Search

The surface characteristics of biomaterials, especially regarding the sustained delivery of bone morphogenetic protein-2 (BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis. In this study, we evaluated the BMP-2 adsorption and release ability of the surface biomimetic hydroxyapatite (HAp) nanostructure on a new HAp-coated genipin-chitosan conjugation scaffold (HGCCS), and the resulting osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. HGCCS exhibited a loading efficiency of 65% (1.30 μg), which is significantly higher than 28% (0.56 μg, p < 0.01) for the genipin cross-linked chitosan framework, as quantified by an enzyme-linked immunosorbent assay. More importantly, we found that the release of BMP-2 from HGGCS sustained for at least 14 days in simulated body fluid in vitro, which is much better than the burst release within 3 days for CGF. Moreover, the BMP-2 release from HGCCS induced an increase in alkaline phosphatase activity as an indicator of osteogenic differentiation of seeded BMSCs for 14 days in vitro. HGCCS also stimulated a high mRNA expression of osteogenic differentiation makers, runt-related transcription factor 2 for 14 days, osteopontin for 3 days, and osteocalcin for 14 days. The results of this study suggest that the surface biomimetic HAp nanostructure of HGCCS used as a delivery system for BMP-2 is capable of promoting osteogenic differentiation in vitro. These findings demonstrated that HAp nanostructure assembled on organic porous scaffold could work as both calcium source and absorption/release platform, which opened a new research avenue for cell growth factor release, and provided a promising strategy for design and preparation of bioactive scaffold for bone tissue engineering.

Objective: This review of literature was aimed to assess in vivo experiments which have evaluated the efficacy of dental pulp stem cells (DPSCs) for bone regeneration., Design: An electronic search of English-language papers was conducted on PubMed database. Studies that assessed the use of DPSCs in bone regeneration in vivo were included and experiments evaluating regeneration of hard tissues other than bone were excluded. The retrieved articles were thoroughly reviewed according to the source of stem cell, cell carrier, the in vivo experimental model, defect type, method of evaluating bone regeneration, and the obtained results. Further assessment of the results was conducted by classifying the studies based on the defect type., Results: Seventeen papers formed the basis of this systematic review. Sixteen out of 17 experiments were performed on animal models with mouse and rat being the most frequently used animal models. Seven out of 17 animal studies, contained subcutaneous pockets on back of the animal for stem cell implantation. In only one study hard tissue formation was not observed. Other types of defects used in the retrieved studies, included cranial defects and mandibular bone defects, in all of which bone formation was reported., Conclusion: When applied in actual bone defects, DPSCs were capable of regenerating bone. Nevertheless, a precise conclusion regarding the efficiency of DPSCs for bone regeneration is yet to be made, considering the limited number of the in vivo experiments and the heterogeneity within their methods., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2+FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9+FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tripolyphosphate (TPP) crosslinked chitosan (CH)-based fibrous matrices have potential as bioactive scaffolds for bone tissue engineering. This study describes mechanical, biomineralization, and in vitro bone cell growth and differentiation properties of CH-TPP (chitosan-tripolyphosphate) fibrous scaffolds and compared with that of uncrosslinked CH one. The hydrated CH-TPP scaffolds were viscoelastic in nature and their compressive strength was ~2.9 MPa, which is greater than recent polymer experimental bone scaffolds. This improvement in mechanical properties of CH-TPP scaffold may be beneficial toward cancellous bone graft application. Furthermore, CH-TPP fibers supported in vitro biomineralization with phosphate as nucleation site; however, no significant difference in biomineralization morphology was observed with uncrosslinked CH fibers. Interestingly, a significant improvement in cellular responses (>33% increase in cell number based on DNA quantification) was observed when osteoblast like cells were cultured on the CH-TPP scaffolds than that of CH scaffolds without phosphate group. Enhanced osteoblastic differentiation of MG63 cells on CH-TPP scaffolds was also evidenced. Altogether, the results show that the CH-TPP fibrous scaffolds are encouraging for bone tissue engineering., (Copyright © 2012 Wiley Periodicals, Inc.)

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine., (© 2013 The Authors. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender., (© 2013, Copyright the Authors. Artificial Organs © 2013, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Human mesenchymal stem cells (hMSCs) are highly desirable cells for bone engineering due to the inherent multipotent nature of the cells. Unfortunately, there is a high degree of variability, as primary hMSC cultures quickly undergo replicative senescence with loss of proliferative potential as they are continually propagated in cell culture. We sought to reduce the variability of these cells by insertion and expression of human telomerase reverse transcriptase (TERT) to immortalize the cell line. hMSCs were transduced with a lentivirus containing the human TERT gene. The resulting cell line has been propagated through more than 70 population-doubling level (PDL) to date and continues to grow exhibiting the characteristic fibroblastic hMSC phenotype. Expression of TERT mRNA and protein activity was confirmed in the TERT-transduced cells. Mock-transduced hMSCs had almost undetectable levels of TERT mRNA and protein activity and lost proliferation potential at PDL 14. The enhanced growth capacity of the hMSC TERT cells was due to increased cell proliferation and reduced cellular senescence rather than due to inhibition of apoptosis. The multipotent nature of the TERT cells was confirmed by differentiation toward the osteoblastic and adipogenic lineages in vitro. Osteoblastic differentiation was confirmed by both expression of alkaline phosphate and mineral deposition visualized by Alizarin Red staining. Adipogenic differentiation was confirmed by production of lipid droplets, which were detected by Oil Red-O staining. In summary, we have generated a stable hMSC line that can be continually propagated and retains both osteoblastic and adipogenic differentiation potential.

Carcinosarcomas are rare, malignant, biphasic tumors simultaneously comprising carcinoma and sarcoma in a single tumor. We present an extremely rare case of gastric carcinosarcoma with an osteoblastic component that drastically changed its shape within 2 mo. A 59-year-old male patient presented to the emergency outpatient unit with a complaint of black stool. Gastrointestinal endoscopy showed an ulcerated mass in the cardia of the lesser curvature of the stomach. Biopsy specimens revealed only adenocarcinoma. Two months later, the ulcerated lesion drastically changed its shape into an exophytic tumor. Total gastrectomy was performed. In the resected specimen, the gastric tumor contained both adenocarcinoma and sarcoma components with lace-like osteoid. The patient died 7 mo after the operation, and an autopsy was performed. In the autopsy, widespread metastases were present in the liver, lung, lymph nodes and peritoneum. In this report, we describe a case of gastric carcinosarcoma and presume its tumorigenesis based on the autopsy findings.

Objective: Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells (MSCs) has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix (OSX) promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs., Materials and Methods: In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5ìM Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection (MOI) treatment, gene specific methylation of OSX was analyzed by methylation specific PCR (MSP)., Results: The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation (p<0.05), but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards., Conclusion: MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases.

Background: Human dental pulp stem cells (DPSCs) have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1) was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP), and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation., Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21(Cip1), induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide., Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.

Background: Fibrous hamartoma is the key pathology of congenital pseudarthrosis of the tibia (CPT), which was shown to have low osteogenicity and high osteoclastogenicity. This study further investigated the mechanism of impaired osteoblastic differentiation of fibrous hamartoma cells., Methods: Fibroblast-like cells were obtained from enzymatically dissociated fibrous hamartomas of 11 patients with CPT associated with neurofibromatosis type I (NF1). Periosteal cells were also obtained from the distal tibial periosteum of 3 patients without CPT or NF1 as control. The mRNA levels of Wnt ligands and their canonical receptors, such as Lrp5 and β-catenin, were assayed using reverse transcriptase PCR (RT-PCR). Changes in mRNA expression of osteoblast marker genes by rhBMP2 treatment were assayed using quantitative real time RT-PCR. Changes in mRNA expression of transcription factors specifically involved in osteoblastic differentiation by rhBMP2 treatment was also assayed using quantitative real-time RT-PCR., Results: Wnt1 and Wnt3a mRNA expression was lower in fibrous hamartoma than in tibial periosteal cells, but their canonical receptors did not show significant difference. Response of osteoblastic marker gene expression to rhBMP2 treatment showed patient-to-patient variability. Col1a1 mRNA expression was up-regulated in most fibrous hamartoma tissues, osteocalcin was up-regulated in a small number of patients, and ALP expression was down-regulated in most fibrous hamartoma tissues. Changes in mRNA expression of the transcription factors in response to rhBMP2 also showed factor-to-factor and patient-to-patient variability. Dlx5 was consistently up-regulated by rhBMP2 treatment in all fibrous hamartoma tissues tested. Msx2 expression was down-regulated by rhBMP2 in most cases but by lesser extent than control tissue. Runx2 expression was up-regulated in 8 out of 18 fibrous hamartoma tissues tested. Osterix expression was up-regulated in 2 and down-regulated in 3 fibrous hamartoma tissues., Conclusions: Congenital pseudarthrosis of the tibia appears to be caused by fibrous hamartoma originating from aberrant growth of Nf1 haploinsufficient periosteal cells, which failed in terminal osteoblastic differentiation and arrested at a certain stage of this process. This pathomechanism of CPT should be targeted in the development of novel therapeutic biologic intervention.