Differentiation Of Human Periosteum-derived Cells Research Articles

Zinc Sulfate Stimulates Osteogenic Phenotypes in Periosteum-Derived Cells and Co-Cultures of Periosteum-Derived Cells and THP-1 Cells.

Coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption maintains both mechanical integrity and mineral homeostasis. Zinc is required for the formation, mineralization, growth, and maintenance of bones. We examined the effects of zinc sulfate on osteoblastic differentiation of human periosteum-derived cells (hPDCs) and osteoclastic differentiation of THP-1 cells. Zinc sulfate enhanced the osteoblastic differentiation of hPDCs; however, it did not affect the osteoclastic differentiation of THP-1 cells. The levels of extracellular signaling-related kinase (ERK) were strongly increased during osteoblastic differentiation in zinc sulfate-treated hPDCs, compared with other mitogen-activated protein kinases (MAPKs). Zinc sulfate also promoted osteogenesis in hPDCs and THP-1 cells co-cultured with the ratio of one osteoclast to one osteoblast, as indicated by alkaline phosphatase levels, mineralization, and cellular calcium contents. In addition, the receptor activator of nuclear factor kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio was decreased in the zinc sulfate-treated co-cultures. Our results suggest that zinc sulfate enhances osteogenesis directly by promoting osteoblastic differentiation and osteogenic activities in osteoblasts and indirectly by inhibiting osteoclastic bone resorption through a reduced RANKL/OPG ratio in co-cultured osteoblasts and osteoclasts.

PCL/HA Hybrid Microspheres for Effective Osteogenic Differentiation and Bone Regeneration.

The purpose of this study is to develop a bioactive bone graft based on polycaprolactone (PCL, synthetic polymer; used in clinical practices as a grafting material for craniofacial bone defects) and hyaluronic acid (HA, bioactive natural polymer; known as a promoting substance for bone regeneration) that would be fabricated by clinically available procedures (mild condition without toxic chemicals) and provide bioactivity for sufficient period, and thus effectively induce bone reconstruction. For this, PCL/HA hybrid microspheres were produced by a spray-precipitation technique using clinically adapted solvents. The HA was stably and evenly entrapped in the PCL/HA hybrid microspheres. It was demonstrated that the PCL/HA hybrid microspheres provide an appropriate environment for proliferation and osteogenic differentiation of human periosteum-derived cells (hPDCs) (in vitro) and allow significantly enhanced bone regeneration (in vivo) compared with PCL microspheres without HA. The PCL/HA hybrid microspheres can be a simple but clinically applicable bioactive bone graft for large-sized bone defects.

Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked Structure for Bone Regeneration.

Sustained release of bioactive molecules from delivery systems is a common strategy for ensuring their prolonged bioactivity and for minimizing safety issues. However, residual toxic reagents, the use of harsh organic solvents, and complex fabrication procedures in conventional delivery systems are considered enormous impediments toward clinical use. Herein, we describe bone morphogenetic protein-2 (BMP-2)-immobilized porous polycaprolactone particles with unique leaf-stacked structures (LSS particles) prepared using clinically feasible materials and procedures. The BMP-2 immobilized in these LSS particles is continuously released up to 36 days to provide an appropriate environment for osteogenic differentiation of human periosteum-derived cells and new bone formation. Thus, the leaf-stacked structures of these LSS particles provide a simple but clinically applicable platform for effectively delivering a variety of bioactive molecules, such as growth factors, hormones, cytokines, peptides, etc.

Lin28a enhances in vitro osteoblastic differentiation of human periosteum-derived cells.

Despite a capacity for proliferation and an ability to differentiate into multiple cell types, in long-term culture and with ageing, stem cells show a reduction in growth, display a decrease in differentiation potential, and enter senescence without evidence of transformation. The Lin28a gene encodes an RNA-binding protein that plays a role in regulating stem cell activity, including self-renewal and differentiation propensity. However, the effect of the Lin28a gene on cultured human osteoprecursor cells is poorly understood. In the present study, alkaline phosphatase activity, alizarin red-positive mineralization, and calcium content, positive indicators of osteogenic differentiation, were significantly higher in cultured human periosteum-derived cells (hPDCs) with Lin28a overexpression compared with cells without Lin28a overexpression. Lin28a overexpression by hPDCs also increased mitochondrial activity, which is essential for cellular proliferation, as suggested by a reduced presence of reactive oxygen species and significantly enhanced lactate levels and ATP production. Our results suggest that, in hPDCs, the Lin28a gene enhances osteoblastic differentiation and increases mitochondrial activity. Although Lin28a is known as a marker of undifferentiated human embryogenic stem cell, there is limited evidence regarding the influence of Lin28a on osteoblastic differentiation of cultured osteoprecursor cells. This study was to examine the impact of Lin28a on osteogenic phenotypes of human periosteum-derived cells. Their phenotypes can be similar to those of mesenchymal stem cells. Our results suggest that the Lin28a gene enhances the osteoblastic differentiation of human periosteum-derived cells. In addition, the Lin28a gene increases mitochondrial activity in human periosteum-derived cells.

ID: 1058 Effective bone formation through BMP-2-immobilized highly porous GBR membrane

Sound healing of large bone defects is critical challenges in most of clinical fields. In general healing process of bone regeneration, the rapid infiltration of connective tissue, whereas relatively slow bone regeneration in bone defect leads to the incomplete bone formation. To solve this drawback, guide bone regeneration (GBR) membrane which could prevent rapid infiltration of connective tissue into bone defect, thus GBR membrane is feasible for compact bone regeneration in clinical fields. In recent, the most researchers believed that bioactive molecules-grafted GBR membranes may enhance the bone regeneration. To allow graft of bioactive molecules from porous membrane, chemically modified scaffolds are commonly used. This modification leads to sufficient interaction with active sites of bioactive molecules to stable the immobilization of bioactive molecules in the body. However it is hard to apply to clinical applications because of the toxicity of chemical residue used for the modification. In this study, we developed a GBR membrane with leaf-stacked structure which can allow sustained release of bone morphogenetic protein-2 (BMP-2) without any additional modification. The morphology, mechanical property, BMP-2 release profile, osteogenic differentiation of human periosteum-derived cells, and new bone formation efficiency of the BMP-2-loaded GBR membrane compared with commercial product were investigated.

Effects of Osteogenic-Conditioned Medium from Human Periosteum-Derived Cells on Osteoclast Differentiation.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration.

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

Analysis of Gene Expression Signatures for Osteogenic 3D Perfusion-Bioreactor Cell Cultures Based on a Multifactorial DoE Approach

The use of multifactorial design of experiments (DoE) in tissue engineering bioprocess development will contribute to the robust manufacturing of tissue engineered constructs by linking their quality characteristics to bioprocess operating parameters. In this work, perfusion bioreactors were used for the in vitro culture and osteogenic differentiation of human periosteum-derived cells (hPDCs) seeded on three-dimensional titanium (Ti) alloy scaffolds. A CaP-supplemented medium was used to induce differentiation of the cultured hPDCs. A two-level, three-factor fractional factorial design was employed to evaluate a range of bioreactor operating conditions by changing the levels of the following parameters: flow rate (0.5–2 mL/min), cell culture duration (7–21 days) and cell seeding density (1.5 × 103–3 × 103 cells/cm2). This approach allowed for evaluating the individual impact of the aforementioned process parameters upon a range of genes that are related to the osteogenic lineage, such as collagen type I, alkaline phosphatase, osterix, osteopontin and osteocalcin. Furthermore, by overlaying gene-specific response surfaces, an integrated operating process space was highlighted within which predetermined values of the six genes of interest (i.e., gene signature) could be minimally met over the course of the bioreactor culture time.