Reg Gene Expression in Periosteum after Fracture and Its In Vitro Induction Triggered by IL-6.

The periosteum is a thin membrane that surrounds the outer surface of bones and participates in fracture healing. However, the molecular signals that trigger/initiate the periosteal reaction are not well established. We fractured the rat femoral bone at the diaphysis and fixed it with an intramedullary inserted wire, and the expression of regenerating gene (Reg) I, which encodes a tissue regeneration/growth factor, was analyzed. Neither bone/marrow nor muscle showed RegI gene expression before or after the fracture. By contrast, the periosteum showed an elevated expression after the fracture, thereby confirming the localization of Reg I expression exclusively in the periosteum around the fractured areas. Expression of the Reg family increased after the fracture, followed by a decrease to basal levels by six weeks, when the fracture had almost healed. In vitro cultures of periosteal cells showed no Reg I expression, but the addition of IL-6 significantly induced Reg I gene expression. The addition of IL-6 also increased the cell number and reduced pro-apoptotic gene expression of Bim. The increased cell proliferation and reduction in Bim gene expression were abolished by transfection with Reg I siRNA, indicating that these IL-6-dependent effects require the Reg I gene expression. These results indicate the involvement of the IL-6/Reg pathway in the osteogenic response of the periosteum, which leads to fracture repair.

The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell-derived endoderm.

We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)-derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex(-/-) EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha-fetoprotein (Afp) expression. In contrast, stage-specific forced expression of Hex in EBs from wild-type ESCs led to the up-regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c-kit(+) endoderm-enriched EB-derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte-related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm-induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein alpha. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs.

Secretory osteocalcin as a nondestructive osteogenic marker of tissue-engineered bone.

BACKGROUND AND PURPOSE: The constructs of mesenchymal stem cells and ceramics form bone tissue after implantation. Therefore, the constructs can include cultured bone (tissue-engineered bone) as bone grafts. However, the selection of constructs, prior to implantation, with high osteogenic potential is still difficult. We used a rat model to measure the secretory osteocalcin level in culture medium to verify that monitoring osteocalcin levels enables the selection of constructs with high osteogenic potential. METHODS: We prepared constructs of rat hydroxyapatite/cells and used different cell passages of P-1 and P-3 as well as different cell numbers: 1 × 10(5) and 1 × 10(6) cells/ml suspension. These constructs were cultured for 14 days under osteoinductive or nonosteoinductive conditions and implanted subcutaneously in the recipient rat. Secretory osteocalcin in the culture medium was measured using an enzyme-linked immunosorbent assay system during the culture period until day 14, and the osteocalcin content of the harvested construct at 4 weeks was also measured. RESULTS AND CONCLUSION: All constructs except the hydroxyapatite/P-3 construct showed abundant bone formation by histology and both high secretory osteocalcin level in the medium and high osteocalcin content after implantation. Our study revealed that secretory osteocalcin level in vitro was related to osteocalcin content in vivo. The study clearly showed that measuring secretory osteocalcin is a nondestructive method of assessing the osteogenic potential of tissue-engineered bone. One can choose tissue-engineered bone with high osteogenic potential by integrating secretory osteocalcin measurement into the process of bone-tissue regeneration.

Development of a high-specificity enzyme-linked immunosorbent assay (ELISA) system for the quantification and validation of intact rat osteocalcin.

Osteocalcin (OC) exhibits hard tissue-specific expression and binding activity to hydroxyapatite. Therefore, measurement of secreted OC is a very useful index for evaluating osteoblastic differentiation in regenerative bone. In the present study, we established a high-specificity sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of intact rat OC, which could be useful for validating tissue-engineered bone samples nondestructively and continuously. The range of detection with the sandwich ELISA system was 0.1-100 ng OC/mL of cell culture media or rat sera. No cross-reactivities were detected with OCs from other species, including human, bovine and mouse OCs, and other mammalian sera, which would contain the corresponding endogenous OCs. The intra- and inter-assay coefficients of variation were < or =4.9% and

Raloxifene: its ossification-promoting effect on female mesenchymal stem cells.

BACKGROUND: Raloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts' differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation. METHODS: Female and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 10-6 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed. RESULTS: On histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects. CONCLUSIONS: 10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

Impact of metabolic syndrome on brachial-ankle pulse wave velocity in Japanese.

The aim of this study was to determine the effect of metabolic syndrome on brachial-ankle pulse wave velocity (baPWV) by using the new guidelines for diagnosis of this syndrome in Japan. We examined 525 men and women without a history of cardiovascular disease or cancer, and an ankle-brachial index < 0.9. The baPWV was measured using a device (Form PWV/ABI) that simultaneously monitored bilateral brachial and ankle pressure wave forms. Metabolic syndrome was defined as a waist circumference > or = 85 (90) cm in men (women) and two or more of the following risk factors: hypertension, dyslipidemia, and glucose intolerance diagnosed by a 75 g oral glucose tolerance test. The baPWV showed a significant linear relationship with waist circumference, waist-to-hip ratio, body fat, systolic and diastolic blood pressure, triglycerides, fasting glucose, 2-h-postload glucose, fasting insulin, and glycosylated hemoglobin-A1c, after adjusting for sex and age. These factors were also strongly related to fasting insulin levels. When subjects were classified into six groups based on waist circumference and the number of risk factors for metabolic syndrome (0, 1, and > or =2), we found that more risk factors clearly increased the odds ratios for an elevated baPWV in those subjects in the highest quartile of the baPWV distribution in multivariate logistic models. An increase in odds ratio was observed despite a normal waist circumference and may well have been due to increased fasting insulin and blood pressure levels. An increase in the number of risk factors for metabolic syndrome was highly correlated with an increased baPWV, probably due to insulin resistance.

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.

The total amount of DNA damage determines ultraviolet-radiation-induced cytotoxicity after uniformor localized irradiation of human cells.

We have recently developed a micropore ultraviolet irradiation technique. An isopore membrane filter with 3 microm diameter pores shields ultraviolet C radiation from cultured human fibroblasts, leading to partial irradiation within the cells with an average of about three exposed areas per nucleus. This study addressed the question of whether the spatial distribution of DNA damage within a cell nucleus is important in triggering ultraviolet-induced cytotoxicity. We have examined whether there are differences in cytotoxicity between partially ultraviolet-irradiated cells and uniformly irradiated cells after equal amounts of DNA damage were induced in the cell nuclei. We first determined DNA damage formation in normal human fibroblasts using an enzyme-linked immunosorbent assay. We found that 5 J per m2 ultraviolet irradiation produced an equivalent amount of cyclobutane pyrimidine dimers and (6-4) photoproducts per cell as 100 J per m2 with the membrane filter shield. At those doses, we found that both types of ultraviolet irradiation induced similar levels of cytotoxicity as assessed by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Both types of ultraviolet-irradiated cells also had similar cell-cycle distribution and apoptosis as measured by flow cytometry. Moreover, no significant differences in repair kinetics for either type of photolesion were observed between the two different ultraviolet treatments. Similar results were obtained in Cockayne syndrome cells that are defective in transcription-coupled nucleotide excision repair. Present results indicate that in the range of photoproducts studied, the spatial distribution of DNA damage within a cell is less important than the amount of damage in triggering ultraviolet-induced cytotoxicity.

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone.

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).

Prediction of bone mineral density from vitamin D receptor polymorphisms is uncertain in representative samples of Japanese Women. The Japanese Population-based Osteoporosis (JPOS) Study.

BACKGROUND: Vitamin D receptor (VDR) gene polymorphisms have been inconsistently associated with bone mineral density (BMD). To precisely evaluate the associations between three VDR gene polymorphisms and BMD, we performed a large-scale representative study of the Japanese female population. METHODS: Fifty women were randomly selected from each of the 5-year age stratified populations (15-79 years) in each of the three municipalities examined, as a part of the Japanese population-based osteoporosis (JPOS) baseline study in 1996. In the study, BMD at the lumbar spine, hip, and distal forearm were measured using dual energy X-ray absorptiometry (DXA). Two polymorphisms were determined in the VDR gene locus identified by the restriction endonucleases ApaI and TaqI through a novel allele discrimination method using two different allele-specific fluorescence probes. The other VDR gene polymorphism was identified by the restriction endonuclease FokI using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Changes in BMD were determined in a follow-up study 3 years after the baseline study. RESULTS: After the exclusion of women who had any medical or menstrual history affecting BMD, 1434 women were analysed. The annual per cent changes in BMD at the lumbar spine over 3 years in subjects with tt genotype in the TaqI polymorphism were different from other genotypes, both in the women who were premenopausal at the follow-up survey (F-premenopausal women) and in the women who were postmenopausal at the baseline survey (B-postmenopausal women). However, the effects of tt genotype on change in BMD were opposite in the two groups. In addition, these associations or tendencies were not observed at the different skeletal sites. CONCLUSIONS: This study revealed that none of the individual VDR gene polymorphisms displayed consistent association with baseline BMD or BMD change. Therefore, the effect of the VDR genotype on bone mass is negligible in Japanese women.

Osteogenic potential of cultured bone/ceramic construct: comparison with marrow mesenchymal cell/ceramic composite.

Osteogenesis occurs in porous hydroxyapatite (HA) when porous HA blocks combined with marrow mesenchymal cells are grafted in vivo. In vitro bone formation occurs in HA pores when HA combined with marrow cells is cultured in osteogenic medium containing dexamethasone. This cultured bone/HA construct possesses higher osteogenic ability when it is grafted in vivo. In the present study, we compared the osteogenic potential of a cultured bone/HA construct with that of a marrow mesenchymal cell/HA composite. Marrow cells were obtained from the femoral bone shaft of 7-week-old, male Fischer 344 rats and were cultured in T-75 flasks. Cells were concentrated, then frozen and stored in liquid nitrogen for 6 months. The cryopreserved cells were then thawed and prepared for subculture in porous HA (5 x 5 x 5 mm, Interpore 500) and for implantation with porous HA. After 2 weeks of subculture, three cultured bone/HA constructs were separately implanted in the right side of the back of each syngeneic 7-week-old male Fischer rat, and three thawed cell/HA composites (without subculture) were separately implanted in the left side. These implants were harvested at 2 or 4 weeks postimplantation, and prepared for histological, biochemical, and genetic analysis. Alkaline phosphatase activity and osteocalcin content of cultured bone/HA constructs were much higher than those of the cell/HA composites at 2 and 4 weeks postimplantation. Histological examination and gene expression data agreed with these findings. The culture technique discussed herein should facilitate the development of biosynthetic bone implants with higher osteogenic capacity.

Osteogenic differentiation of cultured rat and human bone marrow cells on the surface of zinc-releasing calcium phosphate ceramics.

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of beta-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 microg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 microM of free ZnCl(2) (6.5 microg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl(2)addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery.

Recombinant growth/differentiation factor-5 (GDF-5) stimulates osteogenic differentiation of marrow mesenchymal stem cells in porous hydroxyapatite ceramic.

To evaluate the growth/differentiation factor-5 (GDF-5) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), we subcutaneously implanted five different kinds of hydroxyapatite (HA) ceramic implants: HA alone, GDF-5/HA composites (GDF/HA), MSCs/HA composites, the MSCs/HA composites supplemented with GDF-5 (GDF/MSCs/HA), and recombinant bone morphogenetic protein-2 (BMP/MSCs/HA). Neither the HA alone nor the GDF/HA composites exhibited any bone formation at any time after implantation. At 4 weeks, the MSCs/HA composites exhibited a certain amount of bone formation in some pore areas. In contrast, at 2 weeks, the GDF/MSCs/HA composites exhibited histologically obvious de novo bone formation together with active osteoblasts in many pore areas and additional bone formation at 4 weeks. In the de novo formed bone, neither chondrocytes nor endochondral bone was detected. The GDF/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels and the high level of expression was well maintained even at 4 weeks. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. The results indicate that GDF-5 synergistically enhances de novo bone formation capability of MSCs/HA composite and suggest that tissue-engineered GDF/MSCs/HA composites could be used as bone graft substitutes.

Gender difference regarding selenium penetration into the mouse brain.

A sex difference in the penetration of selenium into the brain was observed using lipopolysaccharide (LPS)-injected mice. The selenium concentration increased in the brains of sodium selenite-injected LPS-treated female mice, but not males. The selenium concentration peaked when selenite was injected 3 h after the injection of LPS into female mice. In addition, selenium in the brain increased when a dosage of 30 micromol/kg and more of selenite was injected into LPS-treated female mice. Also, the selenium concentration in the brain increased and peaked 2-3 h after selenite injection; 24 h later, the level was similar to the Se-only group. The penetration of selenium into the brain was inhibited by pretreatment with aminoguanidine, an inhibitor of nitric oxide synthetase. From the present results, selenium more easily penetrated into the brains of female mice compared to males after LPS treatment, and nitric oxide may have affected the penetration. However, the sex difference mechanism for selenium penetration needs further investigation.

REG Iα gene expression is linked with the poor prognosis of lung adenocarcinoma and squamous cell carcinoma patients via discrete mechanisms.

The aim of the present study was to evaluate the effects of the REG Iα and REG Iß genes on lung cancer cell lines, and thereafter, the expression of REG family genes (REG Iα, REG Iß, REG III, HIP/PAP and REG IV) in lung cancer in relation to patient prognosis was evaluated. Lung adenocarcinoma (AD) and squamous cell carcinoma (SCC) cell lines expressing REG Iα or REG Iß (HLC-1 REG Iα/Iß and EBC-1 REG Iα/Iß) were established, and cell number, cell invasive activity, and anchorage-independent cell growth were compared with these variables in the control cells. The expression levels of REG family genes were evaluated by real-time RT-PCR in surgically resected lung cancers, and disease-specific survival (DSS) curves were generated. The HLC-1 REG Iα/Iß cell line showed significant increases in cell number and anchorage-independent cell growth compared with the control cells. EBC-1 REG Iα/Iß cells showed significant increases in cell invasive activity and anchorage-independent cell growth as compared with the control cells. Except for the REG Iß gene, expression of other REG family genes was observed in the surgically resected samples; however, DSS was significantly worse only in stage I patients who were positive for REG Iα expression than in patients who were negative for REG Iα expression. The effects of REG Iα on AD and SCC cells were different in the in vitro study, and a correlation between REG Iα expression and patient prognosis was noted in the in vivo study. Therefore, overexpression of REG Iα is a risk factor for poor prognosis caused by discrete mechanisms in AD and SCC patients.

Osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs) seeded on irradiated allogenic bone.

Allogenic bone grafting, a technique used in orthopaedic surgery, has several problems, including low osteogenic activity. To overcome the problem, this study aimed to determine whether in vivo osteogenesis could be enhanced using allogenic irradiated bone grafts after seeding with autologous bone marrow-derived mesenchymal stem cells (BMSCs). The allogenic bone cylinders were extracted from ACI rats and sterilized by irradiation. Donor BMSCs were obtained from fresh Fischer 344 (F344) rat bone marrow by cell culture. The allogenic bone with or without BMSCs were transplanted subcutaneously into syngeneic F344 rats. At 4 weeks after transplantation, high alkaline phosphatase (ALP) activity, bone-specific osteocalcin mRNA expression and newly formed bone were detected in the allogenic bone with BMSCs. The origin of the newly formed bone was derived from cultured donor BMSCs. However, none of these identifiers of osteogenesis were detected in either the fresh or the irradiated allogenic bone without BMSCs. These results indicate the availability of autologous BMSCs to heighten the osteogenic response of allogenic bone. Our present tissue-engineering method might contribute to a wide variety of allogenic bone grafting techniques in clinical settings.

Carbon nanotubes induce bone calcification by bidirectional interaction with osteoblasts.

Multi-walled carbon nanotubes (MWCNTs) promote calcification during hydroxyapatite (HA) formation by osteoblasts. Primary cultured osteoblasts are incubated with MWCNTs or carbon black. After culture for 3 weeks, the degree of calcification is very high in the 50 µg mL(-1) MWCNT group. Transmission electron microscopy shows needle-like crystals around the MWCNTs, and diffraction patterns reveal that the peak of the crystals almost coincides with the known peak of HA.

Engineering bioartificial tracheal tissue using hybrid fibroblast-mesenchymal stem cell cultures in collagen hydrogels.

We aimed at providing the first in vitro and in vivo proof-of-concept for a novel tracheal tissue engineering technology. We hypothesized that bioartificial trachea (BT) could be generated from fibroblast and collagen hydrogels, mechanically supported by osteogenically-induced mesenchymal stem cells (MSC) in ring-shaped 3D-hydrogel cultures, and applied in an experimental model of rat trachea injury. Tube-shaped tissue was constructed from mixtures of rat fibroblasts and collagen in custom-made casting molds. The tissue was characterized histologically and mechanically. Ring-shaped tissue was constructed from mixtures of rat MSCs and collagen and fused to the tissue-engineered tubes to function as reinforcement. Stiffness of the biological reinforcement was enhanced by induction of osteogeneic differentiation in MSCs. Osteogenic differentiation was evaluated by assessment of osteocalcin (OC) secretion, quantification of calcium (Ca) deposit, and mechanical testing. Finally, BT was implanted to bridge a surgically-induced tracheal defect. A three-layer tubular tissue structure composed of an interconnected network of fibroblasts was constructed. Tissue collapse was prevented by the placement of MSC-containing ring-shaped tissue reinforcement around the tubular constructs. Osteogenic induction resulted in high OC secretion, high Ca deposit, and enhanced construct stiffness. Ultimately, when BT was implanted, recipient rats were able to breathe spontaneously.

The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue.

Mesenchymal stem cells (MSCs) can give rise to osteoblasts and have therefore been suggested as a cell source for bone engineering. Here we hypothesized that MSC osteoblastic differentiation and maturation can be supported by three-dimensional cultures in collagen hydrogels (hydrogel culture) to ultimately give rise to mechanically robust bone-like tissue. We first compared the osteoblastic differentiation efficiency of MSCs using osteoinductive supplements (ß-glycerophosphate, vitamin C, and dexamethasone) in a hydrogel culture and in a two-dimensional culture (2D culture) by assessing surrogate parameters for osteoblastic differentiation, including osteocalcin (OC) secretion and calcium (Ca) deposition. We next constructed ring-shaped bone-like tissues using MSCs in the hydrogel cultures, and assessed their mechanical (strain-strain analysis), biochemical/molecular (OC secretion, Ca deposition, and Runx2/osterix mRNA levels), and morphological (von Kossa staining) properties. OC secretions and Ca depositions were significantly higher in the hydrogel cultures than those in the 2D cultures, suggesting better osteoblastic differentiation and maturation in the hydrogel cultures. Collagen hydrogel-based ring-shaped bone-like tissues conditioned with osteoinductive supplements developed enhanced biomechanical properties, including high tissue stiffness and ultimate burst strength, superior molecular/biochemical properties, and morphological signs typically found in mineralized bone. These results may be exploited not only to generate bioartificial bone, but also to elucidate the basic mechanisms of bone physiology.

Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model.

Orthopedic surgeons have long been troubled by cases involving nonunion of fractured bones. This study aimed to enhance bone union by cell sheet transplantation of mesenchymal stem cells. A nonunion model was made in rat femur, and rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create a cell sheet that could be scraped off as a single sheet. Cell sheets were transplanted onto fractured femurs without a scaffold in the model. X-ray and histological analysis were performed at 2, 4 and 8 weeks. Ultrasonography and biomechanical analysis were performed at 8 weeks. X-ray photographs and histological sections showed callus formation around the fracture site in the cell sheet-transplanted group (sheet group). Bone union was obtained in the sheet group at 8 weeks. By contrast, the control group (without sheet transplantation) showed nonunion of the femur. The results of pullout evaluation in the vertical direction of the femur in the sheet group were significantly better than that of the control group. Analysis of the origin of de novo formed bone using the Sry gene, which was used as a marker for donor cells, showed that transplanted cells without scaffolds could survive and differentiate into osteogenic lineage cells in vivo. These results showed that the femoral fracture in our model was completely cured by the transplantation of a cell sheet created by tissue engineering techniques. Thus, we think that cell sheet transplantation can contribute to hard tissue reconstruction in cases involving nonunion, bone defects and osteonecrosis.