JP5099309B2 - Cultured bone production equipment - Google Patents

Description

The present invention relates to a cultured bone manufacturing apparatus .

  Conventionally, when there is a bone defect in bone fractures due to bone tumor curettage, trauma, etc., or when there is a bone defect, autologous bone transplantation and allogeneic bone transplantation have been performed as surgical treatment methods. The problem remains. For example, a bone repairing method is performed by repairing a bone by filling a bone defect with a bone grafting material. However, when the bone defect covers a wide area, it is difficult to treat with this technique.

  In recent years, regenerative medicine has been attempted for bone defects. In other words, there is a technique in which bone marrow is collected from a patient, artificially proliferated with osteoblasts based on mesenchymal stem cells contained in the collected bone marrow, and then returned to the patient's body again. Has been done. In this case, to promote differentiation from bone marrow collected from patients themselves to osteoblasts, and after sufficient growth, return them to the patient's body, thus promoting bone formation without causing immune reactions such as rejection Is expected.

  Also, development of a system for culturing such cells has been attempted (see, for example, Patent Documents 1 and 2). If differentiation from bone marrow cells to osteoblasts can be promoted, fewer bone marrow cells can be collected from the patient and returned to the patient in a short time after collection, thus shortening the treatment period It becomes.

On the other hand, low-power ultrasound (Low-Intensity
Pulsed Ultrasound: hereinafter abbreviated as LIPUS. ) It is known that pulse stimulation has a function of promoting fracture repair (see, for example, Patent Document 3). A treatment device having LIPUS conditions (frequency 1.5 MHz, repetition frequency 1 kHz, pulse width 200 μs, output value 30 mW / cm ² ) that brings about such an action has been developed as an ultrasonic fracture treatment device by Exogen, USA.

  This ultrasonic fracture treatment device is used in clinical trials for healing promotion effects such as tibial shaft fracture (for example, see Non-Patent Document 1), distal radius fracture (for example, Non-Patent Document 2), and animal models. The effectiveness (for example, refer nonpatent literature 3) is illuminated. These ultrasonic treatments use non-invasive and non-thermal effects, and the minute mechanical stimulation generated on the bone surface by the sound pressure of ultrasonic waves affects the fracture site and surrounding cells. It is considered.

  As a study to elucidate these mechanisms, LIPUS has reported that expression of factors such as COX-2 related to enhanced osteogenesis in early osteoblasts has been enhanced, and that cross-linking of collagen molecules has been enhanced. There is a report that this has led to an increase in bone strength (see, for example, Non-Patent Documents 4 and 5). In addition, various researchers are examining the effects of ultrasonic stimulation on osteoblasts, bone cells, chondrocytes, etc. under a number of ultrasonic conditions, but details of the mechanism have not yet been clarified. Furthermore, in a three-dimensional chondrocyte culture model, there is a report that ultrasonic stimulation promotes the differentiation of chondrocytes (see, for example, Non-Patent Document 6), but the details are unknown.

A regenerative medicine business in which mesenchymal stem cells collected from a patient's bone marrow fluid are cultured and then differentiated into bone cells on a bone filling material and provided as cultured bone has been put into practice. As such a bone prosthetic material, β-tricalcium phosphate (β-TCP) is commercially available, and is applied to the regenerative medicine technology by utilizing the property of being replaced by the bone itself while being absorbed by the living body in the orthopedic field.
JP 2004-119 A JP 2004-16045 A U.S. Pat. No. 4,530,360 JBone and Joint Surg, 76A, p26-34, 1994 JBone and Joint Surg, 79A, p961-973, 1997 JBone Mineral Res, vol.16, p671-680, 2001 NaruseK et al., J Bone Mineral Res, vol.18, p360-369, 2003 SaitoM et al., Bone, vol.35, p644-655, 2004 NishikoriT et al., John Wiley & Sons Inc., p201-206, 2001

However, the method of seeding bone marrow cells collected from a patient on a bone filling material, adding an osteoinductive agent to differentiate into osteoblasts, and returning them to the patient when a sufficient amount is reached requires time for culturing. Therefore, there are inconveniences such as delayed treatment time and delayed bone regeneration.
The present invention has been made in view of the above-described circumstances, and provides a cultured bone production apparatus that can further promote differentiation from bone marrow cells to osteoblasts and quickly repair a bone defect of a patient. The purpose is that.

  In order to achieve the above object, the present inventors conducted extensive studies on a method for promoting the differentiation of bone marrow cells into osteoblasts. As a result, the present inventors conducted ultrasound on bone marrow cells cultured in a bone grafting material. The following means are provided by finding a method of promoting differentiation into osteoblasts by irradiating with.

In the reference example of the present invention, a bone differentiation inducer is added to bone marrow cells seeded on a bone grafting material, and the frequency is 1.3 to 2 MHz, the repetition period is 100 to 1 kHz, the burst width is 10 to 2000 μs, and the spatiotemporal average output Provided is a method for producing cultured bone that is cultured while being irradiated with an ultrasonic pulse of 100 mW / cm ² or less.

In the above reference example, it is preferable that the ultrasonic pulse has a frequency of 1.5 MHz, a repetition period of 1 kHz, a burst width of 200 μs, and a spatiotemporal average output of 30 mW / cm ² .
Moreover, in the said invention, it is preferable that the said bone grafting material is a molded object which consists of a beta tricalcium phosphate porous body.
Furthermore, in the above reference example , the bone differentiation inducer is preferably dexamethasone and β-glycerophosphate.

The present invention is a cultured bone production apparatus for producing cultured bone from bone marrow fluid, a cell extraction unit for extracting bone marrow cells from the bone marrow fluid, and primary culture for culturing the bone marrow cells extracted by the cell extraction unit Part, an enzyme container that stores the proteolytic enzyme that is put into the primary culture part and separates the cultured bone marrow cells, a filling material container that contains a bone filling material, and the proteolytic enzyme from the primary culture part The bone marrow cells separated by the above are loaded, and the bone grafting material is loaded from the filling material container so that the bone marrow cells are seeded on the bone grafting material, and a medium further containing a bone differentiation inducer is provided. A secondary culture unit for culturing the bone marrow cells seeded on the bone grafting material by being charged, and the secondary culture unit has a frequency of 1. for the bone marrow cells seeded on the bone grafting material. 3 to 2 MHz Repetition period 100～1.0KHz, burst width 10Myuesu～2000myuesu, provides a cultured bone manufacturing apparatus comprising an ultrasonic oscillator for irradiating a space-time average power 100 mW / cm ² or less of the ultrasonic pulses.
In the above invention, the ultrasonic pulse preferably has a frequency of 1.5 MHz, a repetition period of 1.0 kHz, a burst width of 200 μs, and a spatiotemporal average output of 30 mW / cm ² .
Moreover, in the said invention, it is preferable that the said bone grafting material is a molded object which consists of a beta tricalcium phosphate porous body.
Moreover, in the said invention, it is preferable that the said bone differentiation inducer is a dexamethasone and (beta) glycerophosphate.
Moreover, in the said invention, it is good also as providing the test | inspection part which test | inspects the presence or absence of the infection of the said bone marrow fluid or the said cultured bone.

  According to the present invention, the bone marrow cells seeded on the bone grafting material can be differentiated into osteoblasts at an early stage, and a mature cultured bone that can be filled in a bone defect of a patient in a short period of time can be produced at an early stage. The treatment period can be shortened and the burden on the patient can be reduced.

Hereinafter, a culture apparatus 100 and a manufacturing method according to an embodiment of the present invention will be described with reference to FIGS. 1 and 2.
The culture apparatus 100 according to the present embodiment includes a culture vessel 103 containing a bone filling material 101 seeded with bone marrow cells and a culture medium 102 to which a bone differentiation-inducing agent is added, and a culture vessel 103 disposed below the culture vessel 103. And an ultrasonic irradiation device 104 that irradiates bone marrow cells with ultrasonic waves U.

  The culture tank 103 includes a container portion 105 that stores the culture medium 102 and a lid portion 106 that is disposed so as to close the upper opening of the container portion 105. The container portion 105 and the lid portion 106 are made of a material that is not toxic to the living body. In particular, the container portion 105 is made of a transparent material or a translucent material such as polyethylene or polystyrene. The bottom surface of the container part 105 is formed with a thickness of about 1 mm.

  The lid portion 106 covers the entire outer periphery of the upper opening of the container portion 105, and has a flange portion 108 that is disposed with a gap between the container portion 105 and the spacer 107, and an inner portion of the flange portion 108. And a convex portion 109 which is inserted into the container portion 105 and brought into contact with the liquid level of the culture medium 102 stored in the container portion 105. At least the convex portion 109 of the lid portion 106 is made of an ultrasonic absorber, for example, silicone rubber.

  The ultrasonic irradiation device 104 stores sterilized water 110, accommodates the culture tank 103 in the vicinity of the water surface, for example, a constant temperature water tank 111 that maintains the water temperature at a culture temperature of 37 ° C., and the constant temperature water tank 111. And an ultrasonic oscillator 112 that irradiates the ultrasonic pulse U toward the bottom surface of the culture vessel 103. When the ultrasonic oscillator 112 is operated to emit the ultrasonic pulse U, the ultrasonic pulse U propagates through the sterilized water 110 and is incident on the culture tank 103 from the bottom surface of the container portion 105 of the culture tank 103, and the culture tank. Ultrasonic vibration is applied to bone marrow cells on the bone prosthetic material 101 accommodated in 103.

The ultrasonic oscillator 112 emits an ultrasonic pulse U having a frequency of 1.3 to 2 MHz, a repetition period of 100 to 1 kHz, a burst width of 10 to 2000 μs, and a spatiotemporal average output of 100 mW / cm ² or less.

The bone grafting material 101 constitutes cultured bone by the bone marrow cells carried thereon being induced to differentiate into osteoblasts. For example, high purity β tricalcium phosphate (β-TCP) porous For example, a body formed into a cubic block shape is used.
The medium 102 is an osteogenic medium containing dexamethasone or β-glycerophosphate (β-GP) as a bone differentiation inducer.

In order to manufacture cultured bone using the culture apparatus 100 according to this embodiment configured as described above, the bone formation medium 102 is stored in the container portion 105 of the culture tank 103, and bone marrow cells are stored in the bone filling material 101. The seeded material is put into the container portion 105 of the culture tank 10 and the upper opening is covered with the lid portion 106. And this culture tank 103 is accommodated in the constant temperature water tank 111 which stored the sterilized water 110, and the ultrasonic oscillator 112 is operated.

  The ultrasonic pulse U emitted from the ultrasonic oscillator 112 propagates through the sterilized water 110 and enters the culture tank 103. In the present embodiment, since the bottom surface of the container part 105 constituting the culture tank 103 is formed as thin as about 1 mm, reflection at the interface between the sterilized water 110 and the container part 105 does not occur so much, and the ultrasonic pulse U Is smoothly incident on the culture tank 103 and irradiated to the bone marrow cells seeded on the bone grafting material 101.

  Since the culture tank 103 is accommodated in the constant temperature water tank 111, it is maintained at an appropriate culture temperature of 37 ° C. and differentiation of bone marrow cells into osteoblasts by the action of the ultrasonic pulse U emitted from the ultrasonic oscillator 112. Is promoted efficiently. As a result, there is an effect that differentiation induction into osteoblasts can be greatly promoted compared to conventional cases, and cultured bone can be obtained at an early stage.

Example 1
Next, examples of the cultured bone manufacturing method according to the above-described embodiment will be described.
In this embodiment, a 6-hole culture dish (hereinafter also referred to as culture dish 105) is used as the container part 105 of the culture tank 103, and silicone rubber is molded as the cover part 106 covering the upper part of the culture dish 105. An ultrasonic absorption plate was used. A frame 113 was placed in the constant temperature water tank 111, and the constant temperature water tank 111 was supported near the surface of the sterilized water 110 by the frame 113. In the frame 113, an ultrasonic oscillator 112 is disposed below the culture dish 105 at a position corresponding to each of the six wells 105 a of the culture dish 105. The ultrasonic output conditions were a frequency of 1.5 MHz, a repetition period of 1.0 kHz, a burst width of 200 μs, and a spatiotemporal average output of 30 mW / cm ² . Reference numeral 114 denotes a cable for supplying electric power to the ultrasonic oscillator 112.

As bone marrow cells, bone marrow cells derived from rat femur were used. Specifically, bone marrow cells were collected from a 6-week-old femur of Fischer334 (rat), and primary culture was performed in a 75 mm ² culture dish. The medium at this time uses α-MEM and 15% FCS and does not contain a differentiation inducer. Bone marrow cells obtained by trypsin treatment were prepared to a concentration of 10 ⁶ cells / mL, and a 5-mm square (125 mm ³ ) and a porosity of 75% β-TCP block (Osferion / Olympus Biomaterials stock) 101) was immersed for 2 hours.

Thereafter, a complex of bone marrow cells and β-TCP (hereinafter referred to as complex 101) is taken out from the medium, placed on a 6-well culture dish 105, and an osteogenic medium (α-MEM, 15% FCS, 10 ⁸ mM). The following two groups cultured in dexamethasone (hereinafter referred to as DEX) and 10 mM β-glycerophosphate (hereinafter referred to as β-GP) 102 were set.
(1) Ultrasonic irradiation group: a group in which ultrasonic pulse U stimulation was performed in a 37 ° C. incubator for the complex + DEX + β-GP.
(2) Control group: a group that is left to stand in a 37 ° C. incubator with respect to the complex + DEX + β-GP and does not perform ultrasonic stimulation.
For the ultrasonic irradiation group, ultrasonic pulse U stimulation (20 minutes / day) was performed by operating the ultrasonic oscillator 112 after one day of culture.

The examination items in this example are listed below.
(1) DNA amount The collected βTCP block 101 was crushed with liquid nitrogen, homogenized with PBS / NP-40 solution, the dsDNA amount was quantified using Hoechist 33258, and the number of cells was calculated.
(2) Alkaline phosphatase (ALP) ALP activity was measured by the Kind-King method using the solution after homogenation as described above.
(3) Osteocalcin (OC) level (mRNA level and protein level)
The mRNA expression level was analyzed by PT-PCR method using a specific device primer. The amount of OC protein was quantified by the EIA method using the recovered βTCP block 101 deashed with 20% formic acid and the extract obtained by desalting and freeze-drying.
(4) Collagen content (mRNA level and protein content)
The mRNA expression level was analyzed by the PT-PCR method using a specific primer. The amount of collagen protein was obtained by hydrolyzing βTCP block 101 with 6N hydrochloric acid at 110 ° C. for 24 hours, and then determining the amount of hyroxyroline by the HPLC method and converting it to the amount of collagen.
(5) Collagen crosslink formation amount (Immature type crosslinks: DHLNL, HLNL, LNL, mature crosslinks: Pyridinoline, Deoxypyridinoline)
After βTCP block 101 was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, immature and mature cross-links were separated and quantified by HPLC, and calculated as per unit collagen amount.

Table 1 shows the relationship between the number of stimulations of the ultrasonic pulse U and the ratio (%) of the ultrasonic stimulation group to the control group.
As analysis samples, 24 hours after one ultrasonic stimulation, 24 hours after three stimulations (3 days), 24 hours after seven stimulations (7 days), and 14 stimulations (2 weeks) Samples 24 hours after and 24 hours after 21 stimulations (3 weeks) were used.

  Before conducting this study, we examined the differentiation state from bone marrow cells to osteoblasts by ultrasonic stimulation without adding differentiation inducers such as DEX. However, induction of osteocalcin and induction of collagen cross-linking pattern to bone type were observed. I couldn't.

  In the control group, an osteoblast differentiation tendency was observed in about 2 weeks of culture. From this result, maturation of the calcified substrate was recognized about one week earlier than the control group by ultrasonic stimulation. DEX, which is a differentiation inducer, is effective in inducing differentiation of bone marrow cells into osteoblasts, but it has been found that the longer the culture period, the more effective.

  Therefore, also in this result, no significant difference was observed due to ultrasonic stimulation after the second week of culture, but the amount of osteocalcin and the amount of collagen up to that time were significantly different in the complex 101 in the ultrasonic stimulation group. Is accumulated within. From this, it was suggested that a substrate with high maturity, that is, a substrate with very high bone quality, could be obtained even if the substrate mass was the same. As described above, it has been clarified that the differentiation of bone marrow cells in the bone graft material 101 in the presence of a differentiation inducer into osteoblasts is promoted by ultrasonic stimulation.

(Example 2)
The same test as in Example 1 was carried out, cultured for 21 days, collected over time and analyzed. In addition, collagen cross-linking patterns (immature cross-linking: (DHLNL + HLNL) / LNL, mature cross-linking: DPD / PYD) were added as examination items.
A photomicrograph of the 14th day of culture is shown in FIG. FIG. 4A shows the ultrasonic irradiation group, and FIG. 4B shows the control group. According to this, it can be seen that the bone matrix is formed around the TCP in the ultrasonic irradiation group.

5 shows the number of cells, FIG. 6 shows the amount of collagen, FIG. 7 shows the ALP activity, FIG. 8 shows the amount of osteocalcin, FIG. 9 shows the amount of immature cross-linking, FIG. 10 shows the amount of mature cross-linking, and FIG. FIG. 12 shows mature cross-linked “bone pattern”.
There is no significant difference in the number of cells between the ultrasonic irradiation group and the control group, but the difference between the two is significant in other analysis results. The amount of immature crosslinking was determined by the sum of DHLNL, HLNL and LNL, and the amount of mature crosslinking was determined by the sum of PYD and DPD.

In the immature bridge “bone pattern”, when (DHLNL + HLNL) / LNL is 9 or more, it can be said to be a bone pattern. In the mature bridge “bone pattern”, (DPD / PYD) is 0.2. That's the bone pattern. According to FIGS. 11 and 12, it takes 21 days for the control group to shift to the bone pattern, but the bone pattern is very early on the seventh day due to ultrasonic irradiation. I understand.
As a result, it was found that the “amount” and “quality” of collagen can be increased at an early stage by ultrasonic irradiation, and a bone-type collagen matrix can be induced.

Here, the porous body as the bone grafting material 101 will be described.
Many materials such as calcium phosphate and collagen have been applied to scaffold materials for bone tissue regeneration using cells. Among them, β-TCP is extremely useful because it has a resorbability and serves as a scaffold for bone formation and has a feature of completely replacing bone. Furthermore, in order to allow cells to enter the interior, they must effectively attach, proliferate and differentiate to the scaffold material. Therefore, in the present invention, it is desirable to use a porous body made of β-TCP as the bone grafting material 101. Further, the porosity is desirably 60 to 80%, and most preferably 75%. A porosity of 75% is the most suitable pore property for applying ultrasonic stimulation.

Moreover, the culture bone manufacturing apparatus 1 to which the culture apparatus 100 according to the present embodiment is applied will be described.
The cultured bone production apparatus 1 supplies bone marrow fluid 2 collected from a patient, a bone filling material 101 to which cells contained in the bone marrow fluid 2 are attached, and culture media 4 and 102, so that cultured cells are The adhered bone 5 and data indicating the result of infection test in each step are output.

More specifically, the cultured bone production apparatus 1 includes a storage unit 6, a cell extraction unit 7, a cell culture unit 8 a 8 b, a sealing unit 9, and an inspection unit 10 in an aseptic room 11.
The storage unit 6 includes a plurality of containers that separately store the bone filling material 101 and the culture media 4 and 102, respectively. The medium 4 is composed of, for example, MEM (Minimal Essential Medium), FBS (Fatal Bovine Serum: fetal bovine serum) and an antibiotic as shown in the figure, and in a state where these are mixed. Or in separate containers 12, 13, 14. Moreover, the container which stores the culture medium 4 is accommodated in the refrigerator which is not shown in figure, for example, is preserve | saved at 4 degreeC. The antibiotic is, for example, a penicillin antibiotic. The bone marrow fluid 2 is supplied to the cell extraction unit 7.

  The cell extraction unit 7 is, for example, a centrifuge, and collects bone marrow fluid 2 having a high specific gravity from the bone marrow fluid 2 by receiving the bone marrow fluid 2 from a container containing the bone marrow fluid 2 and turning it. Be able to.

The bone marrow culture part is composed of a primary culture part 8a and a secondary culture part 8b. As shown in the figure, the primary culture unit 8a is connected to each of the containers containing the cell extraction unit 7 and the culture medium 4. The primary culture unit 8a includes a culture vessel 15 that receives the bone marrow cells extracted in the cell extraction unit 7 and the medium 4 stored in the container, and a stirring unit that stirs the bone marrow cells and the medium 4 in the culture vessel 15. (Not shown) and means 16 for maintaining the bone marrow cells and the culture medium 4 mixed with stirring at a predetermined temperature (eg, 37 ± 0.5 ° C.) and CO ₂ concentration (eg, 5%). The bone marrow cells can be cultured under a constant culture condition for a predetermined time. A container 17 for storing a proteolytic enzyme such as trypsin is connected to the primary culture unit 8a.

  Thereby, in the culturing process in the primary culture unit 8a, by supplying trypsin into the culture vessel 15, bone marrow cells adhering to the bottom surface of the culture vessel 15 can be detached from the culture vessel 15. In the figure, reference numeral 18 denotes a centrifuge for extracting bone marrow cells mixed in trypsin.

The culture apparatus 100 according to this embodiment described above is used for the secondary culture unit 8b.
The secondary culture unit 8b is connected to the primary culture unit 8a, and is also connected to a container (not shown) that houses the bone grafting material 101. Similar to the primary culture section 8a, the secondary culture section 8b also includes a culture vessel 103, a stirring means (not shown), and a culture condition maintaining means 20.

The primary culture unit 8a and the secondary culture unit 8b are provided with medium exchange means 27 and 28 for periodically exchanging the medium in the culture vessels 15 and 19. The medium exchange means 27 and 28 include, for example, container rotating means (not shown) for rotating the culture containers 15 and 103 around the horizontal axis. That is, the culture medium 15 can be discharged from the culture containers 15 and 103 by tilting or inverting the culture containers 15 and 103 by rotating the culture containers 15 and 103 around the horizontal axis by operating the container rotating means. ing.
In addition, the medium exchange means 27 and 28 are provided with a waste medium container 29 for accommodating the discharged medium 102 ′.

The sealing unit 9 is connected to the secondary culture unit 8b, and is configured to seal and output the cultured bone produced in the secondary culture unit 8b in the sealed container 30. In the figure, reference numeral 31 denotes a specimen extraction unit for taking a specimen for examination from cultured bone as a final product.
In addition, the test unit is a fully automatic real-time PCR (Polymerase Chain) connected to the bone marrow fluid 2 container, the waste medium container 29 of the primary culture unit 8a, the waste medium container 29 of the secondary culture unit 8b, and the sample extraction unit 31.
Reaction) equipment. In this fully automatic real-time PCR apparatus, a primer capable of detecting all of fungi, bacteria, endotoxin, virus, mycoplasma and the like is used.

In this cultured bone manufacturing apparatus 1, the storage unit 6, the cell extraction unit 7, the cell culture units 8a and 8b, the sealing unit 9 and the inspection unit 10 are disposed in the aseptic room 11, and the aseptic room 11 includes, for example, Like the clean room, the inside is set to a constant positive pressure, and the number of dust per unit volume is limited by a particle counter or the like.

Thus, according to this cultured bone manufacturing apparatus 1, a secondary culture unit that uses bone marrow cells collected from bone marrow fluid and cultures by adding a bone differentiation-inducing agent on bone filling material 101 such as β-TCP. In 8b, by irradiating a low-power ultrasonic pulse U with a specific frequency, differentiation into osteoblasts is promoted, and cultured bone containing autologous bone cells is produced early for repairing bone defects such as fractures. be able to.

It is a longitudinal cross-sectional view which shows the culture apparatus which concerns on one Embodiment of this invention. It is a perspective view which shows the external appearance of the apparatus used for the Example of the culture apparatus of FIG. It is a whole block diagram which shows the culture bone manufacturing apparatus using the culture apparatus which concerns on this embodiment. It is an Example of the manufacturing method of the cultured bone of this invention, Comprising: It is a figure which respectively shows the microscope picture of a culture | cultivation 14th day, (a) ultrasonic irradiation group, (b) control group. It is a graph which shows the change of the cell number in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of the collagen amount in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of ALP activity in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of the amount of osteocalcin in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of immature bridge | crosslinking in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of mature bridge | crosslinking "amount" in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of immature bridge | crosslinking "bone type pattern" in the Example of the manufacturing method of the cultured bone of this invention. It is a graph which shows the change of mature bridge | crosslinking "bone type pattern" in the Example of the manufacturing method of the cultured bone of this invention.

Explanation of symbols

U ultrasonic pulse 100 culture apparatus 101 bone filling material 102 medium 103 culture tank 104 ultrasonic irradiation apparatus

Claims (5)

A cultured bone manufacturing apparatus for manufacturing cultured bone from bone marrow fluid,
A cell extractor for extracting bone marrow cells from the bone marrow fluid;
A primary culture unit for culturing the bone marrow cells extracted by the cell extraction unit;
An enzyme container for storing a proteolytic enzyme that is put into the primary culture unit and peels off the cultured bone marrow cells;
A filling material container for containing a bone filling material;
The bone marrow cells peeled off by the proteolytic enzyme from the primary culture part are charged, and the bone marrow cells are seeded on the bone filler by loading the bone filler from the filler container. Further, a secondary culture unit for culturing the bone marrow cells seeded on the bone filling material by introducing a medium containing a bone differentiation inducer,
The secondary culture section has a frequency of 1.3 to 2 MHz, a repetition period of 100 to 1.0 kHz, a burst width of 10 μs to 2000 μs, and a spatiotemporal average output of 100 mW / cm ² or less on the bone marrow cells seeded on the bone grafting material. A cultured bone manufacturing apparatus including an ultrasonic oscillator for irradiating an ultrasonic pulse. The cultured bone production apparatus according to claim 1, wherein the ultrasonic pulse has a frequency of 1.5 MHz, a repetition period of 1.0 kHz, a burst width of 200 µs, and a spatiotemporal average output of 30 mW / cm ² .   The cultured bone production apparatus according to claim 1 or 2, wherein the bone grafting material is a molded body made of a β-tricalcium phosphate porous body.   The cultured bone production apparatus according to any one of claims 1 to 3, wherein the bone differentiation-inducing agent is dexamethasone and β-glycerophosphate.   The cultured bone production apparatus according to any one of claims 1 to 4, further comprising an inspection unit that inspects for presence or absence of infection of the bone marrow fluid or the cultured bone.

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