Lorenzo Moroni

Cationic polymers and their therapeutic potential

The last decade has witnessed enormous research focused on cationic polymers. Cationic polymers are the subject of intense research as non-viral gene delivery systems, due to their flexible properties, facile synthesis, robustness and proven gene delivery efficiency. Here, we review the most recent scientific advances in cationic polymers and their derivatives not only for gene delivery purposes but also for various alternative therapeutic applications. An overview of the synthesis and preparation of cationic polymers is provided along with their inherent bioactive and intrinsic therapeutic potential. In addition, cationic polymer based biomedical materials are covered. Major progress in the fields of drug and gene delivery as well as tissue engineering applications is summarized in the present review.

Chitosan/Poly(ɛ-caprolactone) blend scaffolds for cartilage repair

Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.

Biofabrication: reappraising the definition of an evolving field

Biofabrication is an evolving research field that has recently received significant attention. In particular, the adoption of Biofabrication concepts within the field of Tissue Engineering and Regenerative Medicine has grown tremendously, and has been accompanied by a growing inconsistency in terminology. This article aims at clarifying the position of Biofabrication as a research field with a special focus on its relation to and application for Tissue Engineering and Regenerative Medicine. Within this context, we propose a refined working definition of Biofabrication, including Bioprinting and Bioassembly as complementary strategies within Biofabrication.

Evaluation of Photocrosslinked Lutrol Hydrogel for Tissue Printing Applications

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.

Endothelial Differentiation of Mesenchymal Stromal Cells

Human mesenchymal stromal cells (hMSCs) are increasingly used in regenerative medicine for restoring worn-out or damaged tissue. Newly engineered tissues need to be properly vascularized and current candidates for in vitro tissue pre-vascularization are endothelial cells and endothelial progenitor cells. However, their use in therapy is hampered by their limited expansion capacity and lack of autologous sources. Our approach to engineering large grafts is to use hMSCs both as a source of cells for regeneration of targeted tissue and at the same time as the source of endothelial cells. Here we investigate how different stimuli influence endothelial differentiation of hMSCs. Although growth supplements together with shear force were not sufficient to differentiate hMSCs with respect to expression of endothelial markers such as CD31 and KDR, these conditions did prime the cells to differentiate into cells with an endothelial gene expression profile and morphology when seeded on Matrigel. In addition, we show that endothelial-like hMSCs are able to create a capillary network in 3D culture both in vitro and in vivo conditions. The expansion phase in the presence of growth supplements was crucial for the stability of the capillaries formed in vitro. To conclude, we established a robust protocol for endothelial differentiation of hMSCs, including an immortalized MSC line (iMSCs) which allows for reproducible in vitro analysis in further studies.

Chitosan Scaffolds Containing Hyaluronic Acid for Cartilage Tissue Engineering

Scaffolds derived from natural polysaccharides are very promising in tissue engineering applications and regenerative medicine, as they resemble glycosaminoglycans in the extracellular matrix (ECM). In this study, we have prepared freeze-dried composite scaffolds of chitosan (CHT) and hyaluronic acid (HA) in different weight ratios containing either no HA (control) or 1%, 5%, or 10% of HA. We hypothesized that HA could enhance structural and biological properties of CHT scaffolds. To test this hypothesis, physicochemical and biological properties of CHT/HA scaffolds were evaluated. Scanning electron microscopy micrographs, mechanical properties, swelling tests, enzymatic degradation, and Fourier transform infrared (FTIR) chemical maps were performed. To test the ability of the CHT/HA scaffolds to support chondrocyte adhesion and proliferation, live-dead and MTT assays were performed. Results showed that CHT/HA composite scaffolds are noncytotoxic and promote cell adhesion. ECM formation was further evaluated with safranin-O and alcian blue staining methods, and glycosaminoglycan and DNA quantifications were performed. The incorporation of HA enhanced cartilage ECM production. CHT/5HA had a better pore network configuration and exhibited enhanced ECM cartilage formation. On the basis of our results, we believe that CHT/HA composite matrixes have potential use in cartilage repair.

Fabrication, Characterization and Cellular Compatibility of Poly(Hydroxy Alkanoate) Composite Nanofibrous Scaffolds for Nerve Tissue Engineering

Tissue engineering techniques using a combination of polymeric scaffolds and cells represent a promising approach for nerve regeneration. We fabricated electrospun scaffolds by blending of Poly (3-hydroxybutyrate) (PHB) and Poly (3-hydroxy butyrate-co-3- hydroxyvalerate) (PHBV) in different compositions in order to investigate their potential for the regeneration of the myelinic membrane. The thermal properties of the nanofibrous blends was analyzed by differential scanning calorimetry (DSC), which indicated that the melting and glass temperatures, and crystallization degree of the blends decreased as the PHBV weight ratio increased. Raman spectroscopy also revealed that the full width at half height of the band centered at 1725 cm−1 can be used to estimate the crystalline degree of the electrospun meshes. Random and aligned nanofibrous scaffolds were also fabricated by electrospinning of PHB and PHBV with or without type I collagen. The influence of blend composition, fiber alignment and collagen incorporation on Schwann cell (SCs) organization and function was investigated. SCs attached and proliferated over all scaffolds formulations up to 14 days. SCs grown on aligned PHB/PHBV/collagen fibers exhibited a bipolar morphology that oriented along the fiber direction, while SCs grown on the randomly oriented fibers had a multipolar morphology. Incorporation of collagen within nanofibers increased SCs proliferation on day 14, GDNF gene expression on day 7 and NGF secretion on day 6. The results of this study demonstrate that aligned PHB/PHBV electrospun nanofibers could find potential use as scaffolds for nerve tissue engineering applications and that the presence of type I collagen in the nanofibers improves cell differentiation.

Critical Steps toward a tissue-engineered cartilage implant using embryonic stem cells.

Embryonic stem (ES) cells are a potential source for cartilage tissue engineering because they provide an unlimited supply of cells that can be differentiated into chondrocytes. So far, chondrogenic differentiation of both mouse and human ES cells has only been demonstrated in two-dimensional cultures, in pellet cultures, in a hydrogel, or on thin biomaterials. The next challenge will be to form cartilage on a load-bearing, clinically relevant-sized scaffold in vitro and in vivo, to regenerate defects in patients suffering from articular cartilage disorders. For a successful implant, cells have to be seeded efficiently and homogenously throughout the scaffold. Parameters investigated were the scaffold architecture, seeding method, and cellular condition. Seeding in a three-dimensional fiber-deposited (3DF) scaffold was more homogenous than in a compression-molded scaffold. The seeding efficiency on bare scaffolds was compromised by the absence of serum in the chondrogenic medium, but could be improved by combining the cells with a gel and subsequent injection into the 3DF scaffolds. However, the viability of the cells was unsatisfactory in the interior of the graft. Cell aggregates, the so-called embryoid bodies (EBs), were seeded with increased survival rate. Mouse ES cells readily underwent chondrogenic differentiation in vitro in pellets, on bare scaffolds, in Matrigel, and in agarose, both as single cells and in EBs. The differentiation protocol requires further improvement to achieve homogenous differentiation and abolish teratoma formation in vivo. We conclude that ES cells can be used as a cell source for cartilage tissue engineering, pending further optimization of the strategy.

Critical factors in the design of growth factor releasing scaffolds for cartilage tissue engineering

Background: Trauma or degenerative diseases of the joints are common clinical problems resulting in high morbidity. Although various orthopedic treatments have been developed and evaluated, the low repair capacities of articular cartilage renders functional results unsatisfactory in the long term. Over the last decade, a different approach (tissue engineering) has emerged that aims not only to repair impaired cartilage, but also to fully regenerate it, by combining cells, biomaterials mimicking extracellular matrix (scaffolds) and regulatory signals. The latter is of high importance as growth factors have the potency to induce, support or enhance the growth and differentiation of various cell types towards the chondrogenic lineage. Therefore, the controlled release of different growth factors from scaffolds appears to have great potential to orchestrate tissue repair effectively. Objective: This review aims to highlight considerations and limitations of the design, materials and processing methods available to create scaffolds, in relation to the suitability to incorporate and release growth factors in a safe and defined manner. Furthermore, the current state of the art of signalling molecules release from scaffolds and the impact on cartilage regeneration in vitro and in vivo is reported and critically discussed. Methods: The strict aspects of biomaterials, scaffolds and growth factor release from scaffolds for cartilage tissue engineering applications are considered. Conclusion: Engineering defined scaffolds that deliver growth factors in a controlled way is a task seldom attained. If growth factor delivery appears to be beneficial overall, the optimal delivery conditions for cartilage reconstruction should be more thoroughly investigated.

Combining technologies to create bioactive hybrid scaffolds for bone tissue engineering

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). Poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier Tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. There were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineering.