Beverley Fermor

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose‐derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell‐based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty‐five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage‐specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron‐like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen (∼5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose‐derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two‐fold increase in the rate of protein synthesis and a three‐fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage‐associated matrix molecules, including collagen type II and chondroitin‐4‐sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue‐engineered cartilage.

Interleukin‐1, tumor necrosis factor α, and interleukin‐17 synergistically up‐regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci

OBJECTIVE In osteoarthritis (OA), a combination of biochemical and biomechanical factors may damage both menisci and articular cartilage. Nitric oxide (NO) and prostaglandin E2 (PGE2) have been implicated as mediators of inflammation in OA. The goals of this study were to determine if menisci from patients with OA produce NO and PGE2, and if the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor a (TNFalpha), and IL-17 augment NO and PGE2 production by these tissues. METHODS Menisci were obtained from 17 patients (age 47-75 years) undergoing total knee replacement for OA. Tissue explants were cultured alone or with IL-1beta, IL-17, or TNFalpha, and the release of NO and PGE2 from the tissue as well as the presence of type 2 nitric oxide synthase (NOS2) and cyclooxygenase 2 (COX-2) antigens were measured. RESULTS All menisci constitutively produced NO, and significant increases in NO production were observed in the presence of IL-1beta, TNFalpha, or IL-17 (P < 0.05). The combination of IL-17 and TNFalpha significantly increased NO production compared with either cytokine alone. Basal and cytokine-stimulated NO synthesis was inhibited by the NOS inhibitors NG-monomethyl-L-arginine or N-3-aminoethylbenzylacetamidine (1400W). IL-1beta significantly increased PGE2 production. The combination of IL-1beta and TNFalpha had an additive effect on PGE2 production, while addition of IL-17 to TNFalpha or IL-1beta synergistically enhanced PGE2 production. Inhibition of NO production by 1400W significantly increased IL-1beta-stimulated PGE2 production, and inhibition of PGE2 production by the COX-2 inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide significantly increased IL-17-stimulated NO production. CONCLUSION Menisci from humans with OA spontaneously produced NO and PGE2 in a manner that was synergistically or additively augmented by cytokines. NO and PGE2 exhibited reciprocal regulatory effects on one another, suggesting that pharmaceutical agents designed to inhibit NOS2 or COX-2 production may in fact be influencing both pathways.

The effects of static and intermittent compression on nitric oxide production in articular cartilage explants

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress‐induced upregulation of NOS2, but not NOSI or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biome‐chanical factors may in part regulate NO production in vivo.

Diet-induced obesity differentially regulates behavioral, biomechanical, and molecular risk factors for osteoarthritis in mice

IntroductionObesity is a major risk factor for the development of osteoarthritis in both weight-bearing and nonweight-bearing joints. The mechanisms by which obesity influences the structural or symptomatic features of osteoarthritis are not well understood, but may include systemic inflammation associated with increased adiposity. In this study, we examined biomechanical, neurobehavioral, inflammatory, and osteoarthritic changes in C57BL/6J mice fed a high-fat diet.MethodsFemale C57BL/6J mice were fed either a 10% kcal fat or a 45% kcal fat diet from 9 to 54 weeks of age. Longitudinal changes in musculoskeletal function and inflammation were compared with endpoint neurobehavioral and osteoarthritic disease states. Bivariate and multivariate analyses were conducted to determine independent associations with diet, percentage body fat, and knee osteoarthritis severity. We also examined healthy porcine cartilage explants treated with physiologic doses of leptin, alone or in combination with IL-1α and palmitic and oleic fatty acids, to determine the effects of leptin on cartilage extracellular matrix homeostasis.ResultsHigh susceptibility to dietary obesity was associated with increased osteoarthritic changes in the knee and impaired musculoskeletal force generation and motor function compared with controls. A high-fat diet also induced symptomatic characteristics of osteoarthritis, including hyperalgesia and anxiety-like behaviors. Controlling for the effects of diet and percentage body fat with a multivariate model revealed a significant association between knee osteoarthritis severity and serum levels of leptin, adiponectin, and IL-1α. Physiologic doses of leptin, in the presence or absence of IL-1α and fatty acids, did not substantially alter extracellular matrix homeostasis in healthy cartilage explants.ConclusionsThese results indicate that diet-induced obesity increases the risk of symptomatic features of osteoarthritis through changes in musculoskeletal function and pain-related behaviors. Furthermore, the independent association of systemic adipokine levels with knee osteoarthritis severity supports a role for adipose-associated inflammation in the molecular pathogenesis of obesity-induced osteoarthritis. Physiologic levels of leptin do not alter extracellular matrix homeostasis in healthy cartilage, suggesting that leptin may be a secondary mediator of osteoarthritis pathogenesis.

Nitric Oxide Synthase and Cyclooxygenase Interactions in Cartilage and Meniscus

Rheumatoid arthritis and osteoarthritis are painful and debilitating diseases with complex pathophysiology. There is growing evidence that pro-inflammatory cytokines (e.g., interleukin-1 and tumor necrosis factor alpha) and mediators (e.g., prostaglandins, leukotrienes, and nitric oxide) play critical roles in the development and perpetuation of tissue inflammation and damage in joint tissues such as articular cartilage and meniscus. While earlier studies have generally focused on cells of the synovium (especially macrophages), there is increasing evidence that chondrocytes and meniscal cells actively contribute to inflammatory processes. In particular, it is now apparent that mechanical forces engendered by joint loading are transduced to biological signals at the cellular level and that these signals modulate gene expression and biochemical processes. Here we give an overview of the interplay of cytokines and mechanical stress in the production of cyclooxygenases and prostaglandins; lipoxygenases and leukotrienes; and nitric oxide synthases and nitric oxide in arthritis, with particular focus on the interactions of these pathways in articular cartilage and meniscus.

Oxidative DNA damage in osteoarthritic porcine articular cartilage.

Osteoarthritis (OA) is associated with increased levels of reactive oxygen species. This study investigated if increased oxidative DNA damage accumulates in OA articular cartilage compared with non‐OA articular cartilage from pigs with spontaneous OA. Additionally, the ability of nitric oxide (NO) or peroxynitrite (ONOO−) induced DNA damage in non‐OA chondrocytes to undergo endogenous repair was investigated. Porcine femoral condyles were graded for the stage of OA, macroscopically by the Collins Scale, and histologically by the modified Mankin Grade. Levels of DNA damage were determined in non‐OA and OA cartilage, using the comet assay. For calibration, DNA damage was measured by exposing non‐OA chondrocytes to 0–12 Gray (Gy) of X‐ray irradiation. Non‐OA articular chondrocytes were treated with 0–500 µM of NO donors (NOC‐18 or SIN‐1), and DNA damage assessed after treatment and 5 days recovery. A significant increase (P < 0.01) in oxidative DNA damage occurred in OA chondrocytes in joints with Mankin Grades 3 or greater, compared to non‐OA chondrocytes. The percentage of nuclei containing DNA damage increased significantly (P < 0.001) from early to late grades of OA. An increase of approximately 0.65–1.7 breaks/1,000 kb of DNA occurred in OA, compared to non‐OA nuclei. NOC‐18 or SIN‐1 caused significant DNA damage (P < 0.001) in non‐OA chondrocytes that did not undergo full endogenous repair after 5 days (P < 0.05). Our data suggest significant levels of oxidative DNA damage occur in OA chondrocytes that accumulates with OA progression. Additionally, DNA damage induced by NO and ONOO− in non‐OA chondrocytes does not undergo full endogenous repair. J. Cell. Physiol. 217: 828–833, 2008.

Hypoxia, RONS and energy metabolism in articular cartilage

OBJECTIVE Increased pro-inflammatory cytokines and reactive oxygen and nitrogen species (RONS) occur in osteoarthritis (OA). Oxygen tension can alter the levels of RONS induced by interleukin-1 (IL-1). RONS such as nitric oxide (NO) can alter energy metabolism. The aim of this study was to determine if oxygen tension alters energy metabolism, in articular cartilage, in response to IL-1 or NO and to determine if cell death occurred. DESIGN Porcine articular chondrocytes were incubated with IL-1 or the NO donor NOC-18 for 48 h in either 1, 5 or 20% O(2). Adenosine triphosphate (ATP) levels were measured and immunoblots for adenosine monophosphate-activated protein kinase (AMPK) were done. Protein translation was measured by S6 activation. Senescence and autophagy were determined by increased caveolin or conversion of LC3-I to LC3-II respectively. RESULTS One percent O(2) significantly reduced ATP levels compared with 20% O(2). Five percent O(2) significantly increased ATP levels compared with 20% O(2). One percent O(2) significantly increased phospho-AMPK (pAMPK) protein expression compared with 5 or 20% O(2). Oxygen tension had no effects on pS6, caveolin or LC3-II levels. IL-1-induced NO production was significantly reduced with decreased oxygen tension, and significantly reduced ATP levels at all oxygen tensions, but pAMPK was only significantly increased at 5% O(2). IL-1 significantly reduced pS6 at all oxygen tensions. IL-1 had no effects on caveolin and significantly increased LC3-II at 20% O(2) only. NOC-18 significantly reduced ATP levels at all oxygen tensions, and significantly increased pAMPK at 5% O(2) only, and significantly decreased pAMPK at 1% O(2). NOC-18 significantly reduced pS6 at 1% O(2) and significantly increased caveolin at 5% O(2), and LC3-II at 1% O(2). CONCLUSION Our data suggest 5% O(2) is optimal for energy metabolism and protective to some effects of IL-1 and NO. NO has the greatest effects on ATP levels and the induction of autophagy at 1% O(2).