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Background Mesenchymal stem cells (MSCs), which have the potential to differentiate into cardiomyocytes or vascular endothelial cells, have been used clinically as therapy for cardiomyopathy. In this study, we aimed to evaluate the long-term follow-up results.Methods and Results:We studied 8 patients with symptomatic heart failure (HF) on guideline-directed therapy (ischemic cardiomyopathy, n=3; nonischemic cardiomyopathy, n=5) who underwent intracardiac MSC transplantation using a catheter-based injection method between May 2004 and April 2006. Major adverse events and hospitalizations were investigated up to 10 years afterward. Compared with baseline, there were no significant differences in B-type natriuretic peptide (BNP) (from 211 to 173 pg/mL), left ventricular ejection fraction (LVEF) (from 24% to 26%), and peak oxygen uptake (from 16.5 to 19.2 mL/min/kg) at 2 months. During the follow-up period, no patients experienced serious adverse events such as arrhythmias. Three patients died of pneumonia in the 1st year, liver cancer in the 6th year, and HF in the 7th year. Of the remaining 5 patients, 3 patients were hospitalized for exacerbated HF, 1 of whom required heart transplantation in the 2nd year; 2 patients survived for 10 years without worsening HF. Conclusions The results of this exploratory study of intracardiac MSCs administration suggest further research regarding the feasibility and efficacy is warranted.

Many clinical studies of regenerative medicine using bone marrow-derived mesenchymal stem cells (MSCs) have been conducted globally. We initiated clinical studies using MSCs in 2001, and have now treated over 100 cases with patients aged 0–92 years. In a few cases involving patients with chronic heart failure (CHF), we observed that MSCs proliferated poorly. This contrasts with cell therapy studies wherein MSCs of patients with CHF were used for treatment. The effects of serum on the proliferation of MSCs from donors with normal heart function and with CHF have not been reported. Moreover, whether cell therapy is effective for elderly patients remains uncertain. Therefore, characterization of MSCs from aged donors and/or donors with CHF is urgently required. We retrospectively analyzed the population doubling times (PDTs) of MSCs between the first and second passages. Although we had data for many samples of well expanded MSCs from aged donors, a positive correlation was observed between donor age and PDT. A trend toward reduced variance in PDTs was observed in MSCs were supplemented with fetal bovine serum (FBS) compared to those supplemented with autologous serum. When autologous serum was used, the average PDT of MSCs from donors with CHF was significantly longer than that of MSCs from donors without CHF. In contrast, when FBS was used, similar PDTs were observed in MSCs from donors with and without CHF. Thus, FBS promotes MSC expansion even from donors with CHF, and that MSC-based regenerative medicine might be feasible even for elderly patients. This article is protected by copyright. All rights reserved.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

We fabricated a transparent nonfibrillar collagen gel using gamma irradiation (5 kGy) and cultured rat mesenchymal stem cells (MSCs) on both the gamma-irradiated collagen gel and on unirradiated fibrillar collagen gel. Cells attached well and proliferated with high viability on the surface of both gels. The cells cultured on the gamma-irradiated nonfibrillar gel had a unique elongated shape and adhered to each other in culture. After 21 days of culture in dexamethasone-containing culture medium, the contents of bone-specific osteocalcin and calcium on the gamma-irradiated nonfibrillar gel were 1.4 and 1.9 times higher than those on fibrillar collagen gel, respectively. These data show that osteogenic differentiation of MSCs was promoted more efficiently on the gamma-cross-linked nonfibrillar gel than on the fibrillar gel and demonstrate the potential of the gamma-irradiated collagen gel for use in bone tissue engineering.

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 Reg I 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.

In the human body, cells having self-renewal and multi-differentiation capabilities reside in many tissues and are called adult stem cells. In bone marrow tissue, two types of stem cells are well known: hematopoietic stem cells and mesenchymal stem cells (MSCs). Though the number of MSCs in bone marrow tissue is very low, it can be increased by in vitro culture of the marrow, and culture-expanded MSCs are available for various tissue regeneration.The culture-expanded MSCs can further differentiate into osteogenic cells such as bone forming osteoblasts by culturing the MSCs in an osteogenic medium. This paper discusses osteogenically differentiated MSCs derived from the bone marrow of patients. Importantly, the differentiation can be achieved on ceramic surfaces which demonstrate mineralized bone matrix formation as well as appearance of osteogenic cells. The cell/matrix/ceramic constructs could show immediate in vivo bone formation and are available for bone reconstruction surgery.Currently, MSCs are clinically available for the regeneration of various tissues due to their high proliferation/differentiation capabilities. However, the capabilities are still limited and thus technologies to improve or recover the inherent capabilities of MSCs are needed.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Under osteoconductive conditions, porous calcium phosphate ceramics are known to induce new bone formation within their pores. A critical aspect of the design of porous ceramics is the geometrical features of their pores, with regard to promoting bone formation and mass transfer management in pore networks. However, the pore geometries of common porous ceramics lack clear details. Further, the connections between pores are hard to characterize and thus have not been thoroughly researched. To address these issues, we have developed an original method for fabricating porous ceramics, which we have termed “mosaic-like ceramics fabrication (MLCF).” Using MLCF, pore geometries can be designed and fabricated by each unit, and a network covering all the pores can be fabricated. Furthermore, MLCF can be used to build porous ceramics with custom-made shapes. In this study, we assessed the osteogenic influences of MLCF products (MLPC) composed of hydroxyapatite units on the differentiation of rat bone-marrow-derived mesenchymal stem cells (MSCs) in vitro and in vivo. Two types of commercial porous artificial bone were used as positive controls. MLPC was superior in osteogenic potential, and proved to be a reliable scaffold for bone tissue engineering. Furthermore, this study succeeded in defining the important geometries for osteoconduction. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 3571–3579, 2013.

Many clinical studies of regenerative medicine using bone marrow-derived mesenchymal stem cells (MSCs) have been conducted globally. We initiated clinical studies using MSCs in 2001 and have now treated over 100 cases with patients aged 0-92 years. In a few cases involving patients with chronic heart failure (CHF), we observed that MSCs proliferated poorly. This contrasts with cell therapy studies wherein MSCs of patients with CHF were used for treatment. The effects of serum on the proliferation of MSCs from donors with normal heart function and with CHF have not been reported. Moreover, whether cell therapy is effective for elderly patients remains uncertain. Therefore, characterization of MSCs from aged donors and/or donors with CHF is urgently required. We retrospectively analysed the population doubling times (PDTs) of MSCs between the first and second passages. Although we had data for many samples of well-expanded MSCs from aged donors, a positive correlation was observed between donor age and PDT. A trend towards reduced variance in PDTs was observed in MSCs supplemented with fetal bovine serum (FBS) compared with those supplemented with autologous serum. When autologous serum was used, the average PDT of MSCs from donors with CHF was significantly longer than that of MSCs from donors without CHF. In contrast, when FBS was used, similar PDTs were observed in MSCs from donors with and without CHF. Thus, FBS promotes MSC expansion even from donors with CHF and MSC-based regenerative medicine might be feasible even for elderly patients. Copyright © 2016 John WileySons, Ltd.

The authors previously created HAp or CaCO3 formed on or in agarose gels (HAp and CaCO3 gels, respectively) as biocompatible and biodegradable bone graft materials. However, these gels have limitations for bone regeneration. Mesenchymal stromal cells (MSCs) have osteogenic potential and are considered useful for bone tissue engineering. The purpose of this study was to clarify the osteogenic abilities of MSCs loaded in HAp or CaCO3 gels (MSC/HAp and MSC/CaCO3 gels, respectively) using a rat cranial defect model compared to HAp and CaCO3 gels alone. HAp, CaCO3, MSC/Hap, and MSC/CaCO3 gels were prepared for in vivo analyses and implanted into full-thickness bone defects created in the rat cranium. All samples were assessed radiologically and histologically at 4 and 8 weeks after implantation. Using microfocus-computed tomography, an increase in bone formation was observed in the MSC-loaded gels compared to the gels alone. In addition, peripheral quantitative computed tomography revealed higher bone mineral contents in the MSC-loaded gels compared to the gels alone. After transmission X-ray diffraction analyses, the degree of apatite c-axis orientation as a bone quality index of newly formed bone in the MSC-loaded gels was close to that of living cranial bone. Histologically, more extensive bone formation was detected in the MSC-loaded gels compared to gels alone. Overall, MSC/HAp and MSC/CaCO3 gels showed equivalent efficacy for bone regeneration. These findings demonstrate that loading of MSCs into the gels strengthened their osteogenic ability and improved the quality of the newly formed bone. As a result, MSC-loaded gels could represent viable therapeutic biomaterials for bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.

Tissue-engineered medical products (TEMPs) should be evaluated before implantation. Therefore, it is indispensable to establish evaluation protocols in regenerative medicine. Whether or not such evaluation protocols are reasonable is generally verified through a 'round robin' test. However, the round robin test for TEMPs intrinsically includes a deficiency, because 'identical' specimens can not be prepared for TEMPs. The aim of the study was to assess the feasibility and limitations of the round robin test for TEMPs by using a prepared evaluation protocol. We adopted tissue-engineered cartilage constructs as delivered specimens and a protocol of measuring sGAG content as an evaluation protocol proposed to ISO TC150/SC7, which is an invasive, but usually applied, method, although non-invasive methods are keenly required in evaluating TEMPs. The results showed that: (a) the coefficient of variation (CV) of the measured sGAG contents in intralaboratory tests was ~5% at most; (b) the CV of sGAG content in the scheme where each participating laboratory measured different constructs was comparable with that in the scheme where each participating laboratory measured one half of a construct along with the organizing laboratory; (c) the CV caused by factors other than the specimen was ~15%, comparable to that in reproducible experiments in biomedical fields. Based on these results, the study concludes that a round robin test for a TEMP could be valuable, under the condition that the delivered TEMPs are sufficiently reproducible so that the CV of the measured values is

Bone marrow mesenchymal stem cells (MSCs) have been used for bone tissue engineering due to their osteogenic differentiation capability, but their application is controversial. To enhance their capability, we prepared biodegradable gelatin sponges incorporating β-tricalcium phosphate ceramics (GT sponge), which has been shown to possess excellent controlled drug-release properties. The GT sponge was used as a carrier for both rat MSCs and bone morphogenetic protein-2 (BMP-2) and osteogenic differentiation was assessed by subcutaneous implantation of four different kinds of implants, i.e. GT-alone, MSC-GT composites, BMP-GT composites and BMP-GT composites supplemented with MSCs (BMP-MSC-GT) in rats. Two weeks after implantation, histological sections showed new bone formation in the peripheral parts of the BMP-GT and in almost the total volume of the BMP-MSC-GT implants. After 4 weeks, histology as well as microCT analyses demonstrated extensive bone formation in BMP-MSC-GT implants. Gene expression and biochemical analyses of both alkaline phosphatase and bone-specific osteocalcin confirmed the histological findings. These results indicate that the combination of MSCs, GT and BMP synergistically enhances osteogenic capability and provides a rational basis for their clinical application in bone reconstruction.

Various mesenchymal stromal cells (MSCs) have been applied to regenerative medicine. MSCs derived from periodontal tissue could also be a useful cell source for alveolar bone regeneration. However, only a few attempts of direct comparisons have been made between MSCs from periodontal tissues and those from other somatic tissues. The purpose of this study was to clarify the osteogenic characteristics of mesenchymal stromal cells derived from bone marrow (BMSCs), adipose tissue (ASCs) and periodontal ligament (PDLSCs). BMSCs, ASCs and PDLSCs were isolated from Fisher 344 rats. After 1 week of primary culture, stromal cells were subjected to cell surface analysis and osteogenic differentiation. The cells were subcultured for 2 weeks with and without osteogenic supplements (OS), followed by biochemical and histological analyses. With regard to cell surface antigens, all MSCs were positive for CD29 and CD90 and negative for CD45. With regard to osteogenic differentiation, BMSCs with OS had the highest ALP activity, calcium uptake and osteocalcin content. Without OS, PDLSCs had the highest levels of these bone differentiation markers. RT-PCR analysis and histological analysis showed similar trends. These results indicate that PDLSCs are an ideal candidate for alveolar bone regeneration.

Mesenchymal stromal cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage (7). Especially, the differentiation capability towards osteogenic cells is very well known. We have already used the patient's MSCs for the treatments of osteoarthritis (8), bone necrosis (2) and bone tumor cases (5). In most cases, the MSCs were culture- expanded from patient's fresh bone marrow cells, and then combined with porous ceramics. The MSCs/ceramics composites were further cultured in a medium containing dexamethasone to promote osteogenic differentiation of the MSCs. In this culture condition, we could detect bone forming osteoblasts together with mineralized bone matrix on the ceramics (7); therefore, we could fabricate cultured bone using patient's bone marrow and ceramics. However, the proliferation and differentiation capability of the MSCs are variable and many lose their capabilities after several passages. With the aim of conferring higher capability on human bone marrow MSCs, some of transcription factors could be introduced into the MSCs. This review paper demonstrates the importance of the transcription factors to promote the osteogenesis as well as proliferation capabilities of human MSCs.

Array-based comparative genomic hybridization (aCGH) using bacterial artificial chromosomes (BAC) is a powerful method to analyze DNA copy number aberrations of the entire human genome. In fact, CGH and aCGH have revealed various DNA copy number aberrations in numerous cancer cells and cancer cell lines examined so far. In this report, BAC aCGH was applied to evaluate the stability or instability of cell lines. Established cell lines have greatly contributed to advancements in not only biology but also medical science. However, cell lines have serious problems, such as alteration of biological properties during long-term cultivation. Firstly, we investigated two cancer cell lines, HeLa and Caco-2. HeLa cells, established from a cervical cancer, showed significantly increased DNA copy number alterations with passage time. Caco-2 cells, established from a colon cancer, showed no remarkable differences under various culture conditions. These results indicate that BAC aCGH can be used for the evaluation and validation of genomic stability of cultured cells. Secondly, BAC aCGH was applied to evaluate and validate the genomic stabilities of three patient's mesenchymal stem cells (MSCs), which were already used for their treatments. These three MSCs showed no significant differences in DNA copy number aberrations over their entire chromosomal regions. Therefore, BAC aCGH is highly recommended for use for a quality check of various cells before using them for any kind of biological investigation or clinical application.

HOS cell is a model strain of human osteoblasts derived from human osteosarcoma. We cultured the HOS cells on both the conventional collagen gel (neutral gel), and the gamma-crosslinked collagen gel without collagen fibrils (acidic gel). The shape of HOS cells on the neutral gel was similar to that on the culture dish. However, HOS cells on acidic gel had an elongated shape and attached each other to form a mesh-like pattern. The cells attached to the surface of both gels but scarcely penetrated their depths. We measured the biochemical markers for osteogenic differentiation in the HOS cells cultured on both the neutral gel and the acidic gel. The expressions of alkaline phosphatase and osteocalcin were detected in the HOS cells on both types of collagen gel. Deposition of the calcium also occurred on both gels although it was higher in the neutral gel than the acidic one. These results indicate the importance of collagen for the differentiation of HOS cells, but it is not dependent on the molecular structure (fibril formation) of collagen.

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30–100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

For the assessment of tissue-engineered cartilage, easy and precise evaluation methods are anticipated. We have investigated the use of a fluorescent image analyser and the application of fluorescence intensity measurements to describe and evaluate a tissue-engineered cartilage-like tissue construct. We evaluated the matrix production of a tissue-engineered cartilage-like tissue (TCL) construct in vitro using the image analyser. The autofluorescence intensity of the TCL construct was determined using the image analyser driven by a software program after 1, 2, 3 and 4 weeks of culture. The autofluorescence intensity of the TCL construct gradually increased with increasing culture time. However, there was very little change in the autofluorescence intensity of gels without articular chondrocytes as controls. The relationship between the autofluorescence intensity of the TCL construct and the hydroxyproline content, reflecting the collagen content, was investigated. There was a significant correlation between the two items. Our findings demonstrate the ability of a commercial fluorescent image analyser to evaluate the matrix production of the TCL construct in vitro on the basis of objective data, such as the autofluorescence intensity. Therefore, the image analyser can be used to judge the success or failure of the in vitro culture process of TCL construct without a biopsy. Copyright © 2010 John Wiley & Sons, Ltd.

We have developed a new cell transplantation technique, in which mesenchymal stem cells (MSCs) were cultured and lifted as a cell sheet structure. The sheet formed bone tissue after in vivo transplantation at a subcutaneous site, without the need for any scaffold. The sheet could also be transplanted with a scaffold, such as hydroxyapatite (HA). Transplantation of this sheet/HA construct resulted in extensive bone formation. The purpose of the present study was to analyze the spatial distribution and biological potential of the osteogenic sheet/HA constructs. Histological examination showed that the construct allowed bone formation inside the pores, as well as outside the HA at 2 weeks after transplantation. Interestingly, when two HAs were wrapped in one sheet, the bone on the outside of one HA could bond to the bone on the other HA, resulting in union of two HAs. Biochemical investigation showed that the sheet/HA constructs had higher osteogenic potential than previous methods, such as MSC/HA constructs. Our results indicate that this transplantation technique using MSC sheets has the potential to be a useful tool in bone tissue engineering.

Background aims Cell therapy with mesenchymal stromal cells (MSC) has been reported recently as a promising treatment for severe acute graft-versus-host disease (GvHD). Methods We designed a pilot study to treat severe hepatic or gut GvHD using MSC derived from only the donor and cultured without bovine serum. Because the number of cultured MSC is smaller using this method, we planned to treat patients by intra-arterial regional administration directly to the target organs. Results Three patients were enrolled, and the MSC could be expanded using donor serum. There were no obvious side-effects immediately after arterial injection. The maximum response was partial in one of three patients and did not continue for more than 2 months. Idiopathic pneumonia syndrome developed in two of the three patients. Conclusions A single local arterial MSC injection was unable to save these patients' lives and so might not be more effective than multiple systemic intravenous MSC injection. Further clinical research and additional strategies are required to develop appropriate methods for using MSC to achieve extended remission of GvHD.

Although hydroxyapatite is commonly used as a scaffold for bone regeneration, sponges may be suitable because of the adaptability to the defect. To use as a scaffold, the fiber of sponge would be coated with any adhesive to storage stem cells in the sponges. Fiber in the structure of commercially available sponges was coated by immersion in dextran solution and air dried. After seeding of rat bone marrow cells (rBMCs), the sponges were implanted subcutis of rats for estimate osteogenesis in vivo. The level of osteocalcin was 25.28 ± 5.71 ng/scaffold and that of Ca was 129.20 ± 19.69 μg/scaffold. These values were significantly high- er than those in sponges without dextran coating (p < 0.01). It was thought that rBMCs could be stored on the shelf by dextran deposition in the fiber of the sponge. In vivo examination, dextran induced osteogenesis by rBMCs in many spaces in the inner structure of the sponge.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

Poly-lactic-glycolic acid (PLGA) is a biocompatible as well as biodegradable polymer and used in various medical applications. In this study, we evaluated efficiency of the specially designed three-dimensional porous PLGA as a scaffold for bone augmentation. First, cell attachment/proliferation, differentiation, and mineralization of Fisher 344 rat marrow mesenchymal stem cells (MSCs) cultured on the PLGA scaffold were analyzed. Viable MSCs were impregnated into pore areas of the scaffold and a moderate increase of DNA contents was seen. High alkaline phosphatase, osteocalcin content, and calcium content of MSCs in PLGA scaffolds under osteogenic differentiation conditions were seen after 14 or 21 days of culture. Subsequently, we implanted the PLGA/MSCs composites on rat calvaria bone for 30 days. Newly formed bone was seen in only the composite PLGA/MSCs implantation group, which had been precultured under osteogenic condition. We also demonstrated that the newly formed bone originated from the donor composites. These results demonstrate that the three-dimensional PLGA scaffold can support osteogenic differentiation of MSCs, and the scaffold combined with osteogenic MSCs can be used for in vivo bone tissue augmentation. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res 2009

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.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.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.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.

Summary Objective The aim of this study was to investigate whether human articular cartilage can be quantitatively evaluated using a spectrocolorimeter. Materials and methods Human articular cartilage specimens were analyzed using a spectrocolorimeter after macroscopic evaluation using the Outerbridge classification. The cartilage characteristics were examined, the L *, a *, b * colorimetric system, the spectral reflectance distribution and the yellow/red spectral reflectance percentage (Y/R SRP). Moreover, the results of the spectrocolorimetric evaluation were compared with the histological score described by Mankin et al. Results There were significant differences among the macroscopic four grades in the L *, a * and Y/R SRP values. The spectral reflectance distribution of grade 1 cartilage exhibited a gradual increase in the spectral reflectance ratio as the wavelength increased. The spectral reflectance curves of grades 2 to 4 cartilage had dips at a wavelength of around 580nm. Across all the measured wavelengths, there were lower reflectance ratios with the progression of cartilage degeneration. Moreover, correlations were observed between the spectrocolorimetric values and Mankin score. A strong relationship existed between Mankin score and he Y/R SRP values. Conclusions The present study is the first to clearly demonstrate the relationship between spectrocolorimetric evaluation and the degeneration of human articular cartilage. The spectrocolorimeter may be a new quantitative evaluation tool for articular cartilage with clinical potential.

The aim of the current study was to examine in vitro osteogenic capability and in vivo bone formation of mesenchymal stromal cells (MSCs) on two kinds of calcium phosphate ceramics. MSCs derived from human bone marrow were seeded on either hydroxyapatite (HA) ceramic or beta-tricalcium phosphate (beta-TCP) ceramic and then cultured in a medium supplemented with a donor's serum, vitamin C, beta-glycerophosphate, and dexamethasone. The culture revealed the expression of alkaline phosphatase activity, indicating the osteogenic differentiation of the MSCs on the ceramics (fabrication of tissue-engineered construct). The constructs were then implanted subcutaneously into nude rats for 8 weeks. New bone formation was observed in both types of ceramics, and human-specific Alu sequence was detected by in situ hybridization analysis. Quantitative microcomputed tomography showed that the volume of the new bone in the HA ceramic was greater than that in the beta-TCP ceramic in six of seven cases. These results suggest that human MSCs cultured on ceramics could retain their osteogenic capability even after ectopic implantation and provide a rationale for the use of tissue-engineered constructs derived from a patient's MSCs and calcium phosphate ceramics in bone tissue regeneration.

Mesenchymal stromal cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. Especially, the differentiation capability towards osteogenic/chondrogenic cells is very well known. We have already used the patient's MSCs for the treatments of various patients who have osteoarthritis, bone necrosis and bone tumor. However, the proliferation and differentiation capability of the MSCs are variable and many cells lose their capabilities after several passages. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 gene into the cells and found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. Present paper describes our bone tissue engineering strategy and focuses on the importance of transcription factors for the function of MSCs.

Recently, technology of regenerative medicine which utilizes cells after their proliferation and differentiation process has drawn attention. In order to utilize the technology for clinical application, safety issue of the process as well as usefulness of the cells should be confirmed. We analyzed the issues and succeeded in utilizing the cells after proliferation / differentiation process for the purpose of therapeutic applications.

The transparent bodies of see-through medaka fish has led to their use as models for in vivo cell and tissue imaging. However, these fish may also prove useful as models for pathological processes. Accumulating reports show that pathology in fish is similar to that in mammals, including humans. In this study, pathological characterization, comparison of data from rodents and in vivo pharmacological analyses of isoproterenol (Iso)-treated fish were performed. Larval fish showed cardiac hypertrophy-like changes after exposure to10 microg/ml Iso for more than one day. Hearts of Iso-treated fish had a larger size and a thicker wall, especially in the ventricle, compared with normal hearts. Several messenger RNA levels dose-dependently increased in Iso-treated fish in a manner similar to that in rodents. Symptoms that accompany mammalian cardiac hypertrophy, such as congestion, were also observed. Propranolol (Pro), a non-selective beta-adrenoceptor blocker (AR), significantly protected the increase in heart rate and volume and other simultaneous symptoms induced by Iso exposure. Athletic capability, measured by optomotor response, showed a dose-dependent decrease after Iso treatment, but was protected by Pro. In contrast, metoprolol (Met) and butoxamine (Btx), selective beta1- and beta2-AR blockers, had little effect in this analysis. These results show that Iso-treated fish develop pathologies analogous to those found in mammalian systems, suggesting that medaka fish can be used for investigation of cardiac hypertrophy; however, drug affinity differences should be considered between species.

Novel highly branched biodegradable macromolecular systems have been developed by grafting carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers. Such structures organize into sphere-like nanoparticles that are proposed to be used as carriers to deliver bioactive molecules aimed at controlling the behavior of stem cells, namely their proliferation and differentiation. The nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 1 mg mL -1 , and fluorescent probe labeled nanoparticles were found to be internalized with highly efficiency by both human osteoblast-like cells and rat bone marrow stromal cells, under fluorescence-activated cell sorting and fluorescence microscopy analyses. Dexamethasone (Dex) has been incorporated into CMCht/PAMAM dendrimer nanoparticles and release rates were determined by high performance liquid chromatography. Moreover, the biochemical data demonstrates that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles promote the osteogenic differentiation of rat bone marrow stromal cells, in vitro. The nanoparticles exhibit interesting physicochemical and biological properties and have great potential to be used in fundamental cell biology studies as well as in a variety of biomedical applications, including tissue engineering and regenerative medicine.

Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl4)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl4-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.

To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.

Mesenchymal stem cells (MSCs) derived from human bone marrow have capability to differentiate into cells of mesenchymal lineage. The cells have already been applied in various clinical situations because of their expansion and differentiation capabilities. The cells lose their capabilities after several passages, however. With the aim of conferring higher capability on human bone marrow MSCs, we introduced the Sox2 or Nanog gene into the cells. Sox2 and Nanog are not only essential for pluripotency and self-renewal of embryonic stem cells, but also expressed in somatic stem cells that have superior expansion and differentiation potentials. We found that Sox2-expressing MSCs showed consistent proliferation and osteogenic capability in culture media containing basic fibroblast growth factor (bFGF) compared to control cells. Significantly, in the presence of bFGF in culture media, most of the Sox2-expressing cells were small, whereas the control cells were elongated in shape. We also found that Nanog-expressing cells even in the absence of bFGF had much higher capabilities for expansion and osteogenesis than control cells. These results demonstrate not only an effective way to maintain proliferation and differentiation potentials of MSCs but also an important implication about the function of bFGF for self-renewal of stem cells including MSCs.

A tissue engineering approach using human mesenchymal stem cells (MSCs) can be used to regenerate bone/cartilage tissue. For effective tissue regeneration, cell cultivation under biologically safe conditions is essential. The objective of this study was to determine suitable antibiotic concentrations in culture media for MSCs while maintaining their inherent capabilities for proliferation and osteogenic/chondrogenic differentiation. When human MSCs were cultured with over 200 μg/mL of gentamicin, the rate of cell growth decreased significantly. In contrast, a concentration of 20 μg/mL gentamicin supported cell proliferation and differentiation capability very well and did not change the expression patterns of cell surface antigens (CD44+, CD105+, CD34-, CD45-). The concentration of 20 μg/ml is much higher than that of the MIC (Minimum Inhibition Concentration) of common bacteria, such as Staphylococcus aureus, Haemophilus influenzae and Escherichia coli. Therefore, we recommend 20 μg/mL of gentamicin in culture media for effective tissue regeneration to avoid bacterial contamination and maintain the osteogenic/chondrogenic capability of MSCs.

We evaluated the osteogenic differentiation of mesenchymal stem cells (MSCs) using a new class of synthetic self-assembling peptide hydrogels, RADA 16, as a scaffold for three-dimensional culture. MSCs derived from rat bone marrow were culture-expanded and seeded into the hydrogel and further cultured in osteogenic medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone for 2-4 weeks. High alkaline phosphatase activity and osteocalcin (OC) contents were detected at both the protein and gene expression levels during the culture periods. Both calcium and the OC contents increased over time, indicating the growth of a mineralized extracellular matrix within the hydrogel. Moreover, the process of the growth of the mineralized matrix determined by three-dimensional microarchitecture images was obtained by confocal laser scanning microscopy. The findings show that MSCs can differentiate into mature osteoblasts to form mineralized matrices within the hydrogel scaffold. Importantly, the differentiation can occur three dimensionally within the hydrogel, indicating that RADA 16 can be considered attractive synthetic biomaterial for use in bone tissue engineering.

The purpose of this study was to estimate influence of lysine for osteogenesis in the porous hydroxyapatite (HA) scaffolds with bone marrow cells. The HA scaffolds were soaked in 100mM concentration of lysine solution. They were kept in bone marrow cell suspension at 1×106 cells/ml density. Another HA scaffolds without immersion in lysine solution were kept in the cell suspension at 1×106 or 1×107 cells/ml density. They were respectively implanted into dorsal subcutis of rats for 4 weeks. Serially sectioned paraffin specimens were made and observed histologically. In several sections, total pores and ones with bone were counted. Many pores containing bone were found in1×107 cells/ml concentration group. The significant difference was between 1×107 cells/ml group, the lysine group, and 1×106 cells/ml group. Although more bone formation was seen in lysine group than in 1×106 cells/ml group. There was no significant difference between the groups. Concentration of lysine to add in culture medium or scaffold should be improved respectively.

Hydroxyapatite (HA) ceramics together with various kinds of osteogenic cells have been used in bone tissue engineering. It is well known that the ceramics structure and composition affect cell proliferation / differentiation. In this study, three different types of HA ceramics were used to investigate initial cell attachment followed by osteoblastic differentiation of human mesenchymal stromal cells (MSCs). The results indicated that micro-pore affected the cell attachment and porosity (pore diameter and inter-pore connection) was the key to allow spacious distribution of the viable cells in the ceramics. This study also confirmed that surface pore areas of HA ceramics support the differentiation of human MSCs and thus the ceramics have the capability to regenerate damaged bone tissue.

Porous hydroxyapatite (HA) scaffolds were processed in hyaluronic acid solution. Bone marrow cells obtained from the bone shaft of femurs of Fischer 344 rats at 1×106/ml concentration were seeded in pores of the scaffolds. The scaffolds were implanted in the dorsal subcutaneous tissue of rats for 2, 4, 6 or 8 weeks. Removed HA scaffolds at 2 and 4 week after dorsal subcutaneous implantation were histologically examined. At all experimental periods, osteocalcin in the scaffold was immunochemically measured for the quantitative analysis of osteogenesis by bone marrow cells in the porous HA scaffolds. Moreover, value of alkaline phosphatase (ALP) activity in the scaffolds was measured. Osteocalcin measured in scaffolds without bone marrow cells was 1.3 ng in an average and the ALP activity was 62.2 μmol at 4 week. In hyaluronic acid processed scaffold with bone marrow cells, quantity of osteocalcin increased from 1.6 ng at 2 week to 2.2 ng at 4 week after implantation of the scaffold. Histologically, many pores containing bone in the scaffolds immersed in hyaluronic acid solution were detected. Significant difference of the quantity of osteocalcin was recognized between 2 and 4 week implantation. There was no significant difference in the quantity of osteocalcin between the scaffolds implanted for 4 and 8 weeks. Value of ALP activity of the scaffold implanted for 4 weeks showed significant difference comparing with that implanted for 6 and 8 weeks. From the results of this study, quantitative increase of the bone formation in the pores of HA scaffolds would be able to observe from 6 to 8 weeks after implantation on the scaffolds by immersion in hyaluronic acid solution

The Japanese Orthopaedic Association Committee on Tissue Transplantation and Regenerative Medicine has conducted a nationwide survey of the status of bone grafting in Japan every 5 years from 1985. We report here the status of bone grafting from 2000 to 2004, show the trends in bone grafting from 1985 to 2004, and draw attention to the issues affecting banked bone allografts.Questionnaires devised by the Committee were sent to all educational and training hospitals (2239 institutions) approved by the Japanese Orthopaedic Association.Survey responses were obtained from 1263 institutions (56%). Of these, 875 institutions performed tissue transplantation during this period. A total of 163 564 tissue transplantations were performed, and 134 782 (82.4%) of them were bone grafts. Of the bone grafts, 76 015 (56.4%) were autografts, 53 735 (40%) used a synthetic bone substitute, and 4886 (3.6%) were banked bone allografts. The proportion of synthetic bone substitutes increased, and the proportion of autografts decreased year by year. Synthetic bone substitutes were most frequently used for replacement arthroplasty (31%). Fifty percent of banked bone allografts were performed for joint disorders requiring replacement arthroplasty. During this period, 271 institutions performed banked bone allografts, with 210 preserving allografts in their own institutions. Donor selection criteria, processing and preservation methods, and management of the bone bank were not the same in all banks.Most bone grafts performed in Japan during the four surveys were still autografts. However, the proportion of autografts decreased, and the proportion of synthetic bone substitutes increased. The number of synthetic bone substitutes and banked bone allografts used for replacement arthroplasty increased significantly. However, the total number of banked bone allografts reported in the fourth survey was still low. Quality control of banked bone allografts and management of bone banks were not satisfactory, although they were improved.