The cytocompatibility and early osteogenic characteristics of an injectable calcium phosphate cement.

In this study, the cytocompatibility and early osteogenic characteristics of rat bone marrow cells (RBMCs) on injectable calcium phosphate (CaP) cement (Calcibon) were investigated. In addition to unmodified CaP cement discs, 2 other treatments were given to the discs: preincubation in MilliQ and sintering at different temperatures. After primary culture, RBMCs were dropwise seeded on the discs and cultured for 12 days. The samples were evaluated in terms of cell viability, morphology (live and dead assays and scanning electron microscopy (SEM)), cell proliferation (deoxyribonucleic acid (DNA) analyses), early cell differentiation (alkaline phosphatase (ALP) activity), and physicochemical analyses (xray diffraction (XRD)). The live and dead, DNA, and SEM results showed that Calcibon discs without any additional treatment were not supporting osteoblast-like cells in vitro. There were fewer cells, and cell layers were detached from the disc surface. Therefore, different preincubation periods and sintering temperatures were evaluated to improve the cytocompatibility of the CaP cement. Preincubating discs in MilliQ for periods of 1, 4, 8, and 12 weeks resulted in the hydrolysis of a-tri calcium phosphate (TCP) into an apatite-like structure with some b-TCP, as shown with XRD, but the material was not cytocompatible. Sintering the discs between 8008C and 11008C resulted in conversion of a-TCP to b-TCP with some hydroxyapatite and an increase in crystallinity. Eventually, the discs sintered at 11008C achieved better cell attachment, more-abundant cell proliferation, and earlier differentiation than other sintered (6008C, 8008C, and 10008C), preincubated, and unmodified specimens. On basis of our results, we conclude that in vivo results with CaP-based cements do not guarantee in vitro applicability. Furthermore, unmodified Calcibon is not cytocompatible in vitro, although preincubation of the material results in a more-favorable cell response, sintering of the material at 11008C results in the best osteogenic properties. In contrast to in vivo studies, the Calcibon CaP cement is not suitable as a scaffold for cellbased tissue-engineering strategies

Bone regenerative properties of injectable PGLA-CaP composite with TGF-beta1 in a rat augmentation model.

The aim of this study was to examine the bone augmentation properties of an injectable composite consisting of PLGA microspheres/CaP cement (20/80), and the additional effect of loading PLGA microspheres with TGF-β1 (200 ng). For this purpose, PLGA/CaP composites (control) and PLGA/CaP composites loaded with TGF-β1 (test group) were injected on top of the skulls of 24 Wistar rats. Each rat received 2 materials from the same experimental group, and in total 48 implants were placed (n = 8). After 2, 4, and 8 weeks the results were evaluated histologically and histomorphometrically. The contact length between the implants and newly formed bone increased in time, and was significantly higher for the TGF-β1-loaded composites after 2 weeks. Also, bone formation was significantly higher for the TGF-β1-loaded composites (18.5% ± 3) compared to controls (7.21% ± 5) after 8 weeks of implantation. Immunohistochemical staining demonstrated massive inflammatory infiltrates in both groups, particularly at 2 weeks, which decreased substantially at 4 and 8 weeks. In conclusion, injectable PLGA/CaP composites stimulated bone augmentation in a rat model. The addition of TGF-β1 to the composite significantly increased bone contact at 2 weeks and enhanced new bone formation at 8 weeks

Mechanical evaluation of implanted calcium phosphate cement incorporated with PLGA microparticles

In this study, the mechanical properties of an implanted calcium phosphate (CaP) cement incorporated with 20wt% poly (DL-lactic-coglycolic acid) (PLGA) microparticles were investigated in a rat cranial defect. After 2, 4 and 8 weeks of implantation, implants were evaluated mechanically (push-out test) and morphologically (Scanning Electron Microscopy (SEM) and histology). The results of the push-out test showed that after 2 weeks the shear strength of the implants was 0.4470.44MPa (average7sd), which increased to 1.3471.05MPa at 4 weeks and finally resulted in 2.6072.78MPa at 8 weeks. SEM examination showed a fracture plane at the bone–cement interface at 2 weeks, while the 4- and 8-week specimens created a fracture plane into the CaP/PLGA composites, indicating an increased strength of the bone–cement interface. Histological evaluation revealed that the two weeks implantation period resulted in minimal bone ingrowth, while at 4 weeks of implantation the peripheral PLGA microparticles were degraded and replaced by deposition of newly formed bone. Finally, after 8 weeks of implantation the degradation of the PLGA microparticles was almost completed, which was observed by the bone ingrowth throughout the CaP/PLGA composites. On basis of our results, we conclude that the shear strength of the bone–cement interface increased over time due to bone ingrowth into the CaP/PLGA composites. Although the bone–cement contact could be optimized with an injectable CaP cement to enhance bone ingrowth, still the mechanical properties of the composites after 8 weeks of implantation are insufficient for load-bearing purpose

STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

Contains fulltext : 53235.pdf (publisher's version ) (Open Access)Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium

A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells

<div><p>Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for <i>ex vivo</i> expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize <i>ex vivo</i> Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that <i>ex vivo</i> expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the <i>FOXP3</i> gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation <i>in vivo</i>. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the <i>ex vivo</i> expansion of Treg for clinical immunotherapy.</p></div

TNFR2-agonist preserves the stability of low purity MACS-isolated Treg during <i>ex vivo</i> expansion.

<p>Low purity MACS-isolated human Treg were cultured as described under Materials and Methods. Thereafter, the expanded Treg were harvested, washed, and analyzed for their suppressor capacity in a CFSE-based co-culture suppression assay. ^CtrlTreg, ^RapTreg and ^R/TTreg represent cells expanded in the absence or presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (A) Histograms showing the inhibition of proliferation of Tresp following the addition of graded doses of Treg. The ratios of Treg:Tresp are indicated on the top. Numbers in the histograms show the percentage of divided cells. Cumulative data (N = 6) are shown in the right panel. (B) Flow cytometry of intracellular IL-17A and IFNγ of Treg at the start of the culture (day 0) and after expansion under the indicated conditions. Dot plots showing representative data of N = 4–7 individuals as shown in the cumulative data graph (right panel). Numbers show the percentage of positive cells. Each line represents Treg derived from a specific donor were expanded under the conditions described on the X-axis. (C) Expression of CXCR3, CCR5, CCR6, CCR7, CD62L and CD27 before (day 0) and after expansion (day 16). Numbers show the percentage of positive cells. (D) Bisulphite sequencing of the TSDR of expanded Treg. Each dot represents a single experiment. (E) Expanded Treg were harvested, rested overnight, and then restimulated with anti-CD3/anti-CD28 beads in a 1:2 ratio of beads to cells, in the absence or presence of IL-1β (50 ng/mL) and IL-23 (50 ng/mL) for 2-days. Exogenous rhIL-2 (200 U/mL) was included in the cell cultures. Thereafter, intracellular production of IL-17A was analyzed using flow cytometry. Cumulative data derived from seven different donors are shown. A Friedman plus Dunns post hoc test (A, D, and E) or Kruskal-Wallis plus Dunns post hoc test (B) were used for comparison among multiple groups. Asterisks indicate significant differences. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist plus rapamycin expanded FACS-sorted Treg reveal a high suppressive capacity and less IL-17A and IFNγ producing potential.

<p>High purity FACS-sorted human Treg were stimulated as described in Material and Methods. At day 7 of the cell cultures, expanded Treg were harvested, washed, and analyzed for their suppressive capacity in a CFSE-based co-culture suppression assay. ^CtrlTreg, ^RapTreg and ^R/TTreg represent cells expanded in the absence or presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (A) Histograms showing the inhibition of proliferation of responder cells following the addition of graded doses of Treg. The ratio of Treg:Tconv is indicated on the top. Numbers show the percentage of divided cells. (B) Flow cytometry of intracellular IL-17A and IFNγ of Treg before (day 0) and after expansion under the indicated conditions. Data derived from two different healthy donors are shown. Numbers show the percentage of positive cells. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist facilitates <i>ex vivo</i> expansion of low purity MACS-isolated human Treg.

<p>(A) Schematic overview of low purity MACS-isolated Treg expansion strategy as described in Materials and Methods. (B) Dot plots showing a representative FOXP3 expression pattern after MACS isolation of Treg, as shown in the cumulative data graph (N = 10, right panel). (C) Cumulative graph showing fold expansion of low purity Treg that were stimulated under the conditions as indicated on the X-axis (N = 5). A Friedman test was used for comparison of three groups. (D) Flow cytometry of surface expression of HLA-DR, and intracellular expression of FOXP3 and Helios of MACS-isolated Treg before (input) and after expansion under the indicated conditions. Numbers within the quadrant gates show the percentage of positive cells. Cumulative data of %FOXP3+Helios+, %FOXP3+HLA-DR+, the median fluorescence intensity (MFI) of Helios and HLA-DR are shown in the right panel, respectively. Wilcoxon paired t-Test and Kruskal-Wallis test were used for comparison between two and multiple groups, respectively. Asterisks indicate significant differences. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist plus rapamycin expanded Treg inhibit inflammation in a humanised mouse model.

<p>(A) Schematic overview of the humanised skin inflammation mouse model used. In brief, SCID mice were transplanted with a human skin graft, 21 days after engraftment, PBS (as a control), or allogeneic human PBMC (huPBMC) only or huPBMC plus Treg of interest (at a ratio of 1:1) were injected intra peritoneally. 26 days later the animals were sacrificed to analyze the mouse spleen and human skin grafts. ^RapTreg and ^R/TTreg refer to low purity MACS-isolated Treg expanded for 16-days in the presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (B) Representative photographs of spleens derived from mice infused with PBS, huPBMC only, or huPBMC plus Treg of interest, 21 days after the skin transplantation. Cumulative data showing the weight of spleens derived from N = 4 mice (right panel; n.d. = not determined). (C) Representative photographs showing histology (HE staining) of human skin grafts. Left panel: 10 x magnification. Right panel shows the epidermal thickness (μm) of human skin grafts following infusion of PBS, huPBMC, huPBMC plus ^RapTreg or huPBMC plus ^R/TTreg. Mean ± SD, N = 6. (D) Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis. A representative photograph of N = 4 as presented in the cumulative data graph is shown (right panel; Mean ± SD). 20 x magnification. Wilcoxon paried t Test was used to compare the group of mice infused with huPBMC only with other groups of mice infused with huPBMC plus Treg of interest. Asterisks indicate significant differences.</p