Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis

Tanikake, Yohei; Akahane, Manabu; Furukawa, Akira; Tohma, Yasuaki; Inagaki, Yusuke; Kira, Tsutomu; Tanaka, Yasuhito

doi:10.3727/096368916x694166

Cited by 17 publications

(14 citation statements)

References 33 publications

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“…Because CPC materials promote bone regeneration mainly by ion elution, we needed to determine the effect of β‐TCP content on the dissolution of calcium and phosphorus ions in the material. The CPC samples were immersed in α‐MEM medium (Gibco, Thermo Fisher Scientific, Inc., NY) at 0.1 g/ml CPC/α‐MEM ratio, placed in a 37°C incubator for 24 hr and then cultured using a calcium colorimetric assay kit (Abcam) and a phosphate colorimetric assay kit (Abcam) to measure the concentration of calcium and phosphorus ions in the medium according to previous methods (Mestres et al, ; Tanikake et al, ). Six parallel controls were established for each set of samples.…”

Section: Methodsmentioning

confidence: 99%

Osteogenic stimulation of human dental pulp stem cells with self‐setting biphasic calcium phosphate cement

Zhuang

Xie

et al. 2019

J Biomed Mater Res

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Calcium phosphate cement (CPC) is widely used as a repair material for bone defects. However, there is no report on the use of biphasic CPC combined with dental pulp stem cells (DPSCs) for bone tissue engineering. The aim of this study was to construct biphasic CPC and to investigate its effect in the osteogenic stimulation of human DPSCs (hDPSCs) and macrophage polarization. Biphasic CPC was developed by adding different ratios of β‐TCP to an equimolar mixture of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA). The biocompatibility, osteogenic capacity, and inflammatory reactions of the cements were examined. Our results show that biphasic CPC containing both HA and β‐TCP was successfully fabricated. The addition of β‐TCP did not increase necrosis or apoptosis of DPSCs. Significant increases in hDPSC mineral deposition and osteogenic marker (ColI and ALP) expression were observed in 20% TCP‐CPC. The addition of β‐TCP did not promote macrophage polarization to a proinflammatory phenotype. In conclusion, biphasic CPC with hDPSCs for bone regeneration was developed for the first time; the biphasic CPC combined with hDPSCs is expected to be used for the repair and regeneration of bone.

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Section: Methodsmentioning

confidence: 99%

Osteogenic stimulation of human dental pulp stem cells with self‐setting biphasic calcium phosphate cement

Zhuang

Xie

et al. 2019

J Biomed Mater Res

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“…On days 8 and 14, total calcium content of the culture medium was measured using the methylxylenol blue method (Calcium E-test Wako Kit; Wako Pure Chemical Industrials, Kyoto, Japan) ( n = 6 for each group). In the past study, very high correlation between the estimated cumulative calcium reduction and the osteogenic activity in vivo (ALP activity and OC content) was repotted ( r > 0.90) [ 19 ]. Therefore, since reduced calcium in the culture medium reflects the amount of calcium deposited, the calcium concentration in the culture medium can be used as a marker of osteogenesis in tissue-engineered bone [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

“…The gene expression level of OC, ALP, Runt-related transcription factor-2 (Runx2), collagen type 1a1 (Col1a1), and collagen type 4a1 (Col4a1) were evaluated [ 19 ]. Total RNA was extracted from the cultured cells (RNeasy Micro Kit (50) 74004; QIAGEN, Hilden, Germany) and converted to cDNA using oligo-dT primers (oligo-dT primers (Promega, USA)) according to the manufacturer’s protocol ( n = 3 for each group).…”

Section: Methodsmentioning

confidence: 99%

In vitro osteogenesis of rat bone marrow mesenchymal cells on PEEK disks with heat-fixed apatite by CO2 laser bonding

Kawasaki

Inagaki

Akahane

et al. 2020

BMC Musculoskelet Disord

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Background Polyether-ether-ketone (PEEK) is increasingly being used for spinal applications. However, because of its biologically inactive nature, there are risks of false joint loosening and sinking. PEEK materials are coated with apatite to enhance the osteoconductive properties. In this study, we aimed to evaluate whether strontium apatite stimulate osteogenesis on the surface of PEEK by using the CO2 laser technique. Methods We prepared non-coated disks, laser-exposed disks without apatite, and four types of apatite-coated by laser PEEK disks (hydroxyapatite (HAP), strontium hydroxyapatite (SrHAP), silicate-substituted strontium apatite (SrSiP), and silicate-zinc-substituted strontium apatite (SrZnSiP)). A part of the study objective was testing various types of apatite coatings. Bone marrow mesenchymal cells (BMSCs) of rats were seeded at a density of 2 × 104/cm2 onto each apatite-coated, non-coated, and laser-irradiated PEEK disks. The disks were then placed in osteogenic medium, and alkaline phosphatase (ALP) staining and Alizarin red staining of BMSCs grown on PEEK disks were performed after 14 days of culture. The concentrations of osteocalcin (OC) and calcium in the culture medium were measured on days 8 and 14 of cell culture. Furthermore, mRNA expression of osteocalcin, ALP, runt-related transcription factor 2 (Runx2), collagen type 1a1 (Col1a1), and collagen type 4a1 (Col4a1) was evaluated by qPCR. Results The staining for ALP and Alizarin red S was more strongly positive on the apatite-coated PEEK disks compared to that on non-coated or laser-exposed without coating PEEK disks. The concentration of osteocalcin secreted into the medium was also significantly higher in case of the SrHAP, SrSiP, and SrZnSiP disks than that in the case of the non-coated on day14. The calcium concentration in the PEEK disk was significantly lower in all apatite-coated disks than that in the pure PEEK disks on day 14. In qPCR, OC and ALP mRNA expression was significantly higher in the SrZnSiP disks than that in the pure PEEK disks. Conclusions Our findings demonstrate that laser bonding of apatite—along with trace elements—on the PEEK disk surfaces might provide the material with surface property that enable better osteogenesis.

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“…Reduced calcium in the medium would reflect the amount of calcium deposited. Therefore, calcium concentration in the culture medium could be employed as a marker of osteogenesis in tissue-engineered bone [19].…”

Section: Methodsmentioning

confidence: 99%

Silicate-substituted strontium apatite nano coating improves osteogenesis around artificial ligament

Egawa

Inagaki

Akahane

et al. 2019

BMC Musculoskelet Disord

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Background Treatment of anterior cruciate ligament injuries commonly involves the use of polyethylene terephthalate (PET) artificial ligaments for reconstruction. However, the currently available methods require long fixation periods, thereby necessitating the development of alternative methods to accelerate the healing process between tendons and bones. Thus, we developed and evaluated a novel technique that utilizes silicate-substituted strontium (SrSiP). Methods PET films, nano-coated with SrSiP, were prepared. Bone marrow mesenchymal cells (BMSCs) from femurs of male rats were cultured and seeded at a density of 1.0 × 10 4 /cm 2 onto the SrSiP-coated and non-coated PET film, and subsequently placed in an osteogenic medium. The osteocalcin concentration secreted into the medium was compared in each case. Next, PET artificial ligament, nano-coated with SrSiP, were prepared. BMSCs were seeded at a density of 4.5 × 10 5 /cm 2 onto the SrSiP-coated, and non-coated artificial ligament, and then placed in osteogenic medium. The osteocalcin and calcium concentrations in the culture medium were measured on the 8th, 10th, 12th, and 14th day of culture. Furthermore, mRNA expression of osteocalcin, alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (Runx2) was evaluated by qPCR. We transplanted the SrSiP-coated and non-coated artificial ligament to the tibiae of mature New Zealand white rabbits. Two months later, we sacrificed them and histologically evaluated them. Results The secretory osteocalcin concentration in the medium on the film was significantly higher for the SrSiP group than for the non-coated group. Secretory osteocalcin concentration in the medium on the artificial ligament was also significantly higher in the SrSiP group than in the non-coated group on the 14th day. Calcium concentration on the artificial ligament was significantly lower in the SrSiP group than in the non-coated group on the 8th, 10th, 12th, and 14th day. In qPCR as well, OC, ALP, BMP2, and Runx2 mRNA expression were significantly higher in the SrSiP group than in the non-coated group. Newly formed bone was histologically found around the artificial ligament in the SrSiP group. Conclusions Our findings demonstrate that artificial ligaments using SrSiP display high osteogenic potential and thus may be efficiently used in future clinical applications.

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Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis

Cited by 17 publications

References 33 publications

Osteogenic stimulation of human dental pulp stem cells with self‐setting biphasic calcium phosphate cement

Osteogenic stimulation of human dental pulp stem cells with self‐setting biphasic calcium phosphate cement

In vitro osteogenesis of rat bone marrow mesenchymal cells on PEEK disks with heat-fixed apatite by CO2 laser bonding

Silicate-substituted strontium apatite nano coating improves osteogenesis around artificial ligament

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