Mesenchymal stem cells (MSCs) are present in low density in bone marrow and culture expansion is necessary to obtain sufficient numbers for many proposed therapies. Researchers have characterized MSC growth on tissue culture plastic (TCP), although few studies have explored proliferation on other growth substrates. Using adult equine MSCs, we evaluated proliferation on fibrinogen-rich precipitate (FRP) surfaces created from blood plasma. When seeded at 1 × 10(4) cells/cm(2) and passaged five times over 10 days, MSCs on FRP in medium containing fibroblast growth factor 2 (FGF2) resulted in a ∼2.5-fold increase in cell yield relative to TCP. In FGF2-free medium, FRP stimulated a 10.4-fold increase in cell yield over TCP after 10 days, although control cultures maintained in FGF2 on TCP demonstrated that the stimulatory effect of FRP was not as lasting as that of FGF2. Chondrogenic cultures demonstrated that FRP did not affect differentiation. On TCP, MSCs seeded at 500 cells/cm(2) experienced a 4.6-fold increase in cell yield over cultures seeded at 1 × 10(4) cells/cm(2) following 10 days of expansion. In 500 cells/cm(2) cultures, FRP stimulating a two-fold increase in cell yield over TCP without affecting differentiation. Low-density FRP cultures showed a more even distribution of cells than TCP, suggesting that FRP may accelerate proliferation by reducing contact inhibition that slows proliferation. In addition, FRP appears capable of binding FGF2, as FRP surfaces pre-conditioned with FGF2 supported greater proliferation than FGF2-free cultures. Taken together, these factors indicate that substrates obtained from simple and inexpensive processing of blood enhance MSC proliferation and promote efficient coverage of expansion surfaces.
BackgroundIt is well documented that osteoarthritis (OA) can develop following traumatic joint injury and is the leading cause of lameness and subsequent wastage of equine athletes. Although much research of injury induced OA has focused on cartilage, OA is a disease that affects the whole joint organ.MethodsIn this study, we investigated the impact of synovial cells on the progression of an OA phenotype in injured articular cartilage. Injured and control cartilage were cultured in the presence of synoviocytes extracted from normal equine synovium. Synoviocytes and cartilage were evaluated for catabolic and anabolic gene expression. The cartilage was also evaluated histologically for loss of extracellular matrix molecules, chondrocyte cell death and chondrocyte cluster formation.ResultsThe results indicate synoviocytes exert both positive and negative effects on injured cartilage, but ultimately protect injured cartilage from progressing toward an OA phenotype. Synoviocytes cultured in the presence of injured cartilage had significantly reduced expression of aggrecanase 1 and 2 (ADAMTS4 and 5), but also had increased expression of matrix metalloproteinase (MMP) -1 and reduced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1). Injured cartilage cultured with synoviocytes had increased expression of both collagen type 2 and aggrecanase 2. Histologic examination of cartilage indicated that there was a protective effect of synoviocytes on injured cartilage by reducing the incidence of both focal cell loss and chondrocyte cluster formation, two major hallmarks of OA.ConclusionsThese results support the importance of evaluating more than one synovial joint tissue when investigating injury induced OA.
ABSTRACT:The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGF). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four-to ninefold increase in extracellular matrix (ECM) synthesis in TGF-free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan-rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGF exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion-conditioned MSCs without additional TGF exposure was less than samples maintained in TGF throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early-stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short-term culture. Keywords: chondrogenesis; mesenchymal stem cell; cartilage tissue engineering While the biochemical conditions for stimulating chondrogenesis of bone marrow-derived mesenchymal stem cells (MSCs) in vitro have been defined, 1 the potential of MSCs to generate neo-cartilage in vivo remains a topic of ongoing research. Animal studies often utilize MSCs from monolayer expansion culture, 2-6 a state in which MSCs are undifferentiated. Long-term follow-up has shown that transplanted undifferentiated MSCs in an adult equine model 6 and human patients 5 have not produced a lasting, hyaline-like tissue. Therefore, it has been suggested that induction of MSC chondrogenesis prior to implantation would promote superior repair. 6 In support of this hypothesis, small animal models have shown an increase in cartilage repair when MSC-seeded scaffolds were cultured in chondrogenic medium before implantation in experimental defects. 7,8 Pre-implantation culture of cell-seeded scaffolds is a means by which MSC chondrogenesis may be stimulated. However, ex vivo scaffold seeding is a step that would be otherwise unnecessary for carriers such as hydrogels, which as liquids can be mixed with cell suspensions and then crosslinked within defects 9 without the need for additional fixation, and using minimally invasive techniques. 6 In order to preserve this feature of injectable scaffolds, pre-implantation differentiation strategies must induce MSC chondrogene...
The threshold strain necessary to model injury in full-thickness cartilage specimens from the trochlear ridges of the distal femur of adult horses was 60% strain at a rate of 100% strain/s. This in vitro model should facilitate study of pathophysiologic changes and therapeutic interventions for osteoarthritis.
Personality ratings of 196 cats were made by their owners using a 5-point Likert scale anchored by 1: not at all and 5: a great deal with 12 items: timid, friendly, curious, sociable, obedient, clever, protective, active, independent, aggressive, bad-tempered, and emotional. A principal components analysis with varimax rotation identified three intepretable components. Component I had high loadings by active, clever, curious, and sociable. Component II had high loadings by emotional, friendly, and protective, Component III by aggressive and bad-tempered, and Component IV by timid. Sex was not associated with any component, but age showed a weak negative correlation with Component I. Older animals were rated less social and curious than younger animals.
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