferably 6 hours or more.
• pre-freezing at a temperature of -40°C or lower, preferably -60°C or lower is appropriate.
• Stabilizers such as sugars and sugar alcohols are known to protect proteins during freezing, so various stabilizers were examined.
• 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 miPSC cell mass equivalent to one 25 cm 2 culture flask was left to stand at 4°C for 1 hour in a PBS solution containing 5 ml of each additive, and then frozen at -80°C for 6 hours.
• Quantitative real-time RT-PCR analysis was performed using Thunderbird SYBR qPCR Mix (Toyobo) and StepOnePlus real-time PCR system (Thermo Fisher Scientific). Expression of the target gene was quantitatively analyzed using the 2- ⁇ Ct method and normalized by the expression of GAPDH.
• the primer sequences used are shown in Table 2. The primers used were primers for human sequences.
• the 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. Paraffin sections 3.5 ⁇ m thick were stained with hematoxylin and eosin (HE) for morphological observation. Galectin-3, a marker molecule for macrophages, was visualized by immunohistochemical staining.
• HE hematoxylin and eosin
• the paraffin sections were deparaffinized by washing twice for 5 minutes with 100% xylene (Fujifilm Wako Pure Chemical Industries, Ltd.) and xylene/ethanol (50/50), and then rehydrated with 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 Chemicals).
• the slides were washed with PBS and treated with 3% hydrogen peroxide (Fujifilm Wako Pure Chemicals) at room temperature for 10 minutes to inhibit endogenous peroxidase activity.
• Mouse macrophage-like cell line J774A.1 (JCRB9108) was obtained from the Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition. The cells were cultured at 37°C, 5% CO 2 , and humidified using 4.5 g/L glucose -containing DMEM (Nacalai Tesque) without sodium pyruvate, 10% FBS (Japan Bioserum), 2 mM L-glutamine (Fujifilm Wako Pure Chemical), penicillin (50 U)/streptomycin (50 ⁇ g/mL). The cells were passaged twice, with the medium being renewed every 3 days.
• the cells were collected and seeded in a 6-well plate at a cell density of 3.0 ⁇ 10 5 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 collected and analyzed for expression of 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-1 ⁇ , Tnf ⁇ , Il-6) or anti-inflammatory (Il-10, Tgf ⁇ 1, Il-11) cytokines was analyzed on days 1 or 3 using real-time RT-PCR analysis. As shown in the upper images of Figure 7, in the groups to which miPS-BGM produced at either temperature was added, cells had gathered and formed colonies on day 1 compared to the growth medium (GM) group. Gene expression of Tgf ⁇ 1 on day 1 after addition of miPS-BGM was significantly higher than that of the GM group.
• Tgf ⁇ 1, Il-10, and Il-11 genes was higher in the miPS-BGM (-40°C) group than in the GM group, miPS-BGM (-10°C), and miPS-BGM (30°C) groups.
• the expression of Il-1 ⁇ , Tnf ⁇ , and Il-6 genes was significantly reduced in all miPS-BGM groups compared to the GM group.
• the expression of Il-1 ⁇ gene was lower in the miPS-BGM (-40°C) group than in the miPS-BGM (-10°C) and miPS-BGM (30°C) groups, although there was no significant difference (lower graph in Figure 7).
• hMSCs were cultured in the presence of miPS-BGM that had been first dried at temperatures of -40°C, -10°C, and 30°C.
• hMSCs and miPS-BGM were mixed and cultured at a cell density of 5 x 10 5 in a growth medium on a dish.
• hMSCs were washed with PBS and then fixed at room temperature using 3.7% paraformaldehyde. The fixed cells were washed with PBS and permeabilized at room temperature for 5 minutes using 0.5% Triton-X (Fujifilm Wako Pure Chemical).
• the effect of proteins released from miPS-BGM on the cell migration 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 5 cells/mL. After 24 hours, the hMSCs were scraped in a straight line using a 200 ⁇ L pipette tip to create a scratch. The cell debris was gently washed with medium, and the medium was replaced with a growth medium in which miPS-BGM ( ⁇ 40°C), miPS-BGM ( ⁇ 10°C), or miPS-BGM (30°C) was suspended at a concentration of 5 mg/mL.
• miPS-BGM maintained higher amounts of bone metabolism-related proteins M-CSF, IL-27, adiponectin/Acrp30, and IL-6, osteoblast-related proteins SPARC, IL-17E, Decorin, TGF- ⁇ 1, osteoprotegerin, IGF-1, osteopontin, osteoactivin/GPNMB, and VCAM-1, and angiogenesis-related proteins PDGF C, endostatin, VEGF, and VEGFC.
• proteome analysis mass analysis
• miPS was 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 be used as the cell mass before freeze-drying.
• the cell mass before freeze-drying and miPS-BGM were evaluated by DIA proteome analysis (LC-MS/MS (DIA), Promega) ( Figure 11). 5,812 proteins were identified, of which 98.7% (5,749 proteins) were common before and after freeze-drying. It was revealed that freeze-drying has almost no effect on the protein composition.
• the microstructure of miPS-BGM produced by the tray freeze dryer is shown by SEM in Figure 12C.
• the miPS-BGM that was dried at each primary drying temperature and then secondarily dried was observed using a scanning electron microscope (SU8000, Hitachi High-Technologies) at an accelerating voltage of 5 kV.
• SU8000 Hitachi High-Technologies
• the miPS-BGM (-40°C) and miPS-BGM (-10°C) samples showed aggregates of spherical structures with a relatively uniform size of about 10 ⁇ m in particle size (average particle size 7 ⁇ m).
• the miPS-BGM (30°C) flake-like structures with an average particle size of 1 to 2 ⁇ m were observed.
• Figure 12D shows the microstructure of miPS-BGM (Patent Document 2) produced by a conventional cooling stage freeze dryer (JFD-320 Freeze Drying Device, JEOL) with the primary drying temperature set to -10°C.
• JFD-320 Freeze Drying Device, JEOL JFD-320 Freeze Drying Device
• EDX was performed on the miPS-BGM produced using the tray freeze dryer to evaluate the elemental composition of each miPS-BGM.
• the SEM-EDX images showed distribution of elements Ca, P, C, and O on the surface layer of the miPS-BGM, suggesting the presence of carbonate apatite.
• 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.
• the ratio for miPS-BGM (30°C) was 0.43 ( Figure 12E).
• EDX was performed on miPS-BGM (Patent Document 2) and human non-demineralized freeze-dried 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, which was lower than the Ca/P ratio (1.41) of miPS-BGM (-10°C) produced using the above-mentioned shelf freeze-dryer.
• the Ca/P ratio of human non-demineralized freeze-dried bone was 1.41, which was similar to the Ca/P ratio of miPS-BGM (-10°C) produced using the shelf freeze-dryer. Therefore, it was suggested that the miPS-BGM produced using the tray freeze dryer has a Ca/P ratio closer to that of natural bone compared to the miPS-BGM in Patent Document 2, making it more suitable as an artificial bone (bone filler).
• the reason why the properties of miPS-BGM are different is thought to be because, although a cooling stage type freeze dryer can freeze-dry samples by placing them on a cooled stage, it is not possible to control the temperature of the drying chamber itself.
• the shelf type freeze dryer used here is able to control the temperature inside the drying chamber by controlling the shelf temperature, and since there is also a sensor inside the chamber, it is possible to control the temperature more accurately. Furthermore, it is thought that this is because the time to reach a vacuum is quick, at 3 Pa or less in 10 minutes, allowing for rapid drying. This shows that temperature control during freeze-drying is important in order to produce a high-performance bone substitute.
• XRD X-ray diffraction
• TEM Transmission Electron Microscopy
• miPS-BGM was embedded in resin, ultrathin sections (thickness 0.05 ⁇ m) were prepared from the resin block, and observed with a scanning TEM (JEM-ARM200F, JEOL Ltd.) at an accelerating voltage of 80 kV.
• a cluster structure of needle-shaped particles was observed from the TEM image of miPS-BGM ( Figure 13B).
• the length of the needle-shaped structure of miPS-BGM (-40°C) was 35 nm on average, which was significantly shorter than the lengths of the needle-shaped structures of miPS-BGM (-10°C) and miPS-BGM (30°C) (48 nm and 64 nm, respectively) (P ⁇ 0.05).
• the length of the needle-shaped structure correlated with the temperature during primary drying (Figure 13C). Therefore, it is possible to distinguish iPS-BGMs with different temperature conditions for primary drying based on the average length of the needle-shaped structure.
• Human iPS cell-derived bone filler 8. Analysis of human iPS cell-derived bone filler 8.1 Analysis of protein components of human iPS cell-derived osteoblast mass Protein components of human iPS cell-derived osteoblast mass before freeze-drying were analyzed. Human iPS osteoblast mass was prepared according to Patent Documents 1 and 3. Human iPS osteoblast mass was collected in an Eppendorf tube and stored in a freezer at -80°C. Human iPS cell mass before the start of osteoblast differentiation induction was used as a comparison. Protein expression and relative quantitative analysis were performed at Kazusa Genome Technologies using DIA proteome analysis (Promega Corporation).
• 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, as well as EGFR-VEGF/PK1.
• tissue contains many proteins related to angiogenesis, such as 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.
• proteins related to angiogenesis such as Angiopoietin-like 1, Angiopoietin-like Factor, PDGF-AA, Angiopoietin-2, VEGF R3, VEGF R1, and VEGF
• proteins related to bone metabolism such as Beta IG-H3, IL-17B R, IFN-beta, Calcitonin, IL-17R, MMP-10, and TNF-beta.
• hiPS-BGM includes COCO, Cadherin-13, Clusterin, C5/C5a, CD30 Ligand/TNFSF8, Kallikrein 5, IL-4, I GFBP-1, IL-1 F8/FIL1 eta, GRO, IL-8, Glut3, IL-29, ALCAM, CNTF, IL-21 R, CD163, IL-5, CD71, IL-21, E It was revealed that they are rich in proteins such as DG-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.
• hiPS-BGM The temperature conditions for primary drying of hiPS-BGM were examined. As with miPS-BGM, preliminary freezing was performed at -80°C, and cell masses were primary freeze-dried at different shelf temperatures to produce cell masses. An extract of hiPS-BGM was added to bone differentiation induction medium, which was used to culture human bone marrow-derived mesenchymal stem cells (hMSCs), and the bone formation induction ability of hiPS-BGM was evaluated based on the degree of differentiation. hMSCs were induced using bone differentiation medium in the same manner as described in 3.1.
• hMSCs human bone marrow-derived mesenchymal stem cells
• hiPS-BGM was suspended in bone differentiation medium at a concentration of 5 mg/mL and pulverized on ice for 2 minutes at 20% ultrasonic amplitude using an ultrasonic pulverizer. After sonication, the suspension was filtered through a syringe filter (pore size 0.22 ⁇ m, Merck) and added to osteoblast differentiation medium for hMSCs. The osteoblast differentiation medium was replaced every 3 days. 14 days after the start of bone differentiation induction, hMSCs were fixed in 10% neutral buffered formalin solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and stained for alkaline phosphatase (ALP) to evaluate the degree of bone differentiation.
• ALP alkaline phosphatase
• freeze-drying inactivates cells and eliminates the risk of tumor formation, and by optimizing the temperature and time, it is possible to produce a bone filler with excellent osteoconductivity and osteoinductive properties.
• Optimal conditions were examined for all three freeze-drying steps: pre-freezing, primary drying, and secondary drying. In each step, the quality changes depending on the processing temperature and time, but the conditions for primary drying are particularly important, and the temperature of the sample must be maintained accurately. Also, from a cost perspective, it is preferable to carry out the three steps in a shorter time and at a higher cooling temperature.

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