Xuehui Wu

Umbilical cord Wharton's Jelly: a new potential cell source of mesenchymal stromal cells for bone tissue engineering.

Although bone marrow-derived mesenchymal stromal cells (BMSCs) are a main cell source for tissue-engineered bone (TEB), the clinical use of BMSCs is restricted due to the invasive bone marrow aspiration procedure and the decline in available number of mesenchymal stromal cells (MSCs) and differentiation potential with increasing age. Umbilical cord-derived MSCs (UCMSCs) are likely to be a promising alternative cell source for TEB due to their higher availability and potential to proliferate and differentiate. To assess this possibility, we studied bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation and activation of signaling pathways in UCMSCs and BMSCs. UCMSCs showed a phenotype and differentiation potential similar to that of BMSCs. After 14 days of BMP2 treatment, the overall expression of several osteogenic-specific phenotypes (type I collagen, osteopontin, and osteocalcin) was similar for UCMSCs and BMSCs. The signaling pathway by which BMP2 induced differentiation of both cell types involved the membrane receptor-initiated signals including SMADs, P38, and extracellular regulated kinase. The similar characteristics of BMP2-induced osteogenic differentiation of UCMSCs and BMSCs in vitro would support the use of UCMSCs in TEB.

Effects of Pulsed Electromagnetic Field Frequencies on the Osteogenic Differentiation of Human Mesenchymal Stem Cells

The purpose of this study was to evaluate the effect of different frequencies of pulsed electromagnetic fields on the osteogenic differentiation of human mesenchymal stem cells. Third-generation human mesenchymal stem cells were irradiated with different frequencies of pulsed electromagnetic fields, including 5, 25, 50, 75, 100, and 150 Hz, with a field intensity of 1.1 mT, for 30 minutes per day for 21 days. Changes in human mesenchymal stem cell morphology were observed using phase contrast microscopy. Alkaline phosphatase activity and osteocalcin expression were also determined to evaluate human mesenchymal stem cell osteogenic differentiation.Different effects were observed on human mesenchymal stem cell osteoblast induction following exposure to different pulsed electromagnetic field frequencies. Levels of human mesenchymal stem cell differentiation increased when the pulsed electromagnetic field frequency was increased from 5 hz to 50 hz, but the effect was weaker when the pulsed electromagnetic field frequency was increased from 50 Hz to 150 hz. The most significant effect on human mesenchymal stem cell differentiation was observed at of 50 hz.The results of the current study show that pulsed electromagnetic field frequency is an important factor with regard to the induction of human mesenchymal stem cell differentiation. Furthermore, a pulsed electromagnetic field frequency of 50 Hz was the most effective at inducing human mesenchymal stem cell osteoblast differentiation in vitro.

A composite demineralized bone matrix--self assembling peptide scaffold for enhancing cell and growth factor activity in bone marrow.

The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic.

Prevascularisation with endothelial progenitor cells improved restoration of the architectural and functional properties of newly formed bone for bone reconstruction

PurposeThe aim of this study was to examine whether the addition of endothelial progenitor cells (EPCs) contributes to restoring the architectural and functional properties of newly formed bone for reconstruction of bone defects.MethodsBone marrow-derived EPCs and mesenchymal stem cells (MSCs) were co-seeded onto demineralized bone matrix (DBM) as a prevascularized tissue-engineered bone (TEB) for the repair of segmental bone defects to evaluate the effects of prevascularization of TEB on ameliorating morphological, haemodynamic and mechanical characteristics.ResultsThe restoration of the intraosseous vasculature and medullary cavity was improved markedly compared to the non-prevascularized groups. The blood supply, biomechanical strength, and bone mineral density of the prevascularized group were significantly higher than those of the non-prevascularized groups during bone reconstruction.ConclusionsThe present study indicates that EPC-dependent prevascularization contributes to bone healing with structural reconstruction and functional recovery and may improve the understanding of correlation between angiogenesis and osteogenesis.

Effects of Initial Cell Density and Hydrodynamic Culture on Osteogenic Activity of Tissue-Engineered Bone Grafts

This study aimed to study the effects of initial cell density and in vitro culture method on the construction of tissue-engineered bone grafts and osteogenic activities. Human mesenchymal stem cells (hMSCs) were seeded onto cubic scaffolds prepared from demineralized bone matrix (DBM) by three methods - static, hydrodynamic, or fibrin hydrogel-assisted seeding. The resulting cell-scaffold constructs were cultured in vitro by static flask culture or hydrodynamic culture. The initial cell density and the subsequent in vitro proliferation and alkaline phosphate activities of the constructs were analyzed. The constructs were also subcutaneously implanted in nude mice to examine their in vivo osteogenic activities. Hydrogel-assisted seeding gave the highest seeding efficiency, followed by hydrodynamic and conventional static seeding. During in vitro culture, hydrodynamic culture produced higher plateau cell densities, alkaline phosphatase (ALP) activities, and extracellular matrix production than static culture. After subcutaneous implantation in nude mice, the implants prepared by the combination of hydrogel-assisted seeding and hydrodynamic culture produced higher wet weight and bone mineral density than implants prepared by other methods. The results suggest that the hydrogel-assisted seeding can substantially increase the initial seed cell density in scaffolds. Subsequent hydrodynamic culture can promote the proliferation and osteoblastic differentiation of the seeded cells. Correspondingly, bone grafts produced by the combination of these two methods achieved the highest osteogenic activity among the three methods employed.

Involvement of toll-like receptor 2 and pro-apoptotic signaling pathways in bone remodeling in osteomyelitis.

Background and Aims: Osteomyelitis is a common manifestation of invasive Staphylococcus aureus infection characterized by bone loss and destruction. We investigated the role of toll-like receptor 2 (TLR2) in bacterial recognition and clearance in response to infection with an osteomyelitis isolate of S. aureus. Methods: Apoptosis was assessed in the osteoblastic cell line MC3T3-E1 by Annexin V-FITC/PI staining and flow cytometry. The expression of TLR2 and apoptosis-related and mitogen-activated protein kinase pathway proteins was assessed by qRT-PCR and western blotting. Alkaline phosphatase (ALP) activity and calcium deposition were assessed by ALP activity assay and Alizarin red staining. Results: S. aureus induced apoptosis, upregulated TLR2 expression, and activated mitogen-activated protein kinase pathways in a time dependent manner. Inhibition of the c-Jun N-terminal kinase (JNK) pathway downregulated TLR2 and suppressed the S. aureus induced activation of pro-apoptotic pathways. Short-hairpin RNA mediated silencing of TLR2 reversed S. aureus induced apoptosis and decrease in ALP activity and calcium deposition, and inhibition of JNK had a similar effect. Conclusion: We showed that osteoblast apoptosis and osteogenic differentiation in response to bacterial invasion are dependent on TLR2 expression and JNK activation, suggesting novel potential therapeutic targets for the treatment of osteomyelitis.

Co-culture with endothelial progenitor cells promotes survival, migration, and differentiation of osteoclast precursors.

In this study, we report the effect of endothelial progenitor cells (EPCs) on the biological behavior of osteoclast precursors in vitro by establishing an indirect co-culture system of mice EPCs and RAW 264.7 monocyte cells. Results show that the survival, migration, and differentiation of osteoclast precursors were greatly enhanced when co-cultured with EPCs. These phenotypic changes coincide with the upregulation of multiple genes affected cell behavior, including phospho-VEGFR-2, CXCR4, phospho-Smad2/3, phospho-Akt, phospho-ERK1, and phospho-p38 MAPK. The results collectively suggest that EPCs could modulate the survival, migration, and differentiation potential of osteoclast precursors, thus providing new insights in understanding of correlation between angiogenesis and bone homeostasis.

C/EBPβ mediates osteoclast recruitment by regulating endothelial progenitor cell expression of SDF-1α.

Integration of tissue-engineered bone grafts with the host bone is vital for the healing of critical-size bone defects. An important aspect of this process is bone resorption, which must be carried out by osteoclasts derived from the host. However, the mechanism underlying recruitment of host osteoclast precursors to graft sites remains unclear. Endothelial progenitor cells (EPCs) mobilize from the bone marrow into the circulation and home to sites of angiogenesis such as tissue remodeling. Since EPCs express SDF-1, and C/EBPβ is known to regulate SDF-1α expression, we hypothesized that EPCs may recruit CXCR4-expressing host osteoclast precursors to the repair area and that this recruitment may be mediated through C/EBPβ signaling. Using an inflammatory EPC model we showed that EPCs upregulate protein levels of both SDF-1α and C/EBPβ. A luciferase assay confirmed that C/EBPβ acts on the SDF-1α promoter in these cells, and that binding is increased under conditions of inflammation, while silencing of C/EBPβ reduces expression of SDF-1 α and C/EBPβ. Using RAW264.7 cells as a model of osteoclastic monocyte precursors, we investigated their responses to migratory factors in EPC conditioned medium. We showed that RAW264.7 cells migrate towards conditioned medium from EPCs treated with IL-1β, an effect which could be abolished by silencing C/EBPβ in EPCs, and was almost completely blocked by silencing CXCR4 in RAW264.7 cells. These findings show that EPCs respond to inflammatory stimuli by signaling to osteoclast precursors via SDF-1, and that C/EBPβ mediates this response.

Genome-wide association study for osteoarthritis

Robert Kirsner and colleagues (Sept 15, p 977) describe a spray formulation of allogeneic neonatal keratinocytes and fi broblasts for the treatment of chronic venous leg ulcers. Such therapies are novel and of major interest. However, although this technique certainly has potential, we have some concerns about the interpretation of the results of Kirsner and colleagues’ study. First, it is not clear whether Kirsner and co-workers tested the viability of the fibroblasts and keratinocytes. Viable cells are crucial in the treatment of ulcers. Second, why are there no representative fi gures to show that wound healing was greater with 0·5×106 cells/mL every 14 days than with vehicle alone? Third, the follow-up (12 weeks) was too short to show the eff ect of spray-applied cell therapy with human allogeneic fi broblasts and keratinocytes on longterm outcomes. Were there any such follow-up fi ndings with regard to this technology? Finally, this spray-applied cell therapy contains compound solutions, including cells and matrix. It would be interesting to know which part is responsible for the clinical eff ects, and whether the mechanisms of action are direct (diff erentiation into myofi broblast or vascular endothelial cells), indirect (cell–cell contact or paracrine eff ects), or both.

Tissue-engineered bone treating simple bone cyst-a new strategy.

BACKGROUND The pathological fracture is a most important complication during bone cyst and can be prevented by early focus clearance and bone grafting. Tissue-engineered bone (TEB) with outstanding osteogenesis is a better choice for bone repair. Here, we firstly reported that TEB was used to heal bone cyst. MATERIALS AND METHODS The clinical data were collected from 23 patients who received bone defect repair separately with TEB or allogeneic bone (Allo-B) after erasion during 2004-2008. Allo-B had been as a control. The healing time and healing quality, the incidence of complications, the safety, and the bone grafting failure rate were compared. RESULTS In TEB group, the follow-up time was 28 ± 15.48 months; nine cases were confirmed healed (3.45 ± 2.01 months), one case was cyst healing with defect, and one case had relapse. In Allo-B, 12 patients were followed up for 28.58 ± 20.44 months; seven cases were confirmed healed (6.75 ± 3.31 mo), four cases were cyst healing with defect, and one case had relapse. After operation, no statistically significant differences in bone healing and incidence of complications were observed between two groups, but the difference in bone healing time was statistically significant (P < 0.05). There was no else tumorigenesis in both groups. CONCLUSIONS In treating simple bone cyst, Allo-B and TEB have considerable efficacy and safety; TEB is superior to Allo-B in respect of healing time; there is no rejection after TEB grafting but certain rejection after Allo-B grafting.