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.

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.

Characterization of time-course morphological features for efficient prediction of osteogenic potential in human mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hBMSCs) represents one of the most frequently applied cell sources for clinical bone regeneration. To achieve the greatest therapeutic effect, it is crucial to evaluate the osteogenic differentiation potential of the stem cells during their culture before the implantation. However, the practical evaluation of stem cell osteogenicity has been limited to invasive biological marker analysis that only enables assaying a single end-point. To innovate around invasive quality assessments in clinical cell therapy, we previously explored and demonstrated the positive predictive value of using time-course images taken during differentiation culture for hBMSC bone differentiation potential. This initial method establishes proof of concept for a morphology-based cell evaluation approach, but reveals a practical limitation when considering the need to handle large amounts of image data. In this report, we aimed to scale-down our proposed method into a more practical, efficient modeling scheme that can be more broadly implemented by physicians on the frontiers of clinical cell therapy. We investigated which morphological features are critical during the osteogenic differentiation period to assure the performance of prediction models with reduced burden on image acquisition. To our knowledge, this is the first detailed characterization that describes both the critical observation period and the critical number of time-points needed for morphological features to adequately model osteogenic potential. Our results revealed three important observations: (i) the morphological features from the first 3 days of differentiation are sufficiently informative to predict bone differentiation potential, both activities of alkaline phosphatase and calcium deposition, after 3 weeks of continuous culture; (ii) intervals of 48 h are sufficient for measuring critical morphological features; and (iii) morphological features are most accurately predictive when early morphological features from the first 3 days of differentiation are combined with later features (after 10 days of differentiation).

Spheroids and organoids: Their implications for oral and craniofacial tissue/organ regeneration.

Spheroids are spherical aggregates of cells. Normally, most of adherent cells cannot survive in suspension; however, if they adhere to each other and grow to a certain size, they can survive without attaching to the dish surface. Studies have shown that spheroid formation induces dedifferentiation and improves plasticity, proliferative capability, and differentiation capability. In particular, spontaneous spheroids represent a selective and efficient cultivation technique for somatic stem cells. Organoids are considered mini-organs composed of multiple types of cells with extracellular matrices that are maintained in three-dimensional culture. Although their culture environment is similar to that of spheroids, organoids consist of differentiated cells with fundamental tissue/organ structures similar to those of native organs. Organoids have been used for drug development, disease models, and basic biological studies. Spheroid culture has been reported for various cell types in the oral and craniofacial regions, including salivary gland epithelial cells, periodontal ligament cells, dental pulp stem cells, and oral mucosa-derived cells. For broader clinical application, it is crucial to identify treatment targets that can leverage the superior stemness of spheroids. Organoids have been developed from various organs, including taste buds, oral mucosa, teeth, and salivary glands, for basic biological studies and also with the goal to replace damaged or defective organs. The development of novel immune-tolerant cell sources is the key to the widespread clinical application of organoids in regenerative medicine. Further efforts to understand the underlying basic mechanisms of spheroids and organoids will lead to the development of safe and efficient next-generation regenerative therapies.

GDFs promote tenogenic characteristics on human periodontal ligament-derived cells in culture at late passages.

Tendon/ligament injures are leading disabilities worldwide. The periodontal ligament (PDL) connects teeth to bone, and is comparable to a tendon/ligament-to-bone insertion. PDL-derived cells (PDLCs) express both osteo/cementogenesis and teno/ligamentogenesis genes. However, an efficient method to induce a tenogenic differentiation of PDLCs has not been thoroughly examined. Therefore, this study tested if growth/differentiation factors (GDFs) enhanced tenogenic characteristics of human PDLCs, as a potential cell source for tendon/ligament engineering. Results demonstrated recombinant GDF-5/GDF-7 inhibited alkaline phosphatase (ALP) activity of PDLCs from passage 3 to 6, while GDF-5 enhanced ALP in dental pulp-derived cells and mesenchymal stem cells. GDF-5 (particularly at 10 ng/ml concentration) induced high expression of both early (scleraxis) and mature (tenomodulin, aggrecan, collagen3) tenogenic genes in P4-6 PDLCs, while inhibiting expression of specific transcription-factors for osteogenic, chondrogenic and adipogenic differentiation. Exogenous GDFs might lead PDLCs being expanded in culture during several passages to highly useful cell source for tendon/ligament engineering.

Characteristic differences among osteogenic cell populations of rat bone marrow stromal cells isolated from untreated, hemolyzed or Ficoll-treated marrow.

BACKGROUND AIMS: Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations. METHODS: Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining. RESULTS: Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium. CONCLUSIONS: These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.

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.

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.

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.

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.

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.

Differential effects of growth differentiation factor-5 on porcine dental papilla- and follicle-derived cells.

In this study, the effect of growth differentiation factor-5 (GDF-5) on the growth and differentiation of porcine dental papilla- and follicle-derived cells was investigated. Furthermore, the effect was compared with that of BMP-2. Recombinant mouse GDF-5 (rmGDF-5) enhanced alkaline phosphatase (ALP) activity in dental papilla-derived cells in a dose-dependent manner, while ALP activity in dental follicle-derived cells was reduced. In rmGDF-5 stimulated dental papilla-derived cells, the expressions of odontoblast-marker genes were up-regulated. Conversely, recombinant human BMP-2 (rhBMP-2) enhanced ALP activity dose-dependently in both dental papilla- and follicle-derived cells. When combined, GDF-5 did not further enhance BMP-2-induced ALP activities. Rather, GDF-5 reduced BMP-2-induced ALP activities in both dental papilla- and follicle-derived cells. This suggests that affinity of GDF-5 to the shared receptors may be higher than that of BMP-2 in both cell types. These observations indicate that GDF-5 regulates differentiation of both dental papilla and follicle during odontogenesis, co-operatively with other growth factors such as BMP-2.

Limited but heterogeneous osteogenic response of human bone marrow mesenchymal stem cells to bone morphogenetic protein-2 and serum.

Although there are numerous reports describing the in vivo bone forming capability of recombinant human bone morphogenetic proteins-2 (rhBMP-2), studies have reported limited effects on human mesenchymal stem cells (hMSCs). However, the reasons for these discrepancies are not well understood. The aim of this study was to investigate the responsiveness of hMSCs to osteoinductive signals, focusing on rhBMP-2 and the effect of serum on that responsiveness. Human MSCs from six donors were analysed. When those cells were treated with osteoinduction medium including dexamethasone (Dex), alkaline phosphatase (ALP) activities increased in all cell lines. On the other hand, rhBMP-2-containing medium failed to increase ALP activity. When five different sera were used for cultivation and induction with rhBMP-2, ALP activities increased in two of them, but not in the others. The expression of BMP-2 antagonist noggin was induced in almost all combinations regardless of the responsiveness to rhBMP-2. On the other hand, the expression of follistatin showed significant variations depending on the serum and cell line. However, the expression did not correlate with the responsiveness to rhBMP-2. The results from this study showed limited but heterogeneous osteogenic response of hMSCs to rhBMP-2 and that the results are affected by the choice of serum. This fact should be concerned for the successful and effective clinical application of rhBMP-2.

Effects of extracellular matrix on differentiation of human bone marrow-derived mesenchymal stem cells into smooth muscle cell lineage: utility for cardiovascular tissue engineering.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into various types of cell, and the extracellular matrix (ECM) is acknowledged to be important for the regulation of cell functions. In this study, we demonstrated the effects of ECMs on the differentiation of human bone marrow-derived MSCs into a smooth muscle cell (SMC) lineage. METHODS: Human MSCs (hMSCs) were cultured on dishes coated with 3 types of ECM including laminin (LM), collagen type IV (Col-IV) and fibronectin for 7 days, and simultaneously cultured on a noncoated dish as a control. Cell numbers of these cultured hMSCs were counted, and their expression of SMC-specific genes and proteins was evaluated. hMSCs were then seeded on LM-coated biodegradable sheets and implanted into rat subcutaneous space. After 2 weeks of implantation, these tissues were evaluated. RESULTS: The number of hMSCs was significantly increased by culture on Col-IV-coated dishes. The expression of SMC-specific genes and proteins (alpha-smooth muscle actin, ASMA; h1-calponin, CALP) in hMSC was significantly upregulated from culture on LM-coated dishes. LM-coated sheets showed a significantly increased expression of ASMA and CALP protein in vivo. Moreover, a fully differentiated marker (SM2) was expressed in the in vivo implanted hMSCs in the course of 2 weeks on the LM-coated sheet. CONCLUSION: These results suggest that the signal transduction of the cell-matrix interaction for the differentiation of hMSCs into SMCs was activated when cultured with LM. LM-coated materials may thus be useful for cardiovascular tissue engineering.

Efficacy of membranous cultured periosteum for the treatment of patients with severe periodontitis: a proof-of-concept study.

Osteogenic cells have been found within periosteal tissue. Periosteal cells will also form a membranous structure under the appropriate culture conditions. We have characterized the osteogenic potential of this membranous cultured periosteum (CP) and have demonstrated that CP can successfully regenerate alveolar bone defects in a canine periodontitis model. The aim of this study is to demonstrate periodontal tissue regeneration by using CP for patients with severe periodontitis. CP was applied in treatments for severe alveolar bone defects for a total of seven teeth among four periodontitis patients. Bone formation was evaluated by dental radiography 4 months after grafting, with a follow-up period of 12 to 15 months. CP was successfully generated and formed a membrane (approximately 4 cm in diameter) about 4 weeks after attachment to the dish. Vertical bone gain (3 to 8 mm) was observed in all grafted areas at 4 months post-surgery. The probing depth was also reduced to its normal depth and remained so beyond one year. Results from the present cases suggest that periodontitis patients with bone defects can benefit from CP treatment. Post-operative evaluation indicates periodontal tissue regeneration after CP treatment, suggesting a broad application for patients with periodontal disease.

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.

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions.

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO((R)) MSC SFM) or conventional serum-containing medium (alpha-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105(++) and CD146(dim). After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.

Novel cell-based therapeutic strategy for ischemic colitis with use of bone marrow-derived mononuclear cells in rats.

PURPOSE: Ischemic colitis is a common disorder of the large bowel. In the clinical setting, some patients suffer refractory ischemic colitis regardless of conventional treatment. Meanwhile, bone marrow-derived mononuclear cells are known to accelerate neovascularization. The purpose of this study was to verify the effects of bone marrow-derived mononuclear cells on ischemic colitis in rats. METHODS: An ischemic colitis model was established by partial obstruction of the rectum and interruption of the marginal vessel in the immunodeficient rat. Bone marrow-derived mononuclear cells from a Wistar rat were injected into the ischemic area one day later than the ischemia (Group MNC). As a control, phosphate-buffered saline was injected in the same manner (Group PBS). Seven days after cell transplantation, each rat was evaluated for histology and colic motility. RESULTS: Compared with Group PBS scores, the Group MNC macroscopic and microscopic colitis severity scores were significantly reduced. Moreover, the density of the capillary and myenteric plexus was significantly higher in Group MNC than in Group PBS (9.55 +/- 0.74 vs. 4.61 +/- 0.22, respectively, P < 0.01; and 8.57 +/- 0.41 vs. 5.93 +/- 0.31, respectively, P < 0.02). The whole-gut transit time was significantly shorter in Group MNC compared with Group PBS (472.7 +/- 17.6 vs. 584.8 +/- 24.0 minutes, respectively, P < 0.01). Transplanted cells were detected in all layers of the intestinal wall; however, these cells did not differentiate into vascular or neural cells. CONCLUSIONS: These results suggest that transplantation of bone marrow-derived mononuclear cells might enhance not only tissue regeneration and angiogenesis but also neurogenesis. Transplantation of bone marrow-derived mononuclear cells may be a useful therapeutic strategy for ischemic colitis.