The fibrous tissues prevalent throughout the body possess an ordered structure that underlies their refined and robust mechanical properties. Engineered replacements will require recapitulation of this exquisite architecture in three dimensions. Aligned nanofibrous scaffolds can dictate cell and matrix organization; however, their widespread application has been hindered by poor cell infiltration due to the tight packing of fibers during fabrication. Here, we develop and validate an enabling technology in which tunable composite nanofibrous scaffolds are produced to provide instruction without impediment. Composites were formed containing two distinct fiber fractions: slow-degrading poly(ε-caprolactone) and water-soluble, sacrificial poly(ethylene oxide), which can be selectively removed to increase pore size. Increasing the initial fraction of sacrificial poly(ethylene oxide) fibers enhanced cell infiltration and improved matrix distribution. Despite the removal of >50% of the initial fibers, the remaining scaffold provided sufficient instruction to align cells and direct the formation of a highly organized ECM across multiple length scales, which in turn led to pronounced increases in the tensile properties of the engineered constructs (nearly matching native tissue). This approach transforms what is an interesting surface phenomenon (cells on top of nanofibrous mats) into a method by which functional, 3D tissues (>1 mm thick) can be formed, both in vitro and in vivo. As such, this work represents a marked advance in the engineering of load-bearing fibrous tissues, and will find widespread applications in regenerative medicine.anisotropy | electrospinning | nanofiber | tissue engineering | meniscus fibrocartilage

Fibroblast-like synoviocytes (FLS) reside in the synovial membrane of diarthrodial joints and are exposed to a dynamic fluid environment that presents both physical and chemical stimuli. The ability of FLS to sense and respond to these stimuli plays a key role in their normal function, and is implicated in the alterations to function that occur in osteoarthritis (OA). The present work characterizes the response of FLS to fluid flow-induced shear stress via real-time calcium imaging, and tests the hypothesis that this response is modulated by interleukin-1α (IL-1α), a cytokine elevated in OA. FLS demonstrated a robust calcium signaling response to fluid shear that was dose dependent upon stress level and required both external and internal calcium sources. Preconditioning with 10 ng/mL IL-1α for 24 hrs heightened this shear stress response by significantly increasing the percent of responding cells and peak magnitude, while significantly decreasing the time for a peak to occur. Intercellular communication via gap junctions was found to account for a portion of the FLS population response in normal conditions, and was significantly increased by IL-1α preconditioning. IL-1α was also found to significantly increase average length and incidence of the primary cilia, an organelle commonly implicated in shear mechanosensing. These findings suggest that the elevated levels of IL-1α found in the OA environment heighten FLS sensitivity to fluid shear by altering both intercellular communication and individual cell sensitivity, which could affect downstream functions and contribute to progression of the disease state.

Objective Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. Method In this study sub-10μm cartilage particles or 1μm latex particles were co-cultured with FLS ± 10 ng/mL interleukin-1α (IL-1 α) or tumor necrosis factor- α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. Results Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. Conclusion The proliferative response of FLS to cartilage wear particles resulted in an overall increase in ECM content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.