Microstructured human fibroblast-derived extracellular matrix scaffold for vascular media fabrication.

In the clinical and pharmacological fields, there is a need for the production of tissue-engineered small-diameter blood vessels. We have demonstrated previously that the extracellular matrix (ECM) produced by fibroblasts can be used as a scaffold to support three-dimensional (3D) growth of another cell type. Thus, a resistant tissue-engineered vascular media can be produced when such scaffolds are used to culture smooth muscle cells (SMCs). The present study was designed to develop an anisotropic fibroblastic ECM sheet that could replicate the physiological architecture of blood vessels after being assembled into a small diameter vascular conduit. Anisotropic ECM scaffolds were produced using human dermal fibroblasts, grown on a microfabricated substrate with a specific topography, which led to cell alignment and unidirectional ECM assembly. Following their devitalization, the scaffolds were seeded with SMCs. These cells elongated and migrated in a single direction, following a specific angle relative to the direction of the aligned fibroblastic ECM. Their resultant ECM stained for collagen I and III and elastin, and the cells expressed SMC differentiation markers. Seven days after SMCs seeding, the sheets were rolled around a mandrel to form a tissue-engineered vascular media. The resulting anisotropic ECM and cell alignment induced an increase in the mechanical strength and vascular reactivity in the circumferential direction as compared to unaligned constructs. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid isothermal substrate microfabrication of a biocompatible thermoplastic elastomer for cellular contact guidance.

The use of microstructured substrates to study and influence cell orientation, which plays an important role in tissue functionality, has been of great interest lately. Silicon and poly(dimethylsiloxane) substrates have typically been used, but long processing times and exogenous protein surface coating, required to enhance cell viability, limit their use as large-scale platforms. There is thus a need for a non-biodegradable biocompatible substrate that allows rapid and low cost microfabrication. In this paper a styrene-(ethylene/butylene)-styrene block co-polymer (SEBS) microstructured by a rapid replication technique using low pressure an isothermal hot embossing approach has been demonstrated. SEBS substrates were treated with oxygen plasma to enhance cell adhesion and sterilized using ethylene oxide gas. While cell adhesion to and proliferation on these substrates was as good as on tissue culture polystyrene, cellular alignment on microstructured SEBS was also very high (97.7±0.5%) when calculated within a 10° angle variation from the longitudinal axis. Furthermore, tissue sheets on microstructured SEBS have been produced and exhibited cellular alignment within the engineered tissue. In addition, these results were obtained without coating the material with exogenous proteins. Such substrates should be helpful in the culture of tissue engineered substitutes with an intrinsic orientation and to elucidate questions in cell biology.

The significance of pore microarchitecture in a multi-layered elastomeric scaffold for contractile cardiac muscle constructs.

Multi-layered poly(glycerol-sebacate) (PGS) scaffolds with controlled pore microarchitectures were fabricated, combined with heart cells, and cultured with perfusion to engineer contractile cardiac muscle constructs. First, one-layered (1L) scaffolds with accordion-like honeycomb shaped pores and elastomeric mechanical properties were fabricated by laser microablation of PGS membranes. Second, two-layered (2L) scaffolds with fully interconnected three dimensional pore networks were fabricated by oxygen plasma treatment of 1L scaffolds followed by stacking with off-set laminae to produce a tightly bonded composite. Third, heart cells were cultured on scaffolds with or without interstitial perfusion for 7 days. The laser-microablated PGS scaffolds exhibited ultimate tensile strength and strain-to-failure higher than normal adult rat left ventricular myocardium, and effective stiffnesses ranging from 220 to 290 kPa. The 7-day constructs contracted in response to electrical field stimulation. Excitation thresholds were unaffected by scaffold scale up from 1L to 2L. The 2L constructs exhibited reduced apoptosis, increased expression of connexin-43 (Cx-43) and matrix metalloprotease-2 (MMP-2) genes, and increased Cx-43 and cardiac troponin-I proteins when cultured with perfusion as compared to static controls. Together, these findings suggest that multi-layered, microfabricated PGS scaffolds may be applicable to myocardial repair applications requiring mechanical support, cell delivery and active implant contractility.

Combined technologies for microfabricating elastomeric cardiac tissue engineering scaffolds.

Polymer scaffolds that direct elongation and orientation of cultured cells can enable tissue engineered muscle to act as a mechanically functional unit. We combined micromolding and microablation technologies to create muscle tissue engineering scaffolds from the biodegradable elastomer poly(glycerol sebacate). These scaffolds exhibited well defined surface patterns and pores and robust elastomeric tensile mechanical properties. Cultured C2C12 muscle cells penetrated the pores to form spatially controlled engineered tissues. Scanning electron and confocal microscopy revealed muscle cell orientation in a preferential direction, parallel to micromolded gratings and long axes of microablated anisotropic pores, with significant individual and interactive effects of gratings and pore design.

Tissue-engineered vascular adventitia with vasa vasorum improves graft integration and vascularization through inosculation.

Tissue-engineered blood vessel is one of the most promising living substitutes for coronary and peripheral artery bypass graft surgery. However, one of the main limitations in tissue engineering is vascularization of the construct before implantation. Such a vascularization could play an important role in graft perfusion and host integration of tissue-engineered vascular adventitia. Using our self-assembly approach, we developed a method to vascularize tissue-engineered blood vessel constructs by coculturing endothelial cells in a fibroblast-laden tissue sheet. After subcutaneous implantation, enhancement of graft integration within the surrounding environment was noted after 48 h and an important improvement in blood circulation of the grafted tissue at 1 week postimplantation. The distinctive branching structure of end arteries characterizing the in vivo adventitial vasa vasorum has also been observed in long-term postimplantation follow-up. After a 90-day implantation period, hybrid vessels containing human and mouse endothelial cells were still perfused. Characterization of the mechanical properties of both control and vascularized adventitia demonstrated that the ultimate tensile strength, modulus, and failure strain were in the same order of magnitude of a pig coronary artery. The addition of a vasa vasorum to the tissue-engineered adventitia did not influence the burst pressure of these constructs. Hence, the present results indicate a promising answer to the many challenges associated with the in vitro vascularization and in vivo integration of many different tissue-engineered substitutes.

Surface topography induces 3D self-orientation of cells and extracellular matrix resulting in improved tissue function.

The organization of cells and extracellular matrix (ECM) in native tissues plays a crucial role in their functionality. However, in tissue engineering, cells and ECM are randomly distributed within a scaffold. Thus, the production of engineered-tissue with complex 3D organization remains a challenge. In the present study, we used contact guidance to control the interactions between the material topography, the cells and the ECM for three different tissues, namely vascular media, corneal stroma and dermal tissue. Using a specific surface topography on an elastomeric material, we observed the orientation of a first cell layer along the patterns in the material. Orientation of the first cell layer translates into a physical cue that induces the second cell layer to follow a physiologically consistent orientation mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in an oriented self-assembled tissue sheet. These self-assembled tissue sheets were then used to create 3 different structured engineered-tissue: cornea, vascular media and dermis. We showed that functionality of such structured engineered-tissue was increased when compared to the same non-structured tissue. Dermal tissues were used as a negative control in response to surface topography since native dermal fibroblasts are not preferentially oriented in vivo. Non-structured surfaces were also used to produce randomly oriented tissue sheets to evaluate the impact of tissue orientation on functional output. This novel approach for the production of more complex 3D tissues would be useful for clinical purposes and for in vitro physiological tissue model to better understand long standing questions in biology.