A perfusion culture system for assessing bone marrow stromal cell differentiation on PLGA scaffolds for bone repair

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration

AN ASSESSMENT OF THE PROPOSED NEW RISK MANAGEMENT PROGRAMS

The purpose of this assessment as outlined in the terms of reference is: "To obtain an assessment by an independent third party of the expected performance of the proposed new business risk management program's proposed New NISA and production insurance relative to the current set of risk management programming, including NISA, CFIP, crop insurance and companion programs." Within this context, the specific mandate and scope is to assess "the extent to which the current and proposed programs meet the objectives set out by Agriculture Ministers for business risk management programming, as follows: · to ensure programs are responsive to demand and that government dollars are directed to areas of need with respect to income stabilization, disaster mitigation, insurance coverage and investment; · to provide equal treatment for farmers across Canada facing similar risk situations; · to minimize the distortion of farmers' production and marketing decisions; · to focus on management of risks related to the stability of the entire farm and to avoid duplication of payments; · to be relatively simple and easy to understand; and · to facilitate long term planning by farmers."Risk and Uncertainty,

Pre-culture of human mesenchymal stromal cells in spheroids facilitates chondrogenesis at a low total cell count upon embedding in biomaterials to generate cartilage microtissues

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs. Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. Statement of significance: In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration.</p

BMP2 and TGF-β Cooperate Differently during Synovial-Derived Stem-Cell Chondrogenesis in a Dexamethasone-Dependent Manner

Recent studies highlighting mesenchymal stem cell (MSC) epigenetic memory suggest that a different differentiation medium may be required depending on the tissue of origin. As synovial-derived stem cells (SDSCs) attract interest we aimed to investigate the influence of TGF-β1, BMP-2 and dexamethasone on SDSC chondrogenesis in vitro. We demonstrate that dexamethasone-free medium led to enhanced chondrogenic differentiation at both the mRNA and matrix level. The greatest COL2A1/COL10A1 ratio was detected in cells exposed to a combination medium containing 10 ng/mL BMP-2 and 1 ng/mL TGF-β1 in the absence of dexamethasone, and this was reflected in the total amount of glycosaminoglycans produced. In summary, dexamethasone-free medium containing BMP-2 and TGF-β1 may be the most suitable when using SDSCs for cartilage tissue regeneration

Optimization of loading protocols for tissue engineering experiments

Tissue engineering (TE) combines cells and biomaterials to treat orthopedic pathologies. Maturation of de novo tissue is highly dependent on local mechanical environments. Mechanical stimulation influences stem cell differentiation, however, the role of various mechanical loads remains unclear. While bioreactors simplify the complexity of the human body, the potential combination of mechanical loads that can be applied make it difficult to assess how different factors interact. Human bone marrow-derived mesenchymal stromal cells were seeded within a fibrin-polyurethane scaffold and exposed to joint-mimicking motion. We applied a full factorial design of experiment to investigate the effect that the interaction between different mechanical loading parameters has on biological markers. Additionally, we employed planned contrasts to analyze differences between loading protocols and a linear mixed model with donor as random effect. Our approach enables screening of multiple mechanical loading combinations and identification of significant interactions that could not have been studied using classical mechanobiology studies. This is useful to screen the effect of various loading protocols and could also be used for TE experiments with small sample sizes and further combinatorial medication studies

A rapid method for the generation of uniform acellular bone explants: a technical note

<p>Abstract</p> <p>Background</p> <p>Bone graft studies lack standardized controls. We aim to present a quick and reliable method for the intra-operative generation of acellular bone explants.</p> <p>Methods</p> <p>Therefore, ovine cancellous bone explants from the iliac crest were prepared and used to test several methods for the induction of cell death. Over night heat inactivation was used as positive treatment control, methods to be investigated included UV light, or X- ray exposure, incubation in a hypotonic solution (salt-free water) and a short cycle of repeated freezing and thawing.</p> <p>Results</p> <p>Viability of treated and 2 days cultured bone explants was investigated by lactate dehydrogenase assay. Non-treated cultured control explants maintained around 50% osteocyte viability, while osteocyte survival after the positive treatment control was abolished. The most dramatic loss in cell viability, together with a low standard deviation, was a repeated cycle of freezing and thawing.</p> <p>Conclusions</p> <p>To summarize, we present a freeze-thaw method for the creation of acellular bone explants, which is easy to perform, not time-consuming and provides consistent results.</p

Shear and Dynamic Compression Modulates the Inflammatory Phenotype of Human Monocytes in vitro

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms

Retroviral-mediated overexpression of human bone morphogenetic protein 2 affects human mesenchymal stem cells during monolayer proliferation: A cautionary note

Background: Retroviral vectors are commonly used for gene transfer applications and they represent an effective way to provide a sustained delivery of a bioactive factor in basic research and tissue engineering applications. Cells that have been transduced with retroviral vectors ex vivo are usually amplified on tissue culture plastic, for a prolonged period of time, in order to obtain sufficient cell numbers prior to the experiment of interest. However, the effect of the transgene product on the transduced cells, during this period of time, is rarely, if ever, investigated. The current study investigated if transduction with a VSG.G pseudotyped retroviral vector expressing human bone morphogenetic protein 2 (Rv.BMP-2) influences the gene expression profile of human bone marrow-derived mesenchymal stem cells (hMSCs) during monolayer proliferation. hMSCs that have been transduced with a VSG.G pseudotyped retroviral vector expressing enhanced green fluorescent protein (Rv.eGFP) served as controls. Results: It was confirmed that Rv.BMP-2 transduced hMSCs produce detectable amounts of bone morphogenetic protein 2 (BMP-2). Gene expression analysis revealed that the hypertrophic marker collagen X was down-regulated by approximately 50% and the chondrogenic marker Aggrecan was elevated almost 9-fold in Rv.BMP-2 transduced hMSCs if compared to Rv.eGFP transduced control cells. Interestingly, the same changes in gene expression were detected when hMSCs were exposed to 100 ng/ml of recombinant human BMP-2 and their gene expression profile was compared to control hMSC. Again, collagen X message was down-regulated and Aggrecan message was up-regulated. Conclusion: These results indicate that, when using integrating vectors and then expanding the cells after transduction, controls need to be carefully planned to ensure the results obtained during the 3D experiments are not due to artefacts created in response to the different cell proliferation conditions employed

A Perfusion Culture System for Assessing Bone Marrow Stromal Cell Differentiation on PLGA Scaffolds for Bone Repair

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment

[Abstract] The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.This research was funded by a grant from the Collaborative Research Partner Acute Cartilage Injury Program of AO Foundation, Davos, Switzerland (Henning Madry, Magali Cucchiarini, David Eglin, Mauro Alini, Martin J. Stoddart. We thank R.J. Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA), X. Xiao (The Gene Therapy Center, University of Pittsburgh, Pittsburgh, PA), and E.F. Terwilliger (Division of Experimental Medicine, Harvard Institutes of Medicine and Beth Israel Deaconess Medical Center, Boston, MA) for providing the genomic AAV-2 plasmid clones and the 293 cell line, and G. Scherer (Institute for Human Genetics and Anthropology, Albert-Ludwig University, Freiburg, Germany) for the human SOX) sequenc