Design and functional testing of a multichamber perfusion platform for three-dimensional scaffolds.

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminary in vitro tests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization.

Tissue engineering based on electrochemical desorption of an RGD-containing oligopeptide.

This paper describes a non-invasive approach for efficient detachment of cells adhered to a gold substrate via a specific oligopeptide. Detachment is effected by an electrical stimulus. The oligopeptide contains cysteine, which spontaneously forms a gold-thiolate bond on a gold surface. This chemical adsorption reaches > 95% equilibrium within 10 min after immersion of a gold-coated substrate in a solution containing the peptide. The peptide is reversibly desorbed from the surface within 5 min of application of a negative electrical potential. By taking advantage of this simple adsorption and desorption mechanism, cells can be grown on an oligopeptide-functionalized gold surface and can be efficiently detached as single cells or cell sheets by application of a negative electrical potential. This approach was also applied to the surface of gold-coated microrods. Capillary-like microchannels were formed in collagen gel by transferring endothelial cells to the internal surfaces of the microchannels. During subsequent perfusion culture, the enveloped endothelial cells migrated into the collagen gel and formed luminal structures, which sprouted from the microchannels. This technique has the potential to provide a fundamental tool for the engineering of thick cell sheets as well as vascularized tissues and organs.

Cell-adhesive and cell-repulsive zwitterionic oligopeptides for micropatterning and rapid electrochemical detachment of cells.

In this study, we describe the development of oligopeptide-modified cell culture surfaces from which adherent cells can be rapidly detached by application of an electrical stimulus. An oligopeptide, CGGGKEKEKEK, was designed with a terminal cysteine residue to mediate binding to a gold surface via a gold-thiolate bond. The peptide forms a self-assembled monolayer through the electrostatic force between the sequence of alternating charged glutamic acid (E) and lysine (K) residues. The dense and electrically neutral oligopeptide zwitterionic layer of the modified surface was resistant to nonspecific adsorption of proteins and adhesion of cells, while the surface was altered to cell adhesive by the addition of a second oligopeptide (CGGGKEKEKEKGRGDSP) containing the RGD cell adhesion motif. Application of a negative electrical potential to this gold surface cleaved the gold-thiolate bond, leading to desorption of the oligopeptide layer, and rapid (within 2 min) detachment of virtually all cells. This approach was applicable not only to detachment of cell sheets but also for transfer of cell micropatterns to a hydrogel. This electrochemical approach of cell detachment may be a useful tool for tissue-engineering applications.

Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix.

Materials based on synthetic polymers can be extensively tailored in their physical properties but often suffer from limited biological functionality. Here we tested the hypothesis that the biological performance of 3D synthetic polymer-based scaffolds can be enhanced by extracellular matrix (ECM) deposited by cells in vitro and subsequently decellularized. The hypothesis was tested in the context of bone graft substitutes, using polyesterurethane (PEU) foams and mineralized ECM laid by human mesenchymal stromal cells (hMSC). A perfusion-based bioreactor system was critically employed to uniformly seed and culture hMSC in the scaffolds and to efficiently decellularize (94% DNA reduction) the resulting ECM while preserving its main organic and inorganic components. As compared to plain PEU, the decellularized ECM-polymer hybrids supported the osteoblastic differentiation of newly seeded hMSC by up-regulating the mRNA expression of typical osteoblastic genes (6-fold higher bone sialoprotein; 4-fold higher osteocalcin and osteopontin) and increasing calcium deposition (6-fold higher), approaching the performance of ceramic-based materials. After ectopic implantation in nude mice, the decellularized hybrids induced the formation of a mineralized matrix positively immunostained for bone sialoprotein and resembling an immature osteoid tissue. Our findings consolidate the perspective of bioreactor-based production of ECM-decorated polymeric scaffolds as off-the-shelf materials combining tunable physical properties with the physiological presentation of instructive biological signals.

SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures.

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications.

Cyclic mechanical stimulation favors myosin heavy chain accumulation in engineered skeletal muscle constructs.

PURPOSE: Since stretching plays a key role in skeletal muscle tissue development in vivo, by making use of an innovative bioreactor and a biodegradable microfibrous scaffold (DegraPol(R)) previously developed by our group, we aimed to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs, obtained by seeding and culturing murine skeletal muscle cells on electrospun membranes. METHODS: Following 5 days of static culture, skeletal muscle constructs were transferred into the bioreactor and further cultured for 13 days, while experiencing a stretching pattern adapted from the literature to resemble mouse development and growth. Sample withdrawal occurred at the onset of cyclic stretching and after 7 and 10 days. Myosin heavy chain (MHC) accumulation in stretched constructs (D) was evaluated by Western blot analysis and immunofluorescence staining, using statically cultured samples (S) as controls. RESULTS: Western blot analysis of MHC on dynamically (D) and statically (S) cultured constructs at different time points showed that, at day 10, the applied stretching pattern led to an eight-fold increase in myosin accumulation in cyclically stretched constructs (D) with respect to the corresponding static controls (S). These results were confirmed by immunofluorescence staining of total sarcomeric MHC. CONCLUSIONS: Since previous attempts to reproduce skeletal myogenesis in vitro mainly suffered from the difficulty of driving myoblast development into an architecturally organized array of myosin expressing myotubes, the chance of inducing MHC accumulation via mechanical conditioning represents a significant step towards the generation of a functional muscle construct for skeletal muscle tissue engineering applications.