methoxy-poly(ethylene glycol) modified poly(L-lactide) enhanced cell affinity of human bone marrow stromal cells by the upregulation of 1-cadherin and delta-2-catenin

Poly(l-lactide) (PLLA), a versatile biodegradable polymer, is one of the most commonly-used materials for tissue engineering applications. To improve cell affinity for PLLA, poly(ethylene glycol) (PEG) was used to develop diblock copolymers. Human bone marrow stromal cells (hBMSCs) were cultured on MPEG-b-PLLA copolymer films to determine the effects of modification on the attachment and proliferation of hBMSC. The mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analyzed using RT-qPCR to understand the underlying mechanisms. It was found that MPEG-b-PLLA copolymer films significantly improved cell adhesion, extension, and proliferation.This was found to be related to the significant upregulation of two adhesion genes, CDH1 and CTNND2, which encode 1-cadherin and delta-2-catenin, respectively, two key components for the cadherin-catenin complex. In summary, MPEG-b-PLLA copolymer surfaces improved initial cell adhesion by stimulation of adhesion molecule gene expression.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT(2B), is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT(2B) receptors by circulating c-kit(+) precursor cells, whereas mice lacking 5-HT(2B) receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT(2B) receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT(2B) receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34(+) or mice c-kit(+) progenitor cells in the presence of a 5-HT(2B) receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT(2B) receptors on bone marrow lineage progenitors is critical for the development of PAH.

Dissecting the prostate cancer stem cell niche inside the bone marrow

Prostate cancer frequently metastasizes to bone, which becomes incurable; yet how cancer cells manage to migrate and grow inside the bone remains unknown. In this study I have discovered that both bone and fat cells within the bone marrow actively promote the survival and expansion of prostate cancer cells, and have subsequently developed approaches that can effectively inhibit these processes. Therefore, my work offers opportunities for the development of new prognostic and therapeutic approaches against metastatic prostate cancer and have the potential for improving the treatment outcome of the patients.

Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.

Solid volume fraction estimation of bone:marrow replica models using ultrasound transit time spectroscopy

The acceptance of broadband ultrasound attenuation (BUA) for the assessment of osteoporosis suffers from a limited understanding of both ultrasound wave propagation through cancellous bone and its exact dependence upon the material and structural properties. It has recently been proposed that ultrasound wave propagation in cancellous bone may be described by a concept of parallel sonic rays; the transit time of each ray defined by the proportion of bone and marrow propagated. A Transit Time Spectrum (TTS) describes the proportion of sonic rays having a particular transit time, effectively describing the lateral inhomogeneity of transit times over the surface aperture of the receive ultrasound transducer. The aim of this study was to test the hypothesis that the solid volume fraction (SVF) of simplified bone:marrow replica models may be reliably estimated from the corresponding ultrasound transit time spectrum. Transit time spectra were derived via digital deconvolution of the experimentally measured input and output ultrasonic signals, and compared to predicted TTS based on the parallel sonic ray concept, demonstrating agreement in both position and amplitude of spectral peaks. Solid volume fraction was calculated from the TTS; agreement between true (geometric calculation) with predicted (computer simulation) and experimentally-derived values were R2=99.9% and R2=97.3% respectively. It is therefore envisaged that ultrasound transit time spectroscopy (UTTS) offers the potential to reliably estimate bone mineral density and hence the established T-score parameter for clinical osteoporosis assessment.

Direct bone marrow HSC transplantation enhances local engraftment at the expense of systemic engraftment in NSG mice

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-beta and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation. Published by Elsevier Ltd.

Substrate Strain and Proliferation & Differentiation of Bone Marrow Mesenchymal Stem Cells

New methods of surface modification of transparent silicone substrate were developed, and a new set of cell culture devices that provide homogeneous substrate strain was designed. Using the new device, effects of cyclic substrate strain on bone marrow mesenchymal stem cells(MSCs) were studied. It was found that cyclic strain influenced proliferation and differentiation of bone marrow MSCs in different ways.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2

Background: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. Methods: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. Results: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNF alpha and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion-and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFa induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. Conclusions: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion-and proliferation-stimulating effects of TNFa, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing

Analysing the contribution of ATM/ATR pathway activation in establishing the premature senescence of E2F1/E2F2-/- bone-marrow-derived macrophages

E2F1 and E2F2 transcription factors have an important role during the regulation of cell cycle. In experiments done with E2F1/E2F2 knockout mice, it has been described that bone-marrow-derived macrophages (BMDM) undergo an early rapid proliferation event related to DNA hyper-replication. As a consequence, DNA damage response (DDR) pathway is triggered and E2F1/E2F2 knockout macrophages enter premature senescence related to G2/M phase arrest. The exact mechanism trough which DNA hyper-replication leads to DDR in absence of E2F1 and E2F2 remains undiscovered. To determine whether the ATR/ATM pathway, the master regulator of G2/M checkpoint, might be the surveillance mechanism in order to regulate uncontrolled proliferation in the DKO model, we monitored and analysis biochemical properties of BMDM cultures in the presence of caffeine, a potent inhibitor of ATM/ATR activity. Our results show that the addition of caffeine abolishes premature senescence in DKO BMDM, stimulates γ-H2AX accumulation and decreases Mcm2 expression.