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 Young's 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.

Novel melt-processable chitosan-polybutylene succinate fibre scaffolds for cartilage tissue engineering.

Novel chitosan/polybutylene succinate fibre-based scaffolds (C-PBS) were seeded with bovine articular chondrocytes in order to assess their suitability for cartilage tissue engineering. Chondrocytes were seeded onto C-PBS scaffolds using spinner flasks under dynamic conditions, and cultured under orbital rotation for a total of 6 weeks. Non-woven polyglycolic acid (PGA) felts were used as reference materials. Tissue-engineered constructs were characterized by scanning electron microscopy (SEM), hematoxylin-eosin (H&E), toluidine blue and alcian blue staining, immunolocalization of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification at different time points. SEM showed the chondrocytes' typical morphology, with colonization at the surface and within the pores of the C-PBS scaffolds. These observations were supported by routine histology. Toluidine blue and alcian blue stains, as well as immunohistochemistry for collagen types I and II, provided qualitative information on the composition of the engineered extracellular matrix. More pronounced staining was observed for collagen type II than collagen type I. Similar results were observed with constructs engineered on PGA scaffolds. These also exhibited higher amounts of matrix glycosaminoglycans and presented a central region which contained fewer cells and little matrix, a feature that was not detected with C-PBS constructs.

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.

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.

Assessment of the suitability of chitosan/polybutylene succinate scaffolds seeded with mouse mesenchymal progenitor cells for a cartilage tissue engineering approach.

In this work, scaffolds derived from a new biomaterial originated from the combination of a natural material and a synthetic material were tested for assessing their suitability for cartilage tissue engineering applications. In order to obtain a better outcome result in terms of scaffolds' overall properties, different blends of natural and synthetic materials were created. Chitosan and polybutylene succinate (C-PBS) 50/50 (wt%) were melt blended using a twin-screw extruder and processed into 5 x 5 x 5 mm scaffolds by compression moulding with salt leaching. Micro-computed tomography analysis calculated an average of 66.29% porosity and 92.78% interconnectivity degree for the presented scaffolds. The salt particles used ranged in size between 63 and 125 mum, retrieving an average pore size of 251.28 mum. Regarding the mechanical properties, the compressive modulus was of 1.73 +/- 0.4 MPa (E(sec) 1%). Cytotoxicity evaluation revealed that the leachables released by the developed porous structures were not harmful to the cells and hence were noncytotoxic. Direct contact assays were carried out using a mouse bone marrow-derived mesenchymal progenitor cell line (BMC9). Cells were seeded at a density of 5 x 10(5) cells/scaffold and allowed to grow for periods up to 3 weeks under chondrogenic differentiating conditions. Scanning electron microscopy analysis revealed that the cells were able to proliferate and colonize the scaffold structure, and MTS test demonstrated cell viability during the time of the experiment. Finally, Western blot performed for collagen type II, a natural cartilage extracellular matrix component, showed that this protein was being expressed by the end of 3 weeks, which seems to indicate that the BMC9 cells were being differentiated toward the chondrogenic pathway. These results indicate the adequacy of these newly developed C-PBS scaffolds for supporting cell growth and differentiation toward the chondrogenic pathway, suggesting that they should be considered for further studies in the cartilage tissue engineering field.

Adhesion, proliferation, and osteogenic differentiation of a mouse mesenchymal stem cell line (BMC9) seeded on novel melt-based chitosan/polyester 3D porous scaffolds.

The aim of the present work was to study the biological behavior of a mouse mesenchymal stem cell line when seeded and cultured under osteogenic conditions onto novel processed melt-based chitosan scaffolds. Scaffolds were produced by compression molding, followed by salt leaching. Scanning electron microscopy (SEM) observations and microCT analysis showed the pore sizes ranging between 250 and 500 microm and the interconnectivity of the porous structure. The chitosan-poly(butylenes succinate) scaffolds presented high mechanical properties, similar to the ones of trabecular bone (E1% approximately 75 MPa). Cytotoxicity assays were carried out using standard tests (accordingly to ISO/EN 10993 part 5 guidelines), namely, MTS test with a 24 h extraction period, revealing that L929 cells had similar metabolic activities to that obtained for the negative control. Cell culture studies were conducted using a mouse mesenchymal stem cell line (BMC9). Cells were seeded onto the scaffold and allowed to proliferate for 3 weeks under osteogenic conditions. SEM observations demonstrated that cells were able to proliferate and massively colonize the scaffolds structure. The cell viability assay MTS demonstrated that BMC9 cells were viable after 3 weeks of culture. The cells clearly evidenced a positive differentiation toward the osteogenic lineage, as confirmed by the high ALP activity levels. Moreover, energy dispersive spectroscopy (EDS) analysis revealed the presence of Ca and P in the elaborated extracellular matrix (ECM). These combined results indicate that the novel melt-based chitosan/polyester scaffolds support the adhesion, proliferation, and osteogenic differentiation of the mouse MSCs and shows adequate physicochemical and biological properties for being used as scaffolds in bone tissue engineering-related strategies.