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.

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The viability of tumour cells shed into the intestinal lumen was determined in 49 patients with carcinoma of the large bowel. Preoperative colorectal lavage was performed in 19 patients and irrigation of the cut ends of the operative specimen in 30 patients. The resulting cell suspensions were centrifuged on a Nycodenz linear density gradient column so that tumour cells, being larger, were concentrated in a band at the top. In 14 of 19 colorectal lavage cases viable tumour cells were recovered, as assessed by their characteristic morphology and ability to exclude trypan blue. A median of 0.78 X 10(6) viable tumour cells was recovered. The median percentage cell viability in the suspension was 92, i.e. 8 per cent of the tumour cells were dead (stained with trypan blue). In eight specimens viability was confirmed by the ability of tumour cells to hydrolyse fluorescein diacetate. In 17 of 30 proximal resection margin irrigations a median of 0.55 X 10(5) viable tumour cells was recovered, with a median percentage viability of 92.5. In 15 specimens the neoplastic cells showed fluorescence. In 21 of 25 distal resection margin irrigations a median of 1.92 X 10(5) viable tumour cells was recovered with a median percentage cell viability of 79.3, and fluorescence was observed in all specimens. The number of viable tumour cells did not correlate with the stage, differentiation, diameter or fixity of the tumour. However, the number of tumour cells recovered from the distal resection margin was inversely related to the distance of the tumour from that margin (Rank Difference Coefficient R = -0.6). Thus viable exfoliated tumour cells were demonstrated in 52 of 74 specimens (70 per cent). Their presence in large numbers at the site of intestinal anastomoses supports a potential role in the aetiology of suture-line recurrence.

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.

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen ( approximately 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.

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‐1β (IL‐1β), tumor necrosis factor α (TNFα), 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‐1β, IL‐17, or TNFα, 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‐1β, TNFα, or IL‐17 (P < 0.05). The combination of IL‐17 and TNFα 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‐1β significantly increased PGE2production. The combination of IL‐1β and TNFα had an additive effect on PGE2 production, while addition of IL‐17 to TNFα or IL‐1β synergistically enhanced PGE2 production. Inhibition of NO production by 1400W significantly increased IL‐1β–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.

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 NOS1 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 biomechanical factors may in part regulate NO production in vivo.