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  • Author or Editor: Hayam Hussein x
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Abstract

OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method.

SAMPLE Monolayer cultures of synovial cells from 4 adult horses.

PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS), and 50 (24 hours after end of LPS) hours. At 50 hours, cell number and viability and prostaglandin (PG) E2, interleukin (IL)-6, matrix metalloproteinase (MMP)-3, and cyclooxygenase (COX)-2 production were measured.

RESULTS LPS challenge exposure induced a significant loss of characteristic synovial cell morphology, decrease in cell viability, and increases in concentrations of PGE2, IL-6, MMP-3, and COX-2. Cells treated with HA or HA-CS-NAG had significantly better viability and morphology scores and lower concentrations of PGE2, MMP-3, IL-6, and COX-2 than untreated LPS challenge-exposed cells. Cells treated with HA had significantly better morphology scores at 50 hours than cells treated with HA-CS-NAG. Cells treated with HA-CS-NAG had significantly superior suppression of LPS-induced production of PGE2, IL-6, and MMP-3 than cells treated with HA alone.

CONCLUSIONS AND CLINICAL RELEVANCE HA and HA-CS-NAG protected synovial cells from the effects of LPS. Treatment with HA-CS-NAG had the greatest anti-inflammatory effect. These results supported the protective potential of HA and HA-CS-NAG treatments.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs).

SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers.

PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 106 cells/0.5 mL) by use of a 30% serum gradient. Samples were evaluated on days 0, 3, 7, and 14. Cell migration, differentiation, and distribution into the synECMs were determined by cell surface marker CD90, viability, histologic morphology, and fluorescence microscopy results and expression of GFP, BMP-2, hyaluronan (HA), and proteoglycan (PG).

RESULTS At day 14, synMSCs were viable and had multiplied 2.5-fold in the synECMs. The synECMs seeded with synMSCs had a significant decrease in CD90 expression and significant increases in HA and PG expression. The synECMs seeded with synMSCs cotransduced with GFP, or BMP-2 had a significant increase in BMP-2 expression.

CONCLUSIONS AND CLINICAL RELEVANCE The synECM seeded with synMSCs or synMSCs cotransduced with GFP, or BMP-2 yielded a bioactive synovial scaffold. Expression of BMP-2 by synMSCs cotransduced to enhance expression of BMP-2 or GFP and an accompanying increase in both HA and PG expression indicated production of anabolic agents and synoviocyte differentiation in the scaffold. Because BMP-2 can promote repair of damaged cartilage, such a bioactive scaffold could be useful for treatment of injured cartilage.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE To evaluate 4 methods for generating decellularized equine synovial extracellular matrix.

SAMPLE Villous synovium harvested from the femoropatellar and medial femorotibial joints of 4 healthy adult horses < 7 years of age. Synovial samples were frozen (−80°C) until used.

PROCEDURES Synovial samples were thawed and left untreated (control) or decellularized with 1 of 4 methods (15 samples/horse/method): incubation in 0.1% peracetic acid (PAA), incubation in 0.1% PAA twice, incubation in 1% Triton X-100 followed by incubation in DNase, and incubation in 2M NaCl followed by incubation in DNase. Control and decellularized samples were examined for residual cells, villous integrity, and collagen structure and integrity by means of histologic examination and scanning electron microscopy; cell viability was evaluated by means of culture and exclusion staining. Decellularization efficiency was assessed by testing for DNA content and DNA fragment size.

RESULTS Incubation in PAA once preserved the synovial villous architecture, but resulted in high DNA content and retention of large (> 25,000 base pair) DNA fragments. Incubation in Triton and incubation in NaCl resulted in low DNA content and short (< 200 base pair) DNA fragments, but destroyed the synovial villous architecture. Incubation in PAA twice resulted in low DNA content and short DNA fragments while retaining the synovial villous architecture.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that of the methods evaluated, incubation in 0.1% PAA twice was the best method for generating decellularized equine synovial extracellular matrix.

Full access
in American Journal of Veterinary Research