Periostin Accelerates Bone Healing Mediated by Human Mesenchymal Stem Cell-Embedded Hydroxyapatite/Tricalcium Phosphate Scaffold

<div><p>Background</p><p>Periostin, an extracellular matrix protein, is expressed in bone, more specifically, the periosteum and periodontal ligaments, and plays a key role in formation and metabolism of bone tissues. Human adipose tissue-derived mesenchymal stem cells (hASCs) have been reported to differentiate into osteoblasts and stimulate bone repair. However, the role of periostin in hASC-mediated bone healing has not been clarified. In the current study, we examined the effect of periostin on bone healing capacity of hASCs in a critical size calvarial defect model.</p><p>Methods and Results</p><p>Recombinant periostin protein stimulated migration, adhesion, and proliferation of hASCs <i>in vitro</i>. Implantation of either hASCs or periostin resulted in slight, but not significant, stimulation of bone healing, whereas co-implantation of hASCs together with periostin further potentiated bone healing. In addition, the number of Ki67-positive proliferating cells was significantly increased in calvarial defects by co-implantation of both hASCs and periostin. Consistently, proliferation of administered hASCs was stimulated by co-implantation with periostin <i>in vivo</i>. In addition, co-delivery of hASCs with periostin resulted in markedly increased numbers of CD31-positive endothelial cells and α-SMA-positive arterioles in calvarial defects.</p><p>Conclusions</p><p>These results suggest that recombinant periostin potentiates hASC-mediated bone healing by stimulating proliferation of transplanted hASCs and angiogenesis in calvarial defects.</p></div

Periostin stimulates chemotaxis migration, adhesion, and proliferation activity of hASCs <i>in vitro</i>.

<p>(A) Dose-dependent effect of periostin on hASC migration. hASCs were loaded in the upper chamber of a chemotaxis chamber apparatus and recombinant periostin with the indicated concentration was placed in the lower chamber, followed by measurement of the number of migrated cells after 12 h incubation. (B) Checkerboard analysis of the periostin-induced migration of hASCs was measured using a chemotaxis chamber. Periostin was placed in either the bottom, top, or both chambers of the chemotaxis system and hASCs were loaded into the upper chamber. The number of migrated cells was quantified after incubation of the cells for 12 h. Data represent mean ± S.D.; *, p < 0.05. (C) 96-well plates were coated with indicated concentration of periostin and adhesion of hASCs onto the plates was determined. (D) Dose dependence of periostin-stimulated proliferation. hASCs were treated with the increasing concentrations of periostin for 3 days and proliferation of cells was determined by MTT assay. Data represent mean ± S.D. *, p < 0.05 vs control.</p

Histological analysis of newly regenerated bone after implantation of hASCs and/or periostin.

<p>(A) H&E staining of calvarial defects eight weeks after implantation of hASCs and/or periostin. Higher magnification images of the regions highlighted by the black box in left panels are shown in right panels. Scale bar = 100 μm. (B) Area of newly regenerated bone tissues was determined in the defected bone and the percentage of bone area per healed area was calculated. Data represent mean ± S.D. (n = 8). *, p < 0.05 vs control.</p

Effects of hASCs and periostin on angiogenesis in calvarial defects.

<p>(A) Immunostaining of endothelial cells and smooth muscle cells in calvarial defects implanted with hASCs and/or periostin. The specimens were immunostained with anti-CD31 (red color) or anti-α-SMA (green color) antibodies, and overlaid images with nuclei (blue color) are shown. Scale bar = 50 μm. The numbers of CD31-positive capillaries (B) and α-SMA-positive blood vessels (C) per high power field were counted. Data represent mean ± S.D. (n = 8). #, p < 0.05; *, p < 0.05 vs control.</p

Effects of hASCs and/or periostin on regeneration of calvarial bone defects.

<p>(A) Masson’s trichrome staining of calvarial defects (blue color: mineralized bone tissue; red color: non-mineralized bone tissue). Higher magnification images of the regions highlighted by the black box in left panels are shown in right panels. (B) The gap of calvarial defects in the Massons’s trichrome staining images was quantified and shown as the distance between the advancing edges. Data represent mean ± S.D. (n = 8). *, p < 0.05 vs control.</p

Effects of periostin on proliferation of implanted hASCs <i>in vivo</i>.

<p>(A) hASCs were labeled with CM-DiI and loaded into HA/TCP scaffold with or without periostin, followed by implantation of the scaffold into the defective calvaria. Two weeks after implantation, tissue specimens were immunostained with anti-PCNA antibody. Overlaid images of CM-DiI-positive hASCs (red color), nuclei (blue color), and PCNA (green color) are shown. Scale bar = 20 μm. (B) The numbers of PCNA- and CM-DiI-double positive cells, which indicate proliferating hASCs, were counted and the percentage of PCNA-positive cells per CM-DiI-positive cells was determined. Data represent mean ± S.D. (n = 8). *, p < 0.05 vs control.</p

Micro-CT analysis of bone regeneration following implantation of hASCs and/or periostin into calvarial defects.

<p>Calvarial defects were implanted with HA/TCP scaffold bearing hASCs and/or periostin, or mock-treated (control). (A) Posterior view of a coronal sliced micro-CT images of the calvaria was captured at week 0 and 8. (B) The distance between the advancing edges was quantified from coronal-section view images of calvarial defects by Micro-CT. Data represent mean ± S.D. (n = 8). *, p < 0.05 vs control.</p

Efficient Production of Retroviruses Using PLGA/bPEI-DNA Nanoparticles and Application for Reprogramming Somatic Cells

<div><p>Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.</p> </div

Transfection efficacy of HEK293FT cells using various nanoparticles.

<p>GFP positive cells were determined by flow cytometry analysis approximately 24 h (B) or 40 h (A) after transfection. (A) HEK293FT cells were transfected with DNA encoding enhanced green fluorescence protein (EGFP) (pEGFP) using PLGA nanoparticles prepared using different protocols. Bright field images (1-3) and fluorescence images (4-6) of cells transfected with PLGA nanoparticles prepared by nanoprecipitation (1,4), double emulsification (2,5), and modified double emulsification (3,6) protocol are shown. 50 µl of PLGA-DNA preparation (100 mg PLGA and 100 µg DNA / ml) was applied to each well of six-well plates. (B) Bright field images (1-3) and fluorescence images (4-6) of cells transfected with PLGA/bPEI-DNA nanoparticles prepared with different molecular weight bPEIs (1, 4; 0.8 kDa, 2, 5; 2 kDa, 3, 6; 25 kDa), using a modified double emulsification protocol are shown. 1 µg of pEGFP was applied to each well of a six-well plate. Scale bars represent 100 µm (*, p < 0.05 by Student’s t-test). n=3.</p

Effects of nanoparticles on viability of HEK293FT cells.

<p>Cells in 24-well plate were subjected to MTT assay after incubation with different amounts of PLGA+DNA (DNA adsorbed on PLGA nanoparticles, 6:1, w/w), bPEI-DNA (3:1, w/w), or PLGA/bPEI-DNA (6:3:1, w/w/w) corresponding to the indicated polymer concentrations (PLGA+bPEI) (left panel). On the right panel, cell viability was tested after incubation with PLGA/bPEI-DNA (6:3:1, w/w/w, 24 well tested) for varying amount of time. n=3.</p