In vivo efficacy proof of concept of a large-size bioprinted dermo-epidermal substitute for permanent wound coverage.

Introduction: An autologous split-thickness skin graft (STSG) is a standard treatment for coverage of full-thickness skin defects. However, this technique has two major drawbacks: the use of general anesthesia for skin harvesting and scar sequelae on the donor site. In order to reduce morbidity associated with STSG harvesting, researchers have developed autologous dermo-epidermal substitutes (DESs) using cell culture, tissue engineering, and, more recently, bioprinting approaches. This study assessed the manufacturing reliability and in vivo efficacy of a large-size good manufacturing practice (GMP)-compatible bio-printed human DES, named Poieskin®, for acute wound healing treatment. Methods: Two batches (40 cm2 each) of Poieskin® were produced, and their reliability and homogeneity were assessed using histological scoring. Immunosuppressed mice received either samples of Poieskin® (n = 8) or human STSG (n = 8) immediately after longitudinal acute full-thickness excision of size 1 × 1.5 cm, applied on the skeletal muscle plane. The engraftment rate was assessed through standardized photographs on day 16 of the follow-up. Moreover, wound contraction, superficial vascularization, and local inflammation were evaluated via standardized photographs, laser Doppler imaging, and PET imaging, respectively. Histological analysis was finally performed after euthanasia. Results: Histological scoring reached 75% ± 8% and 73% ± 12%, respectively, displaying a robust and homogeneous construct. Engraftment was comparable for both groups: 91.8% (SD = 0.1152) for the Poieskin® group versus 100% (SD = 0) for the human STSG group. We did not record differences in either graft perfusion, PET imaging, or histological scoring on day 16. Conclusion: Poieskin® presents consistent bioengineering manufacturing characteristics to treat full-thickness cutaneous defects as an alternative to STSG in clinical applications. Manufacturing of Poieskin® is reliable and homogeneous, leading to a clinically satisfying rate of graft take compared to the reference human STSG in a mouse model. These results encourage the use of Poieskin® in phase I clinical trials as its manufacturing procedure is compatible with pharmaceutical guidelines.

From local to global matrix organization by fibroblasts: a 4D laser-assisted bioprinting approach.

Fibroblasts and myofibroblasts play a central role in skin homeostasis through dermal organization and maintenance. Nonetheless, the dynamic interactions between (myo)fibroblasts and the extracellular matrix (ECM) remain poorly exploited in skin repair strategies. Indeed, there is still an unmet need for soft tissue models allowing to study the spatial-temporal remodeling properties of (myo)fibroblasts.In vivo, wound healing studies in animals are limited by species specificity.In vitro, most models rely on collagen gels reorganized by randomly distributed fibroblasts. But biofabrication technologies have significantly evolved over the past ten years. High-resolution bioprinting now allows to investigate various cellular micropatterns and the emergent tissue organizations over time. In order to harness the full dynamic properties of cells and active biomaterials, it is essential to consider 'time' as the 4th dimension in soft tissue design. Following this 4D bioprinting approach, we aimed to develop a novel model that could replicate fibroblast dynamic remodelingin vitro. For this purpose, (myo)fibroblasts were patterned on collagen gels with laser-assisted bioprinting (LAB) to study the generated matrix deformations and reorganizations. First, distinct populations, mainly composed of fibroblasts or myofibroblasts, were establishedin vitroto account for the variety of fibroblastic remodeling properties. Then, LAB was used to organize both populations on collagen gels in even isotropic patterns with high resolution, high density and high viability. With maturation, bioprinted patterns of fibroblasts and myofibroblasts reorganized into dispersed or aggregated cells, respectively. Stress-release contraction assays revealed that these phenotype-specific pattern maturations were associated with distinct lattice tension states. The two populations were then patterned in anisotropic rows in order to direct the cell-generated deformations and to orient global matrix remodeling. Only maturation of anisotropic fibroblast patterns, but not myofibroblasts, resulted in collagen anisotropic reorganizations both at tissue-scale, with lattice contraction, and at microscale, with embedded microbead displacements. Following a 4D bioprinting approach, LAB patterning enabled to elicit and orient the dynamic matrix remodeling mechanisms of distinct fibroblastic populations and organizations on collagen. For future studies, this method provides a new versatile tool to investigatein vitrodermal organizations and properties, processes of remodeling in healing, and new treatment opportunities.

Automated Buildup of Biomimetic Films in Cell Culture Microplates for High-Throughput Screening of Cellular Behaviors.

An automatic method is established for layer-by-layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid-handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film-coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide-functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell-based assays for regenerative medicine and high-throughput drug screening.