Controlled surface initiated polymerization of N-isopropylacrylamide from polycaprolactone substrates for regulating cell attachment and detachment

Poly(ε-caprolactone) (PCL) substrates were modified with thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) brushes to direct and control cellular attachment and detachment. Prior to brush growth, the surface of PCL was activated by a diamine to allow for initiator coupling. Infrared spectra taken before and after cell culturing demonstrated the covalently attached nature of the PNIPAM brushes. PCL is a biocompatible polymer and to prove that the modifications described above did not change this characteristic property, a cell attachment/detachment study was carried out. The modified substrates showed a lower cell attachment when compared to PCL alone and to PCL films modified with the initiator. The possibility to detach the cells in the form of a sheet was proved using PNIPAM-modified PCL films by lowering the temperature to 25 °C. No relevant detachment was shown by the unmodified or by the initiator modified surfaces. This confirmed that the detachment was temperature dependent and not connected to other factors such as polymer swelling. These functionalized polymeric films can find applications as smart cell culture systems in regenerative medicine applications

In Vitro Bone Formation Associated with Apatite Coated Polylactide

Bone formation onto poly(L-lactide), which was plasma-spray coated with various quantities of hydroxyapatite (0%, 15%, 36% and 100% coverage), was investigated in an in vitro assay. Rat bone marrow cells were grown on the different coatings and the cellular response and elaborated extracellular matrix was examined at the light and electron microscopical level after 1, 2 , 4 and 8 weeks of culture. Proliferation of cells into multilayers was seen on the 0% , 36% and 100% , but not on the 15 % coatings. Coinciding with this was the sparse formation of extracellular matrix on the latter, and its abundant appearance on the former three coatings. Scanning and transmission electron microscopy revealed a mineralized extracellular matrix on the 100% and 36% coatings after 2 and 4 weeks , respectively, and on the 15 % coating after 8 weeks. Mineralization was not observed on uncoated poly(L-lactide). At the interface between hydroxyapatite and the mineralized extracellular matrix, one or more electron dense layers were frequently observed , which showed morphological similarities with structures between these two entities in vivo. The results of this in vitro study show that, in the model used, hydroxyapatite is required to obtain the elaboration of mineralized extracellular matrix on poly(L-lactide)

Distribution and viability of fetal and adult human bone marrow stromal cells in a biaxial rotating vessel bioreactor after seeding on polymeric 3D additive manufactured scaffolds

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiatio

Hydrogels that listen to cells:a review of cell-responsive strategies in biomaterial design for tissue regeneration

The past decade has seen a decided move from static and passive biomaterials to biodegradable, dynamic, and stimuli responsive materials in the laboratory and the clinic. Recent advances towards the rational design of synthetic cell-responsive hydrogels-biomaterials that respond locally to cells or tissues without the input of an artificial stimulus-have provided new strategies and insights on the use of artificial environments for tissue engineering and regenerative medicine. These materials can often approximate responsive functions of a cell's complex natural extracellular environment, and must respond to the small and specific stimuli provided within the vicinity of a cell or tissue. In the current literature, there are three main cell-based stimuli that can be harnessed to create responsive hydrogels: (1) enzymes (2) mechanical force and (3) metabolites/small molecules. Degradable bonds, dynamic covalent bonds, and non-covalent or supramolecular interactions are used to provide responsive architectures that enable features ranging from cell selective infiltration to control of stem-cell differentiation. The growing ability to spatiotemporally control the behavior of cells and tissue with rationally designed responsive materials has the ability to allow control and autonomy to future generations of materials for tissue regeneration, in addition to providing understanding and mimicry of the dynamic and complex cellular niche

The effect of bone marrow aspiration strategy on the yield and quality of human mesenchymal stem cells

Introduction Large inter-donor differences exist in human mesenchymal stem cell (hMSC) yield and the response of these cells to osteogenic stimuli. The source of these differences may be clinical differences in stem cell characteristics between individuals or the aspiration procedure itself

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers : effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterial

3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates

3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM-MSCs) combined with calcium phosphate microparticles and enveloped in a platelet-rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose-derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM-MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM-MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM-MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un-aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM-MSCs (7/9) followed by SVF+rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose-derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect