Cartilage Wear Particles Induce an Inflammatory Response Similar to Cytokines in Human Fibroblast-Like Synoviocytes.

The synovium plays a key role in the development of osteoarthritis, as evidenced by pathological changes to the tissue observed in both early and late stages of the disease. One such change is the attachment of cartilage wear particles to the synovial intima. While this phenomenon has been well observed clinically, little is known of the biological effects that such particles have on resident cells in the synovium. The present work investigates the hypothesis that cartilage wear particles elicit a pro-inflammatory response in diseased and healthy human fibroblast-like synoviocytes, like that induced by key cytokines in osteoarthritis. Fibroblast-like synoviocytes from 15 osteoarthritic human donors and a subset of three non-osteoarthritic donors were exposed to cartilage wear particles, interleukin-1α or tumor necrosis factor-α for 6 days and analyzed for proliferation, matrix production, and release of pro-inflammatory mediators and degradative enzymes. Wear particles significantly increased proliferation and release of nitric oxide, interleukin-6 and -8, and matrix metalloproteinase-9, -10, and -13 in osteoarthritic synoviocytes, mirroring the effects of both cytokines, with similar trends in non-osteoarthritic cells. These results suggest that cartilage wear particles are a relevant physical factor in the osteoarthritic environment, perpetuating the pro-inflammatory and pro-degradative cascade by modulating synoviocyte behavior at early and late stages of the disease. Future work points to therapeutic strategies for slowing disease progression that target cell-particle interactions. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1979-1987, 2019.

A Functional Tissue-Engineered Synovium Model to Study Osteoarthritis Progression and Treatment.

IMPACT STATEMENT: The synovium envelops the diarthrodial joint and plays a key regulatory role in defining the composition of the synovial fluid through filtration and biosynthesis of critical boundary lubricants. Synovium changes often precede cartilage damage in osteoarthritis. We describe a novel in vitro tissue engineered model, validated against native synovium explants, to investigate the structure-function of synovium through quantitative solute transport measures. Synovium was evaluated in the presence of a proinflammatory cytokine, interleukin-1, or the clinically relevant corticosteroid, dexamethasone. We anticipate that a better understanding of synovium transport would support efforts to develop more effective strategies aimed at restoring joint health.

Fibroblast-like synoviocyte mechanosensitivity to fluid shear is modulated by interleukin-1α.

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 10ng/mL IL-1α for 24h 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 cilium, 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.

Transient expression of the diseased phenotype of osteoarthritic chondrocytes in engineered cartilage.

Due to the degradation of osteoarthritic (OA) cartilage in post-traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal-release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time-dependent differences in interleukin (IL)-8, keratinocyte chemoattractant (KC)-like protein, MMP-1, MMP-2, and MMP-3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829-836, 2017.

Sacrificial nanofibrous composites provide instruction without impediment and enable functional tissue formation.

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.

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration.

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.