Masako Ikeuchi

Zinc-releasing calcium phosphate for stimulating bone formation ☆

Zinc-containing tricalcium phosphate (ZnTCP) is biocompatible and bioactive, and functions as an effective zinc carrier. ZnTCP contains a maximum of 12 mol% of zinc. ZnTCP ceramics and composite ceramics of ZnTCP and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 were prepared by sintering at 1100 °C. ZnTCP/HAP continued to release zinc for more than 50 days in 0.9 wt.% sodium chloride solution. ZnTCP/HAP with a zinc content of 1.2 wt.% significantly increased osteoblastic MC3T3-E1 cell proliferation and alkaline phosphatase activity of rat stromal cells in vitro. ZnTCP/HAP with a zinc content of 0.316 wt.% increased bone formation by 51% (n=6; p=0.0509) 4 weeks after implantation in rabbit compared to the control without zinc. A zinc concentration within a noncytotoxic level of a solution does not completely block the crystal growth of apatite in the solution. When ZnTCP is added to calcium phosphate cement, the ZnTCP showed neither inhibitory nor promoting effect on the setting ability of the cement. All these findings suggest that the zinc-containing tricalcium phosphate is a biomaterial that has a pharmaceutical effect of promoting bone formation.

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.

Bone tissue engineering using novel interconnected porous hydroxyapatite ceramics combined with marrow mesenchymal cells : quantitative and three-dimensional image analysis

We developed fully opened interconnected porous calcium hydroxyapatite ceramics having two different pore sizes. One has pores with an average size of 150 μm in diameter, an average 40-μm interconnecting pore diameter, and 75% porosity (HA150). The other has pores with an average size of 300 μm in diameter, an average 60–100-μm interconnecting pore diameter, and 75% porosity (HA300). Because of its smaller pore diameter, HA150 has greater mechanical strength than that of HA300. These ceramics were combined with rat marrow mesenchymal cells and cultured for 2 weeks in the presence of dexamethasone. The cultured ceramics were then implanted into subcutaneous sites in syngeneic rats and harvested 2–8 weeks after implantation. All the implants showed bone formation inside the pore areas as evidenced by decalcified histological sections and microcomputed tomography images, which enabled three-dimensional analysis of the newly formed bone and calculation of the bone volume in the implants. The bone volume increased over time. At 8 weeks after implantation, extensive bone volume was detected not only in the surface pore areas but also in the center pore areas of the implants. A high degree of alkaline phosphatase activity with a peak at 2 weeks and a high level of osteocalcin with a gradual increase over time were detected in the implants. The levels of these biochemical parameters were higher in HA150 than in HA300. The results indicate that a combination of HA150 and mesenchymal cells could be used as an excellent bone graft substitute because of its mechanical properties and capability of inducing bone formation.

In-Situ Visualization and Quantification of Mineralization of Cultured Osteogenetic Cells

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, β-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.

Role of metallothionein isoforms in bone formation processes in rat marrow mesenchymal stem cells in culture.

Temporal changes in mRNAs for metallothionein (MT) isoforms in subcultures of rat marrow mesenchymal stem cells (MSCs) after treatment with dexamethasone were investigated. Both MT-1 and MT-2 mRNA expression in the cultured MSCs with dexamethasone showed maximum levels at d 1, whereas ALP and osteocalcin mRNAs peaked at d 12. MT-3 mRNA was not detected in the cultured MSCs at any time. The expression level of MT-2 mRNA at d 1 was 9.4-fold higher than that of MT-1 mRNA. Finally, osteoblast differentiation and mineralization of MSCs at d 14 was inhibited by the addition of a common antisense oligonucleotide for both MT-1 and MT-2 in the culture medium during the first 4 d. The results suggest that the large amounts of MT-2 are produced in the early stage of subculture of MSCs, and this might regulate their differentiation.

Marrow Mesenchymal Stem Cells Cultured on Alumina Ceramics (From Basic Science to Clinical Applications)

Menchymal stem cells (MSCs) were obtained by culturing fresh r at and human bone marrow cells. The MSCs were then cultured on alumina ceramic disks in the medium supplemented with vitamin C, glycerophosphate and dexamethasone. The high alkaline phospha tase activities (ALP) of the cells on culture dishes as well as ceramic disks were observed. Patterns of ALP stain and mineralization on the dishes were similar to those on alumina cera mic disks. These results showed that alumina ceramic surface well supports the osteogenic differ entiation of rat and human marrow MSCs. The differentiation leads to the appearance of active osteobl asts together with matrix formation; i.e. in vitro bone formation on the ceramic surface. Based on the findings, we init iated clinical trials using thus formed in vitro bone on alumina ceramics. After separation of MSCs from patient’s aspirated bone marrow cells, we cultured the MSCs on the surface of artificial ankle prosthesis made by alumina ceramics and used for the treatments of the same patie nts. Introduction When metals or their alloys are implanted at host sites, a fibr ous membrane encapsulates the implants, with the thickness of the membrane proportional to the degree of metallic corrosion [1]. In contrast, the fibrous membrane is minimal and some areas show regenerated ne w (bo ) tissue in contact with the implanted materials without other types of fibrous tissues inte rvening, when alumina (Al 2O3) ceramics or titanium devices are implanted into the bone defects. However, the interface between the bone and the implants is not strong and the interface detaches easily upon shear and distraction loads. In this regard, when certain types of glass ceramics and calci um phosphate ceramics are implanted, chemical bonding is established between the regenerated bone tissue a nd s rf ce of the implant [2,3]. This bone/ ceramic interface is very strong and stable and, upon loading , breakage usually occurs inside the bone or ceramic, but not at the interface. Such bone/material (gla ss and calcium phosphate ceramics) interaction is referred to as bone bonding and these mate rials are called “Bioactive materials”[2]. Though alumina ceramics can show bone apposition onto thei r surface without fibrous capsular formation, the bone cannot bond to the alumina surface and, thus, alumna is not bioactive [3-5]. Due to the non-bioactive nature of the alumina ceramics, the ce ramics cannot be expected to show stable interface between host bone tissue and the ceramic surface. To overc ome the drawback, we try to load the osteogenic cells on alumina surface prior to im plantation into bone defects. The cells we used are cultured mesenchymal stem cells (MSCs) de rive from bone marrow [4], because MSCs can differentiate into bone forming osteoblasts. The present pape r describes the osteogenic differentiation on the alumina ceramic surface. Key Engineering Materials Online: 2003-05-15 ISSN: 1662-9795, Vols. 240-242, pp 651-654 doi:10.4028/www.scientific.net/KEM.240-242.651

Comparison of Recombinant Growth/Differentiation Factor-5 (rGDF-5) and Bone Morphogenetic Protein-2 (rBMP-2) in the In Vivo Bone Formation in Porous Hydroxyapatite Ceramic

Various recombinant growth factors have been used for promoting osteoblastic differentiation cascade. To compare the growth/differentiation factor-5 (GDF-5) and bone morphogenetic protein-2 (BMP-2) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), the bone formation was assessed by rat subcutaneous implantation of 5 kinds of hydroxyapatite (HA) implants; namely GDF/HA composites, BMP/HA composites, MSCs/HA composites and the MSCs/HA composites supplemented with recombinant mouse GDF-5 (GDF/MSCs/HA) or recombinant human BMP-2 (BMP/MSCs/HA). Neither the GDF/HA nor the BMP/HA composites exhibited any bone formation at any time after implantation. At both 2 and 4 weeks after implantation, obvious de novo bone formation together with active osteoblasts was seen histologically in many pores of the GDF/MSCs/HA and BMP/MSCs/HA composites. The GDF/MSCs/HA and BMP/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. These findings indicate that the two growth factors synergistically enhance de novo bone formation capability of MSCs/HA composites and the importance of ceramic surface to retain and to deliver the molecules of growth factors for the cell differentiation and maturation.