JP2006094720A - Substrate for bone cell culture and method for bone cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and inexpensive culture substrate effective for promoting osteogenesis and provide an assay system useful for the determination of various drug actions and the research of cell function by using cultured bone cell. <P>SOLUTION: The substrate for bone cell culture has an apatite particle layer and a collagen layer laminated on a substrate surface. The invention further provides a method for producing the substrate and a method for bone cell culture using the substrate for bone cell culture. The method for assaying the osteogenetic function of a bone marrow cell comprises the culture of bone marrow cells using the substrate for bone cell culture in the presence of calcein and the determination of the amount of produced calcified tubercles by measuring fluorescent intensity. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bone cell culture substrate, a bone cell culture method using this substrate, and a method for assaying the bone forming ability of cultured bone marrow cells.

  Bone is a supporting tissue that is indispensable for maintaining posture and exercising against gravity. At the same time, it has a function of maintaining homeostasis of body fluids as a calcium storage in vivo. In order to keep the calcium concentration in the extracellular fluid constant while maintaining the morphology and mechanical strength, bone is added on one hand to maintain strength, and on the other hand, bone is broken to supply calcium. ing. Bone formation is carried out by osteoblasts derived from undifferentiated mesenchymal cells, and bone resorption is carried out by osteoclasts derived from hematopoietic stem cells. Bone remodeling through formation and resorption is called remodeling, which is always done throughout the body. The differentiation and function of osteoblasts / osteoclasts responsible for bone formation / resorption are complexly controlled by various cytokines (bioactive substances), hormones, mechanical loads, and the like. However, when this control is abnormal and the dynamic balance between bone formation and bone resorption breaks down, bone diseases such as osteoporosis, marble disease, and osteophyte formation occur.

  Various therapeutic agents have been developed for these bone diseases, which are becoming a problem as society ages. Much research has been conducted in vitro on the mechanism by which such therapeutic agents act on bone cells and on the changes in cell function. Furthermore, investigations have been conducted in vitro on the effects of bone-constituting cells on the function of therapeutic agents such as glucocorticoids used in the treatment of rheumatoid arthritis and asthma, which are secondary to bone weakness. Bone cells are known to be greatly affected by differentiation and function regulation by an adherent substrate, that is, a bone matrix composed of calcium phosphate and collagen.

  However, many of the past drug evaluation studies using cell culture are performed on conventional plastic culture substrates. There are few reports of culturing on bone-like substrates that are closer to the in vivo environment. Further, in such a cell culture system, it takes a long period of 3 to 4 weeks until formation of a calcified nodule which is important in determining the drug efficacy. In experiments to screen large numbers of therapeutic candidates, not only is the burden imposed by long-term culture, but the risk of contamination increases.

JP-A-7-194373 describes a method for culturing bone marrow cells, characterized in that a complex of collagen and calcium phosphate having chemical activity is used as a support for bone marrow cells. However, when a support is prepared by suspending a calcium phosphate powder in a mixture of an aqueous collagen solution, a concentrated liquid medium and a buffer according to the disclosed method and then gelling the collagen, the cells are formed on the gel-like support. Although it is preferable as a material for transplantation into hard tissue defects due to the culture, it is difficult to handle when determining drug efficacy and studying cell functions by staining cell bodies and analyzing cell lysates. was there. Furthermore, the gel-like support must always be kept in a wet state, and there is a problem that it is difficult to store and transport.
The collagen-coated cover glass is treated with alkaline phosphatase and phosvitin, and then immersed in β-glycerophosphate for 7 to 14 days to form an apatite layer. Observations and bone resorption pits have been reported (Formation of apatite-collagen complexes, DoiY, Horiguchi T, Moriwaki Y, Kitago H, Kajimoto T, Iwayama Y, Journal of Biomedical Materials Research, 31 (1 ): 43-49, 1996; Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity, Shibutani T, Iwanaga H, Imai K, Kitago M, Doi Y, Iwayama Y, Journal of Biomedical Materials Research, 50 (2): 153-159, 2000). However, in this system, it takes time to form an apatite / collagen layer and it is difficult to adjust the thickness of the coating. Also, collagen coated PET disks and polymers were immersed in simulated body fluid for 6 days to form an apatite layer (Osteoclastic resorption of bone-like apatite formed on a plastic disk as an in vitro assay system, Matsuoka H, Nakamura T, TakadamaH, Yamada S, Tamura J, Okada Y, Oka M, Kokubo T, Journal of Biomedical Materials Research, 42 (2): 278-285, 1998; Bonelike apatite coating on organic polymers: novel nucleation process using sodium silicate solution, Miyaji F, Kim HM, HandaS, Kokubo T, Nakamura T, Biomaterials, 20 (10): 913-919, 1999), a coral disc in which calcium carbonate is replaced with calcium hydroxide is immersed in a suspension of rat bone marrow cells. After 3 weeks, the bone formation was observed after 3 weeks (Biochemical and histological sequences of membranous ossification in ectopic site, Yoshikawa T, Ohgushi H, Okumura M, Tamai S, Dohi Y, Moriyama T, Calcified Tissue Interna tional, 50 (2): 184-188, 1992).
JP 7-194373 A Journal of Biomedical Materials Research, 31 (1): 43-49, 1996 Journal of Biomedical Materials Research, 50 (2): 153-159, 2000 Journal of Biomedical Materials Research, 42 (2): 278-285, 1998 Biomaterials, 20 (10): 913-919, 1999 Calcified Tissue International, 50 (2): 184-188, 1992

  An object of the present invention is to provide a simple and inexpensive culture substrate that promotes bone formation. It is another object of the present invention to provide an assay system useful for various drug efficacy determinations and cell function studies using cultured bone cells.

  The present inventors have found that a bone matrix-like culture substrate can be formed by laminating a layer of apatite (calcium phosphate) particles, which are bone constituents, and a collagen layer on the substrate surface. That is, the present invention provides a bone cell culture substrate characterized by having an apatite particle layer and a collagen layer laminated on a substrate surface.

  In another aspect, the present invention provides a bone cell culture method characterized by using the bone cell culture substrate of the present invention described above.

  In yet another aspect, the present invention provides a method for producing a bone cell culture substrate, characterized in that an apatite particle layer is formed on the surface of a substrate, and then a collagen layer is formed. Preferably, the apatite particle layer is formed by spraying apatite particles suspended in a volatile solvent. More preferably, a plurality of apatite particle layers and a plurality of collagen layers are layered on the substrate surface.

  The present inventors also easily cultured a large amount of samples by culturing bone cells using the bone cell culture substrate of the present invention and labeling the calcified nodules formed with a fluorescent reagent calcein. It was found that bone formation ability can be evaluated. That is, in another aspect, the present invention relates to a method for assaying the bone forming ability of cultured bone marrow cells. The bone marrow cell culture substrate of the present invention is used to culture bone marrow cells in the presence of calcein. The present invention provides a method characterized by measuring the amount of calcified nodules produced by measuring fluorescence intensity.

  The bone cell culture substrate of the present invention is characterized by having an apatite particle layer and a collagen layer laminated on the substrate surface. As the substrate, a plastic culture plate or glass generally used for cell culture can be used. The surface of these base materials may be surface-modified with an organic solvent or sand blast. Thereby, the adhesive force between the substrate and the coating layer is increased, and the coating layer can be made difficult to peel off. In a preferred embodiment, a round cover glass is used as the substrate. In particular, a round cover glass having a size of φ12 can be used in a 24-well multiplate, which is convenient for an osteogenesis assay or observation with an optical microscope.

  Apatite (calcium phosphate) is the main component of human bones and teeth. As the apatite particles, monoclinic hydroxyapatite (HAP), monoclinic tricalcium phosphate (α-TCP), trigonal tricalcium phosphate (β-TCP), or the like can be used. The preferred range of particle size is 1 μm to 5 μm.

  Collagen is a major protein component constituting an extracellular matrix, and is widely used as a biocompatible material or a support for tissue culture. Collagen purified from cattle, pigs, and the like, and various collagen preparations obtained by further chemical or enzymatic treatment thereof are commercially available, and any of these can be used in the present invention.

  The apatite / collagen-coated substrate of the present invention can be produced by forming an apatite particle layer on the substrate surface and then forming a collagen layer. Preferably, the apatite particle layer is formed by spraying apatite particles suspended in a volatile solvent on the surface of the substrate. As the volatile solvent, absolute ethanol or the like can be used. Since the volatile solvent volatilizes quickly after spraying, the apatite fine particles can be sprayed continuously and can be uniformly coded on the substrate. In particular, it is possible to perform uniform coating by repeatedly using a low-concentration suspension. In addition, when spraying a suspension in which calcium phosphate fine particles were dispersed in a collagen solution in advance, coating unevenness was likely to occur, and satisfactory results were not obtained although coating was attempted while changing the concentration of the collagen solution.

A collagen coating layer is then laminated. A collagen solution is prepared by dissolving or diluting a commercially available collagen powder or solution in water, buffer solution, dilute acid, etc. at a concentration of 0.01-10 mg / ml, and this is applied to a substrate coated with apatite particles. Spray or apply. The preferred amount of collagen laminate is ^{1μg / cm 2 -5μg / cm 2} . Moreover, the strength of the substrate can be further increased by repeating the apatite coating and the collagen coating alternately and overlapping the coating layers. A preferable thickness of the coating layer to be formed is 5 μm to 15 μm. The substrate for skeletal cell culture can be sterilized by ultraviolet irradiation, γ-ray irradiation or the like.

  The apatite / collagen-coated substrate of the present invention may further contain a protein contained in a living bone matrix such as bone sialoprotein, osteopontin, TGF-β and the like.

  In another aspect, the present invention provides a method for culturing bone cells using the bone cell culture substrate thus produced. Examples of bone cells include bone marrow cells, osteoblasts, osteoclasts, and bone cells. Bone cells may be collected from animals by methods well known in the art, or established cell lines may be used. The bone cell culture substrate of the present invention is formed on the culture plate, or a glass or plastic piece with the bone cell culture substrate formed on the surface is placed in the culture plate, the medium is added, and the bone cells are seeded. . As the medium, for example, α-MEM, D-MEM and the like can be used, and an optimal one can be selected according to the origin, species, and purpose of the cell. Suitable culture conditions and passage methods for bone cells are well known in the art.

  Differentiation of osteogenic cells into osteoblasts consists of culturing bone marrow cells isolated from mice on this substrate, alkaline phosphatase (ALP) activity indicating differentiation into osteoblasts, and total protein amount correlated with the number of cells. Can be measured by quantifying each. Furthermore, the production of extracellular matrix can be evaluated by fixing cultured cells and observing the sample with a scanning electron microscope.

  As shown in the Examples below, it has been clarified that when bone marrow cells are cultured using the apatite-collagen-coated substrate of the present invention, differentiation into osteoblasts and production of extracellular matrix are promoted. That is, it was confirmed that the apatite / collagen-coated substrate had an effect of accelerating differentiation from bone marrow cells to osteoblasts and promoting production of extracellular matrix, compared to conventional culture substrates.

  In order to produce bone matrix by culturing bone marrow cells on a conventional plastic culture substrate, it took 3 to 4 weeks after adding β-glycerophosphate, ascorbic acid and dexamethasone to the culture solution. On the other hand, on the culture substrate of the present invention similar to the living bone tissue, the production of extracellular matrix was observed in the culture for about one week without adding these. This suggests that the apatite / collagen-coated substrate of the present invention promotes cell differentiation and functional expression.

  In yet another aspect, the present invention provides a method for assaying the osteogenic potential of cultured bone marrow cells. This method includes culturing bone marrow cells in the presence of calcein of the present invention using a bone cell culture substrate and measuring the amount of calcified nodules produced by measuring fluorescence intensity.

  Calcein is a type of fluorescent reagent. When bone cells are cultured using a culture solution containing this reagent, calcium is chelated to calcein when calcified nodules are formed, and the deposited calcified portion is green. It becomes fluorescent. By utilizing the method of the present invention, it is possible to identify newly formed calcified nodules on the apatite-collagen coating matrix. In addition, if cell culture is performed on an apatite / collagen coating substrate placed in a 24-well multi-plate, a general-purpose plate reader that has been used in various assays in the past can easily convert bone formation ability of a large number of samples as fluorescence intensity. It is possible to evaluate.

  By using the culture substrate and assay system of the present invention, it has become possible to evaluate the bone forming ability in an environment similar to a living body. The culture substrate of the present invention has advantages such that the formation of the apatite / collagen layer can be performed in a short time and the thickness and strength of the coating can be controlled by freely stacking the layers. The culture substrate and assay system of the present invention are useful for screening bone disease treatment drugs and candidate drugs used in clinical practice, and further to early osteosynthesis and bone induction for implant materials such as artificial joints and artificial roots. Application is possible.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Production of apatite / collagen-coated substrate Apatite monoclinic crystals (Wako Pure Chemical Industries) were used as calcium phosphate granules for coating the substrate, and rat-tailed collagen type I (BD Biosciences) derived from rats was used as collagen. First, apatite monoclinic crystals were suspended in 100% ethanol at a concentration of 20, 40 or 60 mg / ml. This suspension was sprayed onto a round cover glass (φ12) with an airbrush. After spraying and air-drying to uniformly coat the cover glass with apatite, 0.15 mg / ml rat tail collagen type I solution diluted with 0.2 M acetic acid is continuously sprayed and air-dried. A collagen coating matrix was formed.

  The prepared apatite / collagen-coated substrate was placed in a 24-well multiplate and sterilized by ultraviolet irradiation for 12 hours. Further, the substrate was washed with PBS (phosphate buffered saline), immersed in a culture solution in advance, and used for cell culture.

  FIG. 1 shows the appearance of the manufactured apatite / collagen coating plate, and FIG. 2 shows an image obtained by observing the surface of the coating substrate by SEM. A substrate coated with apatite / collagen on a round cover glass can be used in a 24-well multiplate frequently used in cell culture experiments (FIG. 1). The substrate surface was uniformly coated with apatite granules having a diameter of about 2 to 3 μm (FIG. 2B).

Bone marrow cell isolation and culture Bone marrow cells were collected from 8-12 week old Jc1: ICR mice (Kyudou). The bone ends of the femur, tibia, and humerus were dissected and the bone marrow was washed out and cultured on a culture dish (φ100, BD Falcon) for 2 hours. After culturing, only non-adherent cells were collected and used for experiments. For all cultures, α-MEM (α minimal essential medium, Gibco BRL) supplemented with 10% iFBS (fetal bovine serum, Gibco BRL, 1% antibiotics (penicillin and streptomycin, Gibco BRL)) was used.

Effect of apatite / collagen-coated substrate on cell differentiation In order to evaluate the effect of the substrate of the present invention on bone formation ability, bone marrow cell culture was performed on an apatite / collagen coating substrate and a glass substrate. Put each substrate to a 24-well multiplate, cells were seeded at a density of l0 ⁶ cells / well. The plate was allowed to stand in an incubator maintained at 37 ° C. and 5% CO ₂ concentration, and cultured for 1 week while exchanging half of the culture solution once every 3 to 4 days.

  After one week of culture, cells were buffered (50 mM Trs-HCl, 0.1% Triton) to compare alkaline phosphatase (ALP) activity indicative of osteoblast differentiation and total protein quantity correlated with cell number. -X100, 0.9% NaCl, pH 7.6). The ALP enzyme contained in the lysate was reacted with pNPP (p-nitrophenyl phosphate), and the intensity of coloration was quantified with a plate reader. The total protein amount was quantified using Bradford reagent (Bio-rad). Furthermore, some samples were fixed with 4% paraformaldehyde after culturing, subjected to stepwise dehydration with ethanol, drying and gold vapor deposition, and subjected to observation with a scanning electron microscope (SEM).

  The results are shown in FIGS. 3 and 4, respectively. There was no significant difference in the total protein content between the culture on the apatite / collagen-coated substrate and the culture on the glass substrate. FIG. 5 and FIG. 6 show the results of culturing over time until 3, 7, 10, and 14 days after cell seeding and quantifying the ALP activity and the total protein amount, respectively. There was no change in total protein throughout the culture period. On the other hand, ALP activity increased with the culture period. On the third day of culture, ALP activity on the apatite / collagen-coated substrate was significantly greater than that on the glass substrate, but no significant difference was observed thereafter.

  SEM photographs of bone marrow cells cultured for 10 days on an apatite / collagen-coated substrate and a glass substrate are shown in FIGS. 7 and 8, respectively. On the glass substrate, osteoblast-like cells differentiated from bone marrow cells were flattened (FIG. 8B), and production of extracellular matrix or the like was not observed. On the other hand, on the apatite / collagen-coated substrate, the cells were three-dimensionally distributed and covered the substrate (FIG. 7B). Osteoblast-like cells differentiated from bone marrow cells are actively producing matrix, and cells buried in the matrix produced by themselves were also observed (FIG. 7C). In living bone tissue, it is known that osteoblasts are buried in the bone matrix produced by them and further differentiated into bone cells, and even in cell culture on the apatite / collagen-coated substrate of the present invention, The cell differentiation and matrix production processes were well reproduced.

Effect of Stimulation Factor Addition In order to evaluate whether or not bone formation ability is promoted by adding a stimulation factor to the substrate of the present invention, a substrate was prepared by adding each of the following factors during apatite / collagen coating.
(A) β-glycerophosphate (10 mM) and ascorbic acid (50 μg / ml)
(B) β-glycerophosphate (10 mM), ascorbic acid (50 μg / ml) and dexamethasone (10 ⁻⁸ M)
(C) Vitamin D3 (10 nM)
The alkaline phosphatase activity and the total protein amount on the 3rd, 7th, 10th, and 14th days of culture were measured over time.

  The results are shown in FIGS. 9 and 10, respectively. There was no significant change in total protein throughout the culture period. On the other hand, ALP activity increased with the culture period. However, the increase of ALP activity was not recognized by the addition of each stimulating factor, but rather the activity decreased.

Quantitative Assay for Calcified Nodules The fluorescent reagent calcein was added to the culture solution at a concentration of 1 μg / m1, and bone marrow cell culture was performed for 3 weeks. Since Ca ²⁺ chelates with calcein when a calcified nodule is formed, the deposited calcified portion emits green fluorescence. After culturing for 3 weeks, the cells were fixed with 4% paraformaldehyde and observed with a fluorescence microscope and a confocal laser microscope. The culture was carried out on a glass substrate, and 10 mM β-glycerophosphate, 50 μg / ml ascorbic acid and 10 ⁻⁸ M dexamethasone were added as factors for promoting calcification.

The results are shown in FIG. 11 (A: control, B: β-glycerophosphate and ascorbic acid added, C: β-glycerophosphate, ascorbic acid and dexamethasone added). In culture to which 10 mM β-glycerophosphate, 50 μg / ml ascorbic acid and 10 ⁻⁸ M dexamethasone were added as factors for promoting calcification, calcified nodules emitting green fluorescence were observed by a fluorescence microscope. Furthermore, the result of having observed the nodule with the confocal laser microscope is shown in FIG. 12 (A: differential interference image, B: calcein fluorescence image, c: three-dimensionally reconstructed calcified nodule image). Observation was performed at a pitch of 2 μm in a direction perpendicular to the culture substrate surface to obtain 18 consecutive slice images. By reconstructing this three-dimensionally, it became possible to evaluate the three-dimensional structure of the calcified nodule (FIG. 12C).

Appearance of apatite / collagen coating plate Electron micrograph of apatite / collagen coating substrate surface Comparison of ALP activity after 1 week of bone marrow cell culture Comparison of total protein after one week of bone marrow cell culture Time course of ALP activity in bone marrow cell culture Time course of total protein in bone marrow cell culture SEM image of bone marrow cells cultured on apatite / collagen coated matrix SEM image of bone marrow cells cultured on glass substrate Changes in ALP activity over time in bone marrow cell culture on substrates supplemented with various stimulating factors Changes in total protein over time in bone marrow cell cultures on substrates supplemented with various stimulating factors Observation of calcified nodules using fluorescent microscope Observation of calcified nodule using confocal laser microscope

Claims (7)

A bone cell culture substrate comprising an apatite particle layer and a collagen layer laminated on a substrate surface. A bone cell culture method comprising the bone cell culture substrate according to claim 1. A method for producing a bone cell culture substrate, wherein an apatite particle layer is formed on a substrate surface, and then a collagen layer is formed. A method for producing a bone cell culture substrate, wherein a plurality of apatite particle layers and a plurality of collagen layers are layered on a substrate surface. The method according to claim 3 or 4, wherein the apatite particle layer is formed by spraying apatite particles suspended in a volatile solvent. A bone cell culture method comprising using the bone cell culture substrate produced by the method according to any one of claims 3-5. A method for assaying bone forming ability of cultured bone marrow cells, which is produced by culturing bone marrow cells on the bone cell culture substrate according to claim 1 in the presence of calcein and measuring fluorescence intensity. A method characterized by measuring the amount of calcified nodule.