A Clinical Study of Alveolar Bone Tissue Engineering Using Autologous Bone Marrow Stromal Cells: Effect of Optimized Cell-Processing Protocol on Efficacy.

(1) Objectives: The effect of cell-processing protocols on the clinical efficacy of bone tissue engineering is not well-known. To maximize efficacy, we optimized the cell-processing protocol for bone-marrow-derived mesenchymal stromal cells for bone tissue engineering. In this study, the efficacy of bone tissue engineering using this modified protocol was compared to that of the original protocol. (2) Materials and Methods: This single-arm clinical study included 15 patients. Cells were obtained from bone marrow aspirates and expanded in culture flasks containing basic fibroblast growth factor. The cells were seeded onto ß-tricalcium phosphate granules and induced into osteogenic cells for two weeks. Then, the cell-scaffold composites were transplanted into patients with severe atrophic alveolar bone. Radiographic evaluations and bone biopsies were performed. The results were compared with those of a previous clinical study that used the original protocol. (3) Results: Panoramic X-ray and computed tomography showed bone regeneration at the transplantation site in all cases. The average bone area in the biopsy samples at 4 months was 44.0%, which was comparable to that in a previous clinical study at 6 months (41.9%) but with much less deviation. No side effects related to cell transplantation were observed. In regenerated bone, 100% of the implants were integrated. (4) Conclusions: Compared to the original protocol, the non-inferiority of this protocol was proven. The introduction of an optimized cell-processing protocol resulted in a comparable quality of regenerated bone, with less fluctuation. Optimized cell-processing protocols may contribute to stable bone regeneration.

Tissue Engineering with Compact Bone-Derived Cell Spheroids Enables Bone Formation around Transplanted Tooth.

BACKGROUND: Although tooth transplantation is a desirable treatment option for congenital defects of permanent teeth in children, transplantation to a narrow alveolar ridge is not feasible. In this study, we investigated the possibility of bone tissue engineering simultaneously with tooth transplantation to enhance the width of the alveolar bone. METHODS: Bone marrow mononuclear cells or cortical bone-derived mesenchymal stromal cell spheroids were seeded onto atelocollagen sponge and transplanted with freshly extracted molars from mice of the same strain. New bone formation around the tooth root was evaluated using micro-computed tomography and histological analysis. Tooth alone, or tooth with scaffold but without cells, was also transplanted and served as controls. RESULTS: Micro-computed tomography showed new bone formation in the furcation area in all four groups. Remarkable bone formation outside the root was also observed in the cortical bone-derived mesenchymal stromal cell group, but was scarce in the other three groups. Histological analysis revealed that the space between the new bone and the root was filled with collagen fibers in all four groups, indicating that the periodontal ligament was maintained. CONCLUSION: This study demonstrates the potential of simultaneous alveolar bone expansion employing bone tissue engineering approach using cortical bone-derived mesenchymal stromal cell spheroids for tooth transplantation. The use of an orthotopic transplantation model may further clarify the feasibility and functional recovery of the transplanted tooth over a longer period.

Clinical Outcome and 8-Year Follow-Up of Alveolar Bone Tissue Engineering for Severely Atrophic Alveolar Bone Using Autologous Bone Marrow Stromal Cells with Platelet-Rich Plasma and ß-Tricalcium Phosphate Granules.

BACKGROUND: Although bone tissue engineering for dentistry has been studied for many years, the clinical outcome for severe cases has not been established. Furthermore, there are limited numbers of studies that include long-term follow-up. In this study, the safety and efficacy of bone tissue engineering for patients with a severely atrophic alveolar bone were examined using autogenous bone marrow stromal cells (BMSCs), and the long-term stability was also evaluated. METHODS: BMSCs from iliac bone marrow aspirate were cultured and expanded. Then, induced osteogenic cells were transplanted with autogenous platelet-rich plasma (PRP) and ß-tricalcium phosphate granules (ß-TCP) for maxillary sinus floor and alveolar ridge augmentation. Eight patients (two males and six females) with an average age of 54.2 years underwent cell transplantation. Safety was assessed by monitoring adverse events. Radiographic evaluation and bone biopsies were performed to evaluate the regenerated bone. RESULTS: The major population of transplanted BMSCs belonged to the fraction of CD34-, CD45dim, and CD73+ cells, which was only 0.065% of the total bone marrow cells. Significant deviations were observed in cell growth and alkaline phosphatase activities among individuals. However, bone regeneration was observed in all patients and the average bone area in the biopsy samples was 41.9% 6 months following transplantation, although there were also significant deviations among each case. No adverse events related to the transplants were observed. In the regenerated bone, 27 out of 29 dental implants were integrated. Dental implants and regenerated bone were stable for an average follow-up period of 7 years and 10 months. CONCLUSIONS: Although individual variations were observed, the results showed that bone tissue engineering using BMSCs with PRP and ß-TCP was feasible for patients with severe atrophic maxilla throughout a long-term follow-up period and was considered safe. However, further studies with a larger number of cases and controls to confirm the efficacy of BMSCs and the development of a protocol to establish a reproducible quality of stem cell-based graft material will be required.

Odontoblast death drives cell-rich zone-derived dental tissue regeneration.

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.

Alliin inhibits adipocyte differentiation by downregulating Akt expression: Implications for metabolic disease.

Obesity is currently an important health problem and is associated with an increased likelihood of various diseases. The efficacies of various natural treatments have been assessed for their utility in treating obesity. Alliin (S-allyl-L-cysteine sulfoxides) is considered the major component of garlic and has a wide range of natural antioxidant properties. However, the direct effects of alliin on obesity have not been well clarified. The present study investigated the effects and possible mechanisms of alliin on adipocyte differentiation. The 3T3-L1 cells were treated with alliin (0-40 µg/ml) during adipogenic differentiation. The effect of alliin on lipid accumulation was evaluated by Oil red O staining. Reverse transcription-quantitative PCR was performed to investigate the expression levels of adipogenic differentiation-related genes. The accumulation of lipid droplets was markedly inhibited following alliin treatment. The expression levels of multiple adipogenic transcription markers, such as CCAAT/enhancer-binding protein (C/EBP) ß, C/EBP α and peroxisome proliferation-activity receptor γ, were markedly decreased following treatment with alliin during adipogenic differentiation. Expression levels of several adipocyte-related genes were subsequently suppressed. Additionally, alliin suppressed PKB/Akt and PI3K expression. These results suggested that alliin exhibits anti-adipogenic activity by downregulating major adipogenic differentiation-related genes and Akt/PI3K expression. Alliin may have a potential therapeutic effect on metabolic disease.

Effect of TNF-α and IL-6 on Compact Bone-Derived Cells.

BACKGROUND: Although bone tissue engineering has already been applied clinically, its regeneration efficacy is not always sufficient. Local inflammatory cytokines are considered as the major factors that induce apoptosis of transplanted cells, thus leading to insufficient new bone formation. In this study, we focused on the effects of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) on differentiation and apoptosis of compact bone-derived cells (CBDCs). METHODS: CBDCs were obtained from mouse legs and cultured. The effects of TNF-α and/or IL-6 on the osteogenic differentiation and apoptosis of CBDCs were analyzed in vitro. To confirm the expression of local inflammatory cytokines in vivo, CBDCs were transplanted to the back of immunocompetent mice. RESULTS: IL-6 exerted inconsistent effects on the expression of the different osteogenic markers tested, while significantly upregulating Fas. By contrast, the addition of TNF-α dramatically reduced the expression of all tested osteogenic markers and increased Fas expression. The highest dose of IL-6 could partially reverse the repressive effect of TNF-α, while the addition of IL-6 further increased Fas expression in CBDCs compared to TNF-α alone. The results from in vivo experiments showed the presence of transplants with and without new bone formation. The transplants without bone formation were characterized by higher IL-6 and lower IL-10 expression than those with bone formation, while the expression of TNF-α did not show notable difference. CONCLUSION: The results of this study suggest an important role for IL-6 in modulating the efficacy of bone tissue engineering, which can affect osteogenic cells both positively and negatively.

Cryopreserved Spontaneous Spheroids from Compact Bone-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering.

Spontaneously formed spheroids from mouse compact bone-derived mesenchymal stromal cells (CB-MSCs) possess enhanced stemness and superior plasticity. In this study, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CB-MSC spheroids were investigated. CB-MSCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells was measured. The expression levels of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and noncryopreserved spheroids were transplanted in mice with a beta-tricalcium phosphate as a scaffold to evaluate the in vivo bone-forming capability. The percentage of living cells was highest when 5% DMSO was used as a cryoprotectant, confirmed by the number of dead cells. The expression of stem cell marker genes and osteogenic differentiation capability were maintained after cryopreservation with 5% DMSO. The cryopreserved spontaneous CB-MSC spheroids showed remarkable new bone formation in vivo, identical to that of the noncryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CB-MSC spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CB-MSC spheroids as ready-to-use tissue-engineered products for bone tissue engineering.

Tooth transplantation with a ß-tricalcium phosphate scaffold accelerates bone formation and periodontal tissue regeneration.

OBJECTIVES: Although tooth transplantation is a useful treatment option as a substitute for a missing tooth, transplantation to a narrow alveolar ridge is not feasible. In this study, we tested a tissue engineering approach simultaneously with tooth transplantation using a scaffold or a combination with cells to accelerate bone formation and periodontal tissue regeneration. MATERIALS AND METHODS: Bone marrow mononuclear cells (BM-MNCs) were harvested from C57BL/6J mice. The upper first or the second molar of 3-week-old C57BL/6J mice and a ß-tricalcium phosphate (ß-TCP) scaffold were transplanted with BM-MNCs (MNC group) or without BM-MNCs (ß-TCP group) into the thigh muscle of syngeneic mice. The tooth alone was also transplanted (control group). After 4 weeks, the transplants were harvested and analyzed. RESULTS: Bone volume was significantly larger in the MNC and the ß-TCP groups than that in the control group, and the newly formed bone was observed on the lateral wall of the root. Compared with the control group, the MNC group showed a larger trabecular thickness and fractal dimension. CONCLUSION: This study showed accelerated bone formation and periodontal tissue regeneration when tooth transplantation was performed with a ß-TCP scaffold. BM-MNCs may accelerate bone maturation, while the effect on bone formation was limited.

Effect of short-term betamethasone administration on the regeneration process of tissue-engineered bone.

Local inflammation at the transplanted site of tissue-engineered bone may cause apoptosis of the transplanted cells, thus negatively affecting bone regeneration. To maximize the efficacy of bone tissue engineering, the local effect of short-term corticosteroid administration at the transplanted site of tissue-engineered bone was studied with respect to the expression of inflammatory cytokines. Compact bone-derived cells from mouse leg bones were isolated, cultured and seeded onto ß-tricalcium phosphate granules. The constructs were transplanted to the back of syngeneic mice. Betamethasone sodium phosphate was administered intraperitoneally to an experimental (betamethasone) group, whereas the same amount of saline was administered to a control group. When betamethasone was administered three times (immediately after operation and 12 hours and 24 hours after transplantation), the number of SP7/osterix-positive osteoblasts was larger in the betamethasone group. Three times of betamethasone administration (immediately after operation and 12 hours and 24 hours after transplantation) did not change the number of apoptotic cells and osteoclasts, but showed a slight upregulation of IL-4 and a downregulation of IL-6. However, 7 doses of betamethasone administration (over 7 consecutive days) increased the number of apoptotic cells and osteoclasts, which was correlated with a downregulation of IL-4 and an upregulation of IL-6. TNF-α expression levels showed no significant differences between the two groups. The results showed beneficial effects of 3 betamethasone administrations for bone regeneration therapy but contrary effects when betamethasone was administered 7 times due to the downregulation of anti-inflammatory cytokines (IL-4) and the upregulation of inflammatory cytokines (IL-6). As a conclusion, our results suggested the importance of the cautious usage of corticosteroids to control local inflammation at transplanted sites in bone tissue engineering.

Intra-Bone Marrow Administration of Mesenchymal Stem/Stromal Cells Is a Promising Approach for Treating Osteoporosis.

Mesenchymal stem/stromal cells (MSCs) are known to be useful for treating local bone diseases. However, it is not known if MSCs are effective for treating systemic bone diseases, as the risk for mortality following intravenous MSC administration has hindered research progress. In this study, we compared the safety and efficacy of intra-bone marrow and intravenous administration of MSCs for the treatment of ovariectomy- (OVX-) induced osteoporosis. Cells capable of forming bone were isolated from the murine compact bones and expanded in culture. Relatively pure MSCs possessing increased potential for cell proliferation, osteogenic differentiation, and inhibition of osteoclastogenesis were obtained by magnetic-activated cell sorting with the anti-Sca-1 antibody. Sca-1-sorted MSCs were administered to OVX mice, which were sacrificed 1 month later. We observed that 22% of the mice died after intravenous administration, whereas none of the mice died after intra-bone marrow administration. With respect to efficacy, intravenous administration improved bone mineral density (BMD) by increasing bone mineral content without affecting bone thickness, whereas intra-bone marrow administration improved BMD by increasing both bone mineral content and bone thickness. These results indicate that intra-bone marrow administration of pure MSCs is a safer and more effective approach for treating osteoporosis.

Enhanced bone regeneration capability of chitosan sponge coated with TiO2 nanoparticles.

Chitosan has been a popular option for tissue engineering, however exhibits limited function for bone regeneration due to its low mechanical robustness and non-osteogenic inductivity. Here we hybridized chitosan with TiO2 nanoparticles to improve its bone regeneration capability. Morphology and crystallographic analysis showed that TiO2 nanoparticles in anatase-type were distributed evenly on the surface of the chitosan sponges. Degradation test showed a significant effect of TiO2 nanoparticles addition in retaining its integrity. Biomineralization assay using simulated body fluid showed apatite formation in sponges surface as denoted by PO4- band observed in FTIR results. qPCR analysis supported chitosan - TiO2 sponges in bone regeneration capability as indicated by DMP1 and OCN gene upregulation in TiO2 treated group. Finally, cytotoxicity analysis supported the fact that TiO2 nanoparticles added sponges were proved to be biocompatible. Results suggest that chitosan-50% TiO2 nanoparticles sponges could be a potential novel scaffold for bone tissue engineering.

Nanochitosan antimicrobial activity against Streptococcus mutans and Candida albicans dual-species biofilms.

OBJECTIVE: Chitosan nanoparticle (nanochitosan) has a broad antimicrobial spectrum against diverse pathogenic microorganisms. However, its effect on dental caries-associated microorganisms, such as Streptococcus mutans and Candida albicans is yet to be explored. These microorganisms are known for causing early childhood caries. Therefore, this study was aimed at investigating nanochitosan inhibition capacity against dual-species biofilms of S. mutans and C. albicans. In this study, nanochitosan antimicrobial activity is reported against mono and dual biofilm species of S. mutans and/or C. albicans at 3 and 18 h incubation time. Nanochitosan inhibition capacity was observed through biofilm mass quantity and cell viability. RESULTS: The present study successfully synthesized nanochitosan with average diameter of approximately 20-30 nm, and also established dual-species biofilms of S. mutans and C. albicans in vitro. With nanochitosan treatment, the cell viability of both microorganisms significantly decreased with the increasing concentration of nanochitosan. There was no significant decrease in biofilm mass both in the dual and single-species biofilms after 3 h of incubation. However, greater inhibition of biofilm was observed at 18 h incubation.

Spontaneously Formed Spheroids from Mouse Compact Bone-Derived Cells Retain Highly Potent Stem Cells with Enhanced Differentiation Capability.

The results from our recent study showed the presence of two distinct spheroid-forming mechanisms, i.e., spontaneous and mechanical. In this study, we focused on the spontaneously formed spheroids, and the character of spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) was explored. Cells from (C57BL/6J) mouse leg bones were isolated, and compact bone-derived cells were cultured after enzymatic digestion. Spontaneous spheroid formation was achieved on a culture plate with specific water contact angle as reported. The expression levels of embryonic stem cell markers were analyzed using immunofluorescence and quantitative reverse transcription polymerase chain reaction. Then, the cells from spheroids were induced into osteogenic and neurogenic lineages. The spontaneously formed spheroids from CBDCs were positive for ES cell markers such as SSEA1, Sox2, Oct4, and Nanog. Additionally, the expressions of fucosyltransferase 4/FUT4 (SSEA1), Sox2, and Nanog were significantly higher than those in monolayer cultured cells. The gene expression of mesenchymal stem cell markers was almost identical in both spheroids and monolayer-cultured cells, but the expression of Sca-1 was higher in spheroids. Spheroid-derived cells showed significantly higher osteogenic and neurogenic marker expression than monolayer-cultured cells after induction. Spontaneously formed spheroids expressed stem cell markers and showed enhanced osteogenic and neurogenic differentiation capabilities than cells from the conventional monolayer culture, which supports the superior stemness.

Characterization of spontaneous spheroids from oral mucosa-derived cells and their direct comparison with spheroids from skin-derived cells.

BACKGROUND: Our group has developed a novel method for spontaneous spheroid formation using a specific low-adherence culture plate with around 90° water contact angle. In this study, this method was applied for oral mucosa-derived cells. First, the feasibility of spontaneous spheroid formation was tested. Next, the characteristics of spontaneous spheroids from oral mucosa- and skin-derived cells were compared with special focus on the stemness and neuronal differentiation capability. METHODS: Oral mucosal cells were obtained from the palate and buccal mucosa of C57BL/6J mice. Similarly, skin cells were obtained from the back of the same mouse strain. Passage 2-3 cells were inoculated into the specific low-adherence culture plates to form spontaneous spheroids. The effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and B27 supplement on spheroid formation and maintenance was assessed. Immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to investigate the expression of pluripotency markers, cell proliferation and apoptosis markers, and neurogenic differentiation markers. RESULTS: Using this culture plate, spontaneous spheroid formation was feasible. This process depended on the presence of serum but was independent of the additives such as bFGF, EGF, and B27 supplement, although they improved the efficiency and were essential for spheroid maintenance. This result was confirmed by the higher expression of Caspase7 in the spheroids cultured without the additives than that with the additives. The spheroids from oral mucosa-derived cells expressed stem cell markers, such as Sox2, SSEA1, Oct4, Nanog, and Nestin. The expression of Sox2 in spheroids from oral mucosal cells was higher than that in spheroids from skin-derived cells. Both spheroid-forming cell types had the ability to differentiate into neural and Schwann cells after neurogenic induction, although significantly higher MAP 2, MBP, Nestin, and Nurr1 gene expression was noted in the cells from oral mucosa-derived spheroids. CONCLUSIONS: The results showed that spontaneous spheroids from oral mucosa-derived cells contain highly potent stem cells, which were as good as skin-derived stem cells. The high expression of certain neuronal marker genes suggests an advantage of these cells for regeneration therapy for neuronal disorders.

Around 90° Contact Angle of Dish Surface Is a Key Factor in Achieving Spontaneous Spheroid Formation.

Following the discovery of the primary culture of neural stem cells, the spheroid culture has been recognized as one of the selective culture methods for somatic stem cells. Since then, various methods were reported to generate spheroids, which can enrich the potent stem cell population. However, the fundamental factors affecting spheroid formation remain unclear. In this study, we focused on the surface property of the culture dishes, in particular, hydrophobicity. Primary mouse skin culture cells were prepared with conventional two-dimensional culture, and then, the cells were transferred to culture dishes with varying hydrophobicity, which was confirmed with the water contact angles. Of these, a culture dish possessing an almost 90° water contact angle was the only one that successfully exhibited spheroid formation. The spheroid formation was spontaneous, efficient, and stable. Since this outcome was achieved with a conventional culture medium with serum, but without any additives such as epidermal growth factor, basic fibroblast growth factor, and B27, the spheroid formation from this process was not affected by serum and was also not dependent on additives. The results from immunofluorescence and quantitative real-time polymerase chain reaction testing showed the expression of embryonic stem cell markers such as SSEA-1, SOX2, OCT4, and Nanog, which confirmed that the spheroids with this method are comparable to those from other methods. This outcome was reproducible and could be applied not only to skin-derived cells but also to oral mucosa-derived cells, cortical bone-derived cells, and 3T3 cells, also suggesting the generality and robustness of this phenomenon.

Effect of Cell Seeding Conditions on the Efficiency of In Vivo Bone Formation.

PURPOSE: To optimize methods for seeding cells on granular-type beta-tricalcium phosphate (ß-TCP). MATERIALS AND METHODS: Bone marrow stromal cells were obtained from rat long bones and cultured in flasks with Minimum Essential Medium, Alpha Modification (αMEM) supplemented with 10% fetal bovine serum (FBS), dexamethasone, ascorbic acid, ß -glycerophosphate, and antibiotics. The influence of differential cell seeding densities and dynamic cell seeding conditions (rotation) was investigated using different sizes of ß -TCP granules and a subcutaneous implantation model. RESULTS: Higher cell seeding densities contributed to efficient in vivo bone formation. The rotational seeding did not affect the efficiency but contributed to the uniformity. Although the granule size did not affect the efficiency under the conditions used in this study, large granules showed more uniform distribution of bone regeneration, while small granules showed nonuniform but dense bone formation. Mixtures of relatively large and small granules may be beneficial for both uniform and efficient bone regeneration. CONCLUSION: These findings may contribute to stable bone tissue engineering with bone marrow stromal cells and ß -TCP granules as a scaffold.

Discontinuation of simvastatin leads to a rebound phenomenon and results in immediate peri-implant bone loss.

Although administration of simvastatin has been reported to promote bone formation, the effect of short-term simvastatin administration is not well known. Following implant installation, 10-week-old male Wistar rats (n = 24) were divided into two groups randomly. The experimental group received 10 mg/kg of simvastatin daily for seven days. Then simvastatin administration was discontinued, and the animals were observed up to 28 days. Animals in the control group underwent the same procedure but received saline instead of simvastatin. All animals were analyzed by micro-computed tomography. Samples at days 14 and 21 were subjected to histological analyses. After seven days of simvastatin administration, more new bone formation around the implant was observed in the simvastatin group compared with the control group. Seven days after simvastatin discontinuation, however, the amount of peri-implant trabecular bone began to decrease. Results from morphometric analysis also showed a reduction in new bone area after day 7, which was lowest at day 14. These results were confirmed by histological analyses. In contrast, both the peri-implant trabecular bone and new bone area were maintained in the control group. Short-term administration of simvastatin may affect implant stability owing to a rebound phenomenon and an immediate loss of peri-implant bone.

Transient Exposure to Hypoxic and Anoxic Oxygen Concentrations Promotes Either Osteogenic or Ligamentogenic Characteristics of PDL Cells.

The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells (MSCs) and this tissue is easily available following teeth removal procedures. However, PDL-derived cells (PDLCs) availability for tissue engineering is limited because they are heterogeneous cells at various differentiation and lineage commitments. Therefore, efficient culture conditions to increase MSCs number are needed to use PDLCs in tissue engineering. Recent reports indicate that low-oxygen conditions amplified stem/progenitor cell numbers and inhibited cell differentiation. Our aim was to establish which low-oxygen culture conditions favored bone or tendon/ligament regeneration in cultured PDLCs. Human PDLCs were cultured and exposed to either hypoxic (O2≤5%) or anoxic (O2<0.1%) oxygen conditions in low-glucose/serum-free media for 24 hours. After 24 h, as expected, cell survival was significantly less in PDLCs exposed to anoxic conditions as compared with cells under normal or hypoxic conditions. PDLCs exposed to hypoxic conditions had the highest percentages for MSC markers (CD105, CD166, Stro-1). For both hypoxic and anoxic conditions, stem cell marker genes (oct4, sox2, p75) were upregulated after 6 h. At 24 h, these stem cell markers were maintained in PDLCs under hypoxic condition. Interestingly under anoxic conditions, expression of scleraxis gene (a key transcription factor for tendo/ligamentogenesis) was upregulated markedly. When hypoxic PDLCs were subcultured into osteogenic medium, in vitro calcification and prominent in vivo bone formation in mice calvaria were observed. When anoxic PDLCs were subcultured into tendo/ligamentogenic medium, expression of aggrecan (a mature tenogenic gene) increased remarkably. No obvious differences were detectable on chondrogenic and adipogenic inducibilities. We propose that transient exposure to low-oxygen during the culture enhanced MSC population in PDL. In addition, different low-oxygen concentrations favored osteogenic or tendo/ligamentogenic inducibilities of cultured PDLCs.

Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

Sonographic appearances of cervical lymph nodes in healthy young Japanese adults: Association with age, sex, and body mass index.

PURPOSE: We investigated whether there was any association between age, sex, and body mass index (BMI) and nodal morphology and vascular pattern in healthy young Japanese adults. METHODS: Three neck regions in 100 healthy subjects, 19-40 years old, were examined by gray-scale and color Doppler sonography. Vascular pattern was classified into three groups: avascular, hilar, or scattered. A linear mixed-effect model was used to identify associations of age, sex, or BMI with the short-axis diameter and the short-to-long axis diameter ratio (S/L). A cumulative link mixed model was used to identify any association between age, sex, BMI, and vascular pattern. RESULTS: In the upper cervical region, a decrease in the short-axis diameter was statistically significantly associated with aging (p = 0.04), and an increase in the short-axis diameter was significantly associated with greater BMI (p < 0.001). An increase in short-axis diameter was significantly associated with female sex (p = 0.02) and higher BMI (p = 0.002) in the submandibular region, whereas it was associated with higher BMI (p = 0.001) in the submental region. A greater S/L was significantly associated with higher BMI and female sex in all regions. The scattered vascular pattern tended to be associated with lower BMI (p = 0.051) in the upper cervical region, but it was significantly associated with higher BMI (p = 0.01) in the submental region. CONCLUSIONS: Nodal morphology and vascular pattern may be associated with age, sex, and BMI.