Engineering Shape to Overcome Contraction: The Role of Polymer-Collagen Hybrids in Advanced Dermal Substitutes.

Collagen gels are the standard dermal equivalents par excellence, however the problem of rapid cell-mediated contraction remains unresolved. Therefore, the development of hybrid constructs (HCs) based on collagen and polymeric scaffolds is proposed to address the mechanical instability that usually limits the formation of new, functional tissue. Equally important, these synthetic structures should be temporary (degradable) while ensuring that cells are well-adapted to the new extracellular environment. In this study, we screened a library of scaffolds made of various polymers, including homopolymers of polycaprolactone (PCL) and poly D,L-lactide (PLA50), their blends (PCL/PLA50), and copolymers (poly(D,L-lactide-co-caprolactone), PCLLA50) to prepare HCs in a layer-by-layer fashion. The properties of polymers and copolymers along with their processability by electrospinning and 3D-printing were evaluated. Then, we assessed the HCs resistance toward cell-mediated contraction as well as the degradation of the polymeric scaffolds. Our results indicate that scaffolds with higher PLA50 content (e.g., PLA50 100%, PCL/PLA50 or PCLLA50, both at 50/50 caprolactone-to-D,L-lactide molar ratio) presented more drawbacks in terms of handleability and processing, while those with greater PCL presence showed structural steadiness and ease to use. All the scaffolds integrated well with the collagen gel to form the corresponding HCs. With few exceptions, the HCs demonstrated good resistance to cell-derived contraction over 3 weeks. Notably, HCs based on PCLLA50 90/10 (both versions, electrospun or 3D-printed) performed best, showing only a 5%-17% area reduction compared to the 93% observed in collagen-only gels. This copolymer displayed hydrolytic degradation depending on its shape, with up to 45% and 65% loss of molecular weight for the electrospun and 3D-printed forms, respectively, correlating with their progressive change in mechanical features. HCs containing PCLLA50 90/10 also exhibited a better fibroblast distribution, enhanced myofibroblastic differentiation, and a three-fold increase in cell proliferation (when the electrospun type was used) compared to collagen controls. These findings were instrumental in selecting a potential HC that might be used for future experiments in vivo.

Electrospun microstructured PLA-based scaffolds featuring relevant anisotropic, mechanical and degradation characteristics for soft tissue engineering.

Electrospun scaffolds combine suitable structural characteristics that make them strong candidates for their use in tissue engineering. These features can be tailored to optimize other physiologically relevant attributes (e.g. mechanical anisotropy and cellular affinity) while ensuring adequate degradation rates of the biomaterial. Here, we present the fabrication of microstructured scaffolds by using a combination of micropatterned electrospinning collectors (honeycomb- or square-patterned) and poly(lactic acid) (PLA)-based copolymers (linear or star-shaped). The resulting materials showed appropriate macropore size and fiber alignment that were key parameters to enhance their anisotropic properties in protraction. Moreover, their elastic modulus, which was initially similar to that of soft tissues, gradually changed in hydrolytic conditions, matching the degradation profile in a 2- to 3-month period. Finally, honeycomb-structured scaffolds exhibited enhanced cellular proliferation compared to standard electrospun mats, while cell colonization was shown to be guided by the macropore contour. Taking together, these results provide new insight into the rational design of microstructured materials that can mimic the progressive evolution of properties in soft tissue regeneration.

Angiogenesis and tissue formation driven by an arteriovenous loop in the mouse.

The rapid vascularisation of biomaterials and artificial tissues is a key determinant for their in vivo viability and ultimately for their integration in a host; therefore promoting angiogenesis and maintaining the newly formed vascular beds has become a major goal of tissue engineering. The arteriovenous loop (AVL) has been an extensively studied platform which integrates microsurgery with cells scaffolds and growth factors to form neotissues. Most AVL studies to date are limited to larger animal models, which are surgically easier to perform, but have inherent limits for the understanding and interrogation of the underlying in vivo mechanisms due the paucity of transgenic models. Here, we demonstrate for the first time in a mouse model the utility of the AVL in the de novo production of vascularized tissue. We also present the combined use of the model with 3D printed chambers, which allow us to dictate size and shape of the tissues formed. This novel platform will allow for an understanding of the fundamental mechanisms involved in tissue generation de novo.

Fibroblast migration correlates with matrix softness. A study in knob-hole engineered fibrin.

The invasion of a matrix by migrating cells is a key step in its remodelling. At least in 2D migration models, cells tend to localize in stiffer areas (durotaxis). Here, we show that mechanical properties affect differently the 3D migration rate: non-proteolytic 3D cell migration is facilitated in softer matrices. In these gels, the modulus was varied by introducing defects in fibres, leaving largely intact the nanostructure. The matrices derive from fibrin via functionalization with a bioinert polymer [poly(ethylene glycol), PEG] through an affinity mechanism identical to that presiding to fibrin own self-assembly. Peptidic end groups on PEG were used to bind fibrinogen globular D regions [GPRP (glycine-proline-arginine-proline) for a holes, GHRP (glycine-histidine-arginine-proline) for b holes; Kd evaluated via isothermal titration calorimetry or fluorescence anisotropy]. In a dose-dependent manner, both PEGylated peptides decreased gel stiffness, but most other properties at a macroscopic [e.g., overall elastic character, strain hardening, and high (>0.5) Poisson ratio] or nano/micro level (fibre dimension and pore size) were largely unaffected, suggesting that the softening effect was due to the introduction of defects within fibres, rather than to differences in the network architecture. In these matrices, the key determinant of fibroblast migration was found to be the elastic modulus, rather than the identity or the dose of the PEGylated peptide; softer materials allowed a faster invasion, even if this meant a higher content of non-adhesive PEG. This does not conflict with fibroblast durotaxis (where stiffness controls accumulation but not necessarily the speed of migration) and indicates a way to fine tune the speed of cell colonization.