Biology subjects

Polkoff, K.

Publications and source records attributed to Polkoff, K..

3 recordsLinked to original sources

bioRxiv2022

Transgenic porcine model reveals two roles for LGR5 during lung development and homeostasis

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+ cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV+/SFTPC+ progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+ cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+ cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns of SHH, FGF, WNT and TGF-{beta} signaling pathway genes. These results support distinct roles for LGR5+ cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.

cell biology↗

bioRxiv2021

Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model

Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. IMPACT STATEMENTLittle is known about the impact of donor age and time in culture on the contraction of the 3D hydrogel-based graft. These results show how cellular phenotypes differ based on donor age and time in culture, which could account for important inconsistencies in biomanufacturing of skin grafts and in vitro models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. Future in vivo studies could help determine the best donor age and time in culture for improved wound healing outcomes or more reproducible in vitro testing constructs.

bioengineering↗

bioRxiv2021

Repair of critical-size porcine craniofacial bone defects using a collagen-polycaprolactone composite biomaterial

Regenerative medicine approaches for massive craniomaxillofacial bone defects face challenges associated with the scale of missing bone, the need for rapid graft-defect integration, and challenges related to inflammation and infection. Mineralized collagen scaffolds have been shown to promote mesenchymal stem cell osteogenesis due to their porous nature and material properties, but are mechanically weak, limiting surgical practicality. Previously, these scaffolds were combined with 3D-printed polycaprolactone mesh to form a scaffold-mesh composite to increase strength and promote bone formation in sub-critical sized porcine ramus defects. Here, we compare the performance of mineralized collagen-polycaprolactone composites to the polycaprolactone mesh in a critical-sized porcine ramus defect model. While there were no differences in overall healing response between groups, our data demonstrated broadly variable metrics of healing regarding new bone infiltration and fibrous tissue formation. Abscesses were present surrounding some implants and polycaprolactone polymer was still present after 9-10 months of implantation. Overall, while there was limited successful healing, with 2 of 22 implants showed substantial levels of bone regeneration, and others demonstrating some form of new bone formation, the results suggest targeted improvements to improve repair of large animal models to more accurately represent craniomaxillofacial bone healing. Notably, strategies to increase osteogenesis throughout the implant, modulate the immune system to support repair, and employ shape-fitting tactics to avoid implant micromotion and resultant fibrosis. Improvements to the mineralized collagen scaffolds involve changes in pore size and shape to increase cell migration and osteogenesis and inclusion or delivery of factors to aid vascular ingrowth and bone regeneration.

bioengineering↗