P. Sol

Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Seeded on Melt Based Chitosan Scaffolds for Bone Tissue Engineering Applications

The purpose of this study was to evaluate the growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded onto new biodegradable chitosan/polyester scaffolds. Scaffolds were obtained by melt blending chitosan with poly(butylene succinate) in a proportion of 50% (wt) each and further used to produce a fiber mesh scaffold. hBMSCs were seeded on those structures and cultured for 3 weeks under osteogenic conditions. Cells were able to reduce MTS and demonstrated increasing metabolic rates over time. SEM observations showed cell colonization at the surface as well as within the scaffolds. The presence of mineralized extracellular matrix (ECM) was successfully demonstrated by peaks corresponding to calcium and phosphorus elements detected in the EDS analysis. A further confirmation was obtained when carbonate and phosphate group peaks were identified in Fourier Transformed Infrared (FTIR) spectra. Moreover, by reverse transcriptase (RT)-PCR analysis, it was observed the expression of osteogenic gene markers, namely, Runt related transcription factor 2 (Runx2), type 1 collagen, bone sialoprotein (BSP), and osteocalcin. Chitosan-PBS (Ch-PBS) biodegradable scaffolds support the proliferation and osteogenic differentiation of hBMSCs cultured at their surface in vitro, enabling future in vivo testing for the development of bone tissue engineering therapies.

Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan-based scaffolds using a flow-perfusion bioreactor.

Native articular cartilage is subjected to synovial fluid flow during normal joint function. Thus, it is believed that the morphogenesis of articular cartilage may be positively regulated by the application of similar stimulation in vitro. In the present study, the effect of fluid flow over the chondrogenic differentiation of human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) was investigated. We intended to find out whether the shear stress caused by perfusion of the medium through the constructs was capable of augmenting the differentiation process. Human BMSCs were isolated from bone marrow aspirates and were characterized by flow cytometry. After expansion, hBM‐MSCs were seeded statically onto fibre mesh scaffolds, consisting of a blend of 50:50 chitosan:poly(butylene terephthalate adipate) (CPBTA). Constructs were cultured in a flow‐perfusion bioreactor for 28 days, using complete medium for chondrogenesis supplemented by TGFβ3. An enhanced ECM deposition and collagen type II production was observed in the bioreactor samples when compared to the static controls. Moreover, it was observed that hBM‐MSCs, in static cultures, take longer to differentiate. ECM accumulation in these samples is lower than in the bioreactor sections, and there is a significant difference in the expression of collagen type I. We found that the flow‐induced shear stress has a beneficial effect on the chondrogenic differentiation of hMSCs. Copyright

Chitosan–poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model

Tissue engineering sustains the need of a three‐dimensional (3D) scaffold to promote the regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population, allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our group evidenced significant growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded and cultured on melt‐based porous chitosan fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of these in vitro 3D cell constructs. In this study, chitosan‐based scaffolds were seeded and cultured in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in osteogenic culture conditions was performed into critical cranial size defects in nude mice. The objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks of implantation, scaffolds were harvested and prepared for micro‐computed tomography (µCT) analysis. Retrieved implants showed good integration with the surrounding tissue and significant bone formation, more evident for the scaffolds cultured and implanted with human cells. The results of this work demonstrated that chitosan‐based scaffolds, besides supporting in vitro proliferation and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects for their use in bone tissue‐engineering therapies. Copyright

In vitro degradation and in vivo biocompatibility of chitosan–poly(butylene succinate) fiber mesh scaffolds

In tissue engineering, the evaluation of the host response to the biomaterial implantation must be assessed to determine the extent of the inflammatory reaction. We studied the degradation of poly(butylene succinate) and chitosan in vitro using lipase and lysozyme enzymes, respectively. The subcutaneous implantation of the scaffolds was performed to assess tissue response. The type of inflammatory cells present in the surrounding tissue, as well as within the scaffold, was determined histologically and by immunohistochemistry. In the presence of lipase or lysozyme, the water uptake of the scaffolds increased. Based on the weight loss data and scanning electron microscopy analysis, the lysozyme combined with lipase had a notable effect on the in vitro degradation of the scaffolds. The in vivo implantation showed a normal inflammatory response, with presence of neutrophils, in a first stage, and macrophages, lymphocytes, and giant cells in a later stage. Vascularization in the surrounding tissue and within the implant increased with time. Moreover, the collagen deposition increased with time inside the implant. In vivo, the scaffolds maintained the structural integrity. The degradation in vitro was faster and greater compared to that observed in vivo within the same time periods.

Melt processing of chitosan-based fibers and fiber-mesh scaffolds for the engineering of connective tissues.

We report the production of chitosan-based fibers and chitosan fiber-mesh structures by melt processing (solvent-free) to be used as tissue-engineering scaffolds. The melt-based approach used to produce the scaffolds does not change their main characteristics, including the surface roughness and microporosity. The porosity, pore size, interconnectivity and mechanical performance of the scaffolds are all within the range required for various tissue-engineering applications. Biological assessments are performed in direct-contact assays. Cells are able to colonize the scaffold, including the inner porous structure. The cells show high indices of viability in all of the scaffold types.

Conditioned medium as a strategy for human stem cells chondrogenic differentiation

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow‐derived mesenchymal stem cells (hBSMCs) and human Whartons jelly‐derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three‐dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage‐related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright

Influence of scaffold composition over in vitro osteogenic differentiation of hBMSCs and in vivo inflammatory response

To understand the role of chitosan in chitosan-poly(butylene succinate) scaffolds (50% wt), 50%, 25%, and 0% of chitosan were used to produce different scaffolds. These scaffolds were in vitro seeded and cultured with human bone marrow stromal cells in osteogenic conditions, revealing that higher percentage of chitosan showed enhanced cell viability over time, adhesion, proliferation, and osteogenic differentiation. Scaffolds were also implanted in cranial defects and iliac submuscular region in Wistar rats, and the results evidenced that chitosan-containing scaffolds displayed mild inflammatory response and good integration with surrounding tissues, showed by connective tissue colonization and the presence of new blood vessels. Scaffolds without chitosan-evidenced necrotic tissue in scaffolds’ interior, proving that chitosan exerts a positive effect over cell behavior and displays a milder host inflammatory response in vivo.

Development of non-orthogonal 3D-printed scaffolds to enhance their osteogenic performance

Three-dimensional (3D)-printed polycaprolactone (PCL)-based scaffolds have been extensively proposed for Tissue Engineering (TE) applications. Currently, the majority of the scaffolds produced are not representative of the complex arrangement of natural structures, since the internal morphologies follow an orthogonal and regular pattern. In order to produce scaffolds that more closely replicate the structure of the extracellular matrix (ECM) of tissues, herein both circular and sinusoidal scaffolds were fabricated and compared to their conventional orthogonal counterparts. This is an innovative, versatile and efficient strategy to 3D print PCL scaffolds with unique curved geometries. The morphology and the mechanical behavior of the scaffolds were assessed. The biological response was analyzed by evaluating the cell seeding efficiency, cell adhesion, proliferation, and osteogenic activity of an osteoblastic-like cell line seeded in these scaffolds. The scaffolds were designed and produced to have a similar porosity of about 56%. The non-orthogonal structures demonstrated lead to higher values of Youngs modulus, both under dry conditions and when immersed in PBS. Moreover, the biological data corroborate that non-orthogonal scaffolds influence the cellular responses in a positive manner, namely in the osteogenic activity when compared with the orthogonal controls. These results suggest that introducing less orthogonal elements, which better mimic the tissue ECM and architecture, may have a positive influence on the cellular behavior, being a potential strategy to address bone tissue engineering applications.

Advanced polymer composites and structures for bone and cartilage tissue engineering

The author P. Sol is grateful to Fundacao para a Ciencia e a Tecnologia (FCT) for the financial support through the grant SFRH/BD/81133/2011 financed by the POPH/FSE programme. The authors would also like to acknowledge the funding through the QREN project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” co-financed by the North Portugal Regional Operational Programme (ON.2, O Novo Norte) under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).