S. Tamai

In vitro bone formation by rat marrow cell culture

Fresh marrow cells were obtained from the femora Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) to leach confluent. After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35 mm well in the presence of FCS, 10 mM beta-glycerophosphate, 82 micrograms/mL ascorbic acid phosphate, and 10(-8)M dexamethasone (Dex). Osteoblastic cells and microscopic mineralized nodules began to appear at about 1 week after the subculture, and at 2 weeks many macroscopic nodules that showed high alkaline phosphatase activity (ALP) and appearance of bone Gla protein (BGP) mRNA were evident. As demonstrated by in situ hybridization, the mRNA was manifested by cuboid-shaped cells (osteoblastic cells). X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) showed the mineralization of fine crystals of hydroxyapatite comparable to natural rat bone mineral. In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show the osteoblastic phenotype expression. These analyses indicate that Dex-induced mineralization using rat bone marrow cell culture is an in vitro counterpart of bone formed in vivo. Such a culture is useful for investigating materials/ osteogenic cells interactions.

Osteogenic differentiation of cultured marrow stromal stem cells on the surface of bioactive glass ceramics

To investigate the significance of apatite-wollastonite-containing glass ceramic (AW ceramic) surfaces and the biological apatite layer formed on these surfaces, rat marrow cell culture, which shows osteogenic differentiation, was carried out on four different culture substrata (control culture dish, two AW ceramics, each having a different surface roughness, and a ceramic on which an apatite layer was formed. A culture period of 2 weeks in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain on all substrata. The stain on the apatite-formed AW ceramic was the most intense, the enzyme activity being about twice that of the control culture dish, which had the lowest stain and activity of the four substrata. Northern blot analysis of bone Gla protein (BGP) showed the same tendency, that is, the amount of BGP mRNA from cultured cells on the apatite-formed AW ceramics was the highest and the mRNA on the control dish was the lowest. These data indicate that the glass ceramic surface promotes osteoblastic differentiation and that the promotion can be further enhanced by the formation of a biological apatite layer on the ceramic surface.

Analysis of gene expression in osteogenic cultured marrow/hydroxyapatite construct implanted at ectopic sites : a comparison with the osteogenic ability of cancellous bone

We investigated the in vivo osteogenic ability of cultured marrow cells subcultured in porous hydroxyapatite. This osteogenic ability was compared with that of cancellous bone grafts. Fresh marrow cells were obtained from young adult rat femora and cultured in a standard medium for 10 days, then trypsinized and used to make constructs of porous hydroxyapatite (HA) and cultured marrow cells. An additional 2-week culture (subculture) was performed for the construct in standard medium with and without the addition of dexamethasone (Dex). The 2-week subcultured constructs then were implanted into subcutaneous sites of syngeneic rats. These implants and the rat cancellous bone were harvested and prepared for gene expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) as well as for histological analysis. ALP and OC mRNAs could be detected in Dex-treated subcultured constructs 1 week after implantation with an increase at 2 weeks, comparable to the observation in cancellous bone. Histological analysis showed active bone formation even 1 week postimplantation. In contrast, the subcultured constructs without the addition of Dex did not show bone formation, and only small levels of ALP and OC mRNAs were found. These results indicate that the bone tissue formed by grafting the Dex-treated construct of cultured marrow cells and hydroxyapatite possesses a high osteoblastic activity comparable to that of viable cancellous bone. Thus the prefabricated osteogenic subcultured marrow/HA construct may be applicable in bone reconstructive surgery.

Osteogenic Phenotype Expression of Allogeneic Rat Marrow Cells in Porous Hydroxyapatite Ceramics

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone‐forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagles minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of β‐glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

Al2O3 doped apatite‐wollastonite containing glass ceramic provokes osteogenic differentiation of marrow stromal stem cells

Fresh marrow cells were obtained from femora of Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) until confluence. After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35-mm well in the presence of FCS, beta-glycerophosphate, and ascorbic acid phosphate on four different culture substrata. The period of subculture was 2 weeks; the substrata used were the culture dish, apatite-wollastonite containing glass ceramic (AW), hydroxyapatite coated AW (HA/AW), and Al2O3 doped AW (Al/AW). The HA coating was attained by the incubation of AW in simulated physiological solution. The glass matrix of AW and HA/AW contained MgO, CaO, P2O5, and SiO2; Al/AW contained Al2O3 in addition to these components. The subculture on Al/AW substratum showed many alkaline phosphatase (ALP) positive nodules and the highest ALP activity. On a Northern blot analysis the housekeeping gene of beta-actin mRNA was evenly detected from the cells cultured on all substrata; however, bone-specific osteocalcin mRNA was only detected from the cells on Al/AW. These results indicate that Al/AW provokes the osteoblastic differentiation of marrow stromal stem cells.

Progenitor endothelial cells on vascular grafts: An ultrastructural study

The morphology of progenitor endothelial cells on vascular graft surfaces is addressed in this report. Such cells were seen to attach to intima-expressed CD34 and Flk-1 antigen and showed positive 5-bromo-2-deoxyuridine (BrdU) uptake. We examined CD34 and Flk-1 antigen-expressing endothelial progenitor cells three-dimensionally using confocal laser scanning microscopy (CLSM). Under detailed CLSM observation, through an ameboid-form cell, these progenitor endothelial cells changed from a globular to a flattened form. We also investigated these morphological changes using scanning electron microscopy. From these results, progenitor endothelial cells were observed not only near the advancing edge of endothelium, but also around the developing intimal site. Their form also changed from globular to flattened as observed in the CLSM results. These morphological changes were seen more frequently near the advancing edge and around the developing intimal site. They attached directly to vascular prosthesis fibers and likewise covered the graft luminal surface. Progenitor endothelial cells in any form had a common surface structure. We conclude from our results that progenitor endothelial cells can attach to graft fibers directly without clotting and directly cover the graft luminal surfaces.

Self-setting hydroxyapatite cement as a carrier for bone-forming cells

To investigate the feasibility of using self-setting hydroxyapatite cement as a carrier for marrow cells having a high osteogenic ability, a porous form of this cement was fabricated and combined with cultured marrow cells. Marrow cells were obtained from the femurs of a seven-week-old male Fischer 344 rat and cultured in Eagles MEM containing 15% fetal bovine serum for ten days before being combined with the porous cement or with Interpore 200 hydroxyapatite as a control. The composites were subcutaneously implanted into syngeneic rats and harvested after six weeks. In both types of implants, active osteoblasts together with bone formation were detected in contact with the pore surfaces. No cartilage formation was observed in any of the pores. Both types of implants with and without marrow cells caused very little foreign body reaction. These results indicate that self-setting hydroxyapatite cement containing marrow cells possesses a high osteogenic ability and may be useful as a bone graft substitute as well as a novel delivery system for bone-forming cells.

Viability of ischemia/reperfused muscles in rat: a new evaluation method by RNA degradation

The rats skeletal muscle viability was evaluated using the muscle viability index (MVI) which reflects the mRNA degradation. To evaluate ischemic injury of the muscle, 24 hind limbs of Fischer rats (three subgroups of eight rats each) were preserved at normothermia for 1, 3 and 6 h and then tibialis anterior muscle was harvested. To investigate ischemia/reperfusion injury, another 48 limbs were transplanted to recipient Fischer rats after the ischemia at normothermia for 1, 3 and 6 h, respectively. The transplanted muscles were harvested on day 3 and day 7 after transplantation. Eight fresh muscles were also harvested and used as control. Total RNA isolated from each muscle was fractionated by electrophoresis and hybridized with 32P‐labelled cDNA of GAPDH, and the radioactivity of intact and degraded GAPDH mRNA was measured. MVI was calculated as follows, MVI = {X/(X + Y)} × 100, where X and Y represent the radioactivities corresponding to intact GAPDH and degraded GAPDH mRNA band, respectively.

The role of serum imbibition for skin grafts

Numerous studies of grafted skin suggest that full-thickness skin grafts are nourished by exudate from the recipient bed called a serum imbibition. However, whether serum imbibition by itself is sufficient for nourishment of skin grafts has not been shown definitely and directly. To clarify the role of serum imbibition, we performed a comparative study between 20 skin grafts and 20 musculocutaneous flaps. The nourishment of the cell in the skin graft is by serum imbibition. That in musculocutaneous flaps is mainly derived from blood supply. We evaluated the nourishment by means of the unique characteristics of the cell cycle. Once cells are put into a synthetic phase, they cannot reverse or stop the progress of the cell cycle. To take advantage of this characteristic of the cell cycle, prewounding methods (40 flaps were lifted once and put back to the original sites prior to the evaluation) were intended for the cells in pre-elevated skin to turn into a proliferating phase. Cells were examined by antibody against proliferating cell nuclear antigen immunohistologically, to determine whether they had turned into the proliferating phase or not. After 3 days, all flaps were reelevated; half (20 flaps) had their muscle layer and the neurovascular bundle removed to make a full-thickness skin graft. The rest (20 flaps) were only lifted. They were sutured back to the original sites. Ten skin grafts and musculocutaneous flaps each were harvested at 3 hours (1st day) and at 11 days (11th day) after the second operation. Bromodeoxyuridine, which is a thymidine analog and is taken into the cells in the synthetic phase, was introduced intraperitoneally 2 hours before the harvest. All flaps and grafts were evaluated histologically and immunohistologically. Proliferating cell nuclear antigen analysis showed that the prewounding method induced the cells of skin grafts and musculocutaneous flaps to proliferate before the implantation. Regarding the bromodeoxyuridine uptake, no significant differences could be seen between skin grafts and musculocutaneous flaps irrespective of their different nourishment. No structural changes, such as degenerative or necrotic, could be seen at the hair follicle and other glands even at the 11th day. Almost all of the layers of skin grafts survived as long as they were checked by light microscopy (hematoxylin and eosin stain). No differences could be seen between musculocutaneous flaps and skin grafts or between the 1st and 11th days in this study. We concluded that serum imbibition is sufficient for nourishment of skin grafts, just as blood supply is sufficient for nourishment of musculocutaneous flaps.