Examination of synovial fluid hyaluronan quantity and quality in stifle joints of dogs with osteoarthritis.

OBJECTIVE: To determine the quantity (concentration) and quality (molecular weight) of synovial fluid hyaluronan with respect to presence and severity of osteoarthritis in stifle joints of dogs. ANIMALS: 21 purpose-bred dogs and 6 clinically affected large-breed dogs (cranial cruciate ligament [CrCL] disease with secondary osteoarthritis). PROCEDURES: Research dogs underwent arthroscopic surgery in 1 stifle joint to induce osteoarthritis via CrCL transection (CrCLt; n=5 stifle joints), femoral condylar articular cartilage groove creation (GR; 6), or meniscal release (MR; 5); 5 had sham surgery (SH) performed. Contralateral stifle joints (n=21) were used as unoperated control joints. Synovial fluid was obtained from research dogs at time 0 and 12 weeks after surgery and from clinically affected dogs prior to surgery. All dogs were assessed for lameness, radiographic signs of osteoarthritis, and pathologic findings on arthroscopy as well as for quantity and quality of hyaluronan. RESULTS: Clinically affected dogs had significantly greater degrees of pathologic findings, compared with dogs with surgically induced osteoarthritis (ie, those with CrCLt, GR, and MR stifle joints), and with respect to lameness scores, radiographic signs of osteoarthritis, pathologic findings on arthroscopy, and synovial fluid hyaluronan concentration. Synovial fluid from stifle joints of dogs with surgically induced osteoarthritis had hyaluronan bands at 35 kd on western blots that synovial fluid from SH and clinically affected stifle joints did not. CONCLUSIONS AND CLINICAL RELEVANCE: Synovial fluid hyaluronan quantity and quality were altered in stifle joints of dogs with osteoarthritis, compared with control stifle joints. A specific hyaluronan protein fragment may be associated with early pathologic changes in affected joints.

Effects of dynamic compressive load on collagen-based scaffolds seeded with fibroblast-like synoviocytes.

Synoviocytes have been speculated to play potential reparative and remodeling roles in vascular meniscal injuries. In addition, synoviocytes may mediate the transformation of intraarticularly placed collagen-based scaffolds into fibrocartilage through exposure to dynamic compressive loads. The objectives of this study were to assess the feasibility of using fibroblast-like synoviocytes (FLS) to engineer meniscal-like fibrocartilage and to better understand the mechanosensitivity of FLS by seeding them onto collagen scaffolds exposed to dynamic compressive loads. Canine FLS were seeded onto disks of four commercially available collagen-based scaffolds (Restore, Permacol, Cuff Patch, and Graff Jacket) and subjected either to one of two levels of intermittent dynamic compressive load or no load. The disks were harvested at 1 and 2 weeks and assessed for cell viability, retention, and infiltration, as well as extracellular matrix production. In general, loading regimens decreased cellularity, and nonloaded Restore grafts retained the most cells across time intervals. Spatial distribution of FLS was optimized in Restore grafts and was overall better in non-crosslinked collagen scaffolds (Restore and Graft Jacket) than cross-linked matrices. Collagen production was noted in association with penetrating FLS clusters in the Restore scaffolds only. The applied biomechanical stimulus did not appear to induce fibrochondrogenesis in any treatment group. These data suggest that Restore scaffolds may foster greater cell retention and infiltration when compared to other commercially available, collagen-based biomatrices.

Effects of supplemental lactoferrin on serum lactoferrin and IgG concentrations and neutrophil oxidative metabolism in Holstein calves.

Lactoferrin (LF) is an iron-binding protein present in both colostrum and secondary granules of polymorphonuclear neutrophils (PMNs). We hypothesized that supplemental LF enhances neutrophil function in neonatal calves. Newborn calves were assigned to receive colostrum (C), colostrum + LF (CLF, 1 g/kg), or milk replacer + LF (MRLF, 1 g/kg). Serum (LF and IgG) and whole blood (neutrophil isolation) samples were obtained prior to treatment (day 0) and at 24 hours and 9 days of age. Serum IgG concentrations (mean +/- SD) in C, CLF, and MRLF calves at 24 hours were 1,911 +/- 994 mg/dL, 2,181 +/- 625 mg/dL, and 0 mg/ dL, respectively. Serum LF concentrations in C, CLF, and MRLF calves on day 0 were 324 +/- 334 ng/mL (range 0-863 ng/mL), 135 +/- 158 ng/mL (range 0-429 ng/mL), and 318 +/- 337 ng/mL (range 0-964 ng/mL), respectively. LF concentrations in C, CLF, and MRLF calves at 24 hours were significantly higher (P < .05), at 1,564 +/- 1,114 ng/mL (range 335-3,628 ng/mL, 2,237 +/- 936 ng/mL (range 31-3,287 ng/mL), and 3,189 +/- 926 ng/mL (range 1,736-4,120 ng/mL), respectively. Cytochrome c reduction in opsonized zymosan-treated or phorbol ester-treated cells was not significantly affected by supplemental LF provided at birth. Oral LF is absorbed in calves but does not alter PMN superoxide production and does not alter IgG absorption.

Characterization of gelatinases in bronchoalveolar lavage fluid and gelatinases produced by alveolar macrophages isolated from healthy calves.

OBJECTIVE: To characterize gelatinases in bronchoalveolar lavage fluid (BALF) and gelatinases produced by alveolar macrophages of healthy calves. SAMPLE POPULATION: Samples of BALF and alveolar macrophages obtained from 20 healthy 2-month-old calves. PROCEDURE: BALF was examined by use of gelatin zymography and immunoblotting to detect gelatinases and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Cultured alveolar macrophages were stimulated with lipopolysaccharide (LPS), and conditioned medium was subjected to zymography. Alveolar macrophage RNA was used for reverse transcriptase-polymerase chain reaction assay of matrix metalloproteinases (MMPs), cyclooxygenase-2, and inducible nitric oxide synthase. RESULTS: Gelatinolytic activity in BALF was evident at 92 kd (14/20 calves; latent MMP-9) and 72 kd (18/20; latent MMP-2). Gelatinolytic activity was evident at 82 kd (10/20 calves; active MMP-9) and 62 kd (17/20; active MMP-2). Gelatinases were inhibited by metal chelators but not serine protease inhibitors. Immunoblotting of BALF protein and conditioned medium confirmed the MMP-2 and -9 proteins. Endogenous inhibitors (ie, TIMPs) were detected in BALF from all calves (TIMP-1) or BALF from only 4 calves (TIMP-2). Cultured alveolar macrophages expressed detectable amounts of MMP-9 mRNA but not MMP-2 mRNA. CONCLUSIONS AND CLINICAL RELEVANCE: Healthy calves have detectable amounts of the gelatinases MMP-2 and -9 in BALF Endogenous inhibitors of MMPs were detected in BALF (ie, TIMP-1, all calves; TIMP-2, 4 calves). Lipopolysaccharide-stimulated alveolar macrophages express MMP-9 but not MMP-2 mRNA. The role of proteases in the pathogenesis of lung injury associated with pneumonia has yet to be determined.

Tilmicosin reduces lipopolysaccharide-stimulated bovine alveolar macrophage prostaglandin E(2) production via a mechanism involving phospholipases.

Tilmicosin is a potent antimicrobial with broad-spectrum activity against the bacterial agents involved in the bovine respiratory disease complex. Recent studies indicate that in addition to being bactericidal, tilmicosin is capable of modulating inflammation in the lung. A series of experiments were designed to determine whether tilmicosin alters alveolar macrophage-prostaglandin E(2) (PGE(2)) production induced by Escherichia coli (O55:B5) lipopolysaccharide (LPS). Twenty-two healthy Holstein bull calves were used to study the effects of LPS-induced PGE(2) production of alveolar macrophages after in vivo or in vitro treatment with tilmicosin. In Experiment 1, tilmicosin was given by subcutaneous injection (15 mg/kg) twice, 48 hours apart, to four calves; four control calves received no treatment. Twenty-four hours after the second treatment, alveolar macrophages were stimulated with LPS in vitro. In Experiment 2, alveolar macrophages from five untreated calves were harvested and treated in vitro with tilmicosin, followed by LPS stimulation. In Experiment 3, the ability of in vitro tilmicosin treatment to alter the expression of LPS-induced cyclooxygenase-2 (COX-2) mRNA was evaluated. In Experiments 4 and 5, secretory phospholipase A(2) activity was examined in untreated calves. Treatment of calves with tilmicosin resulted in reduced LPS-induced alveolar macrophage PGE(2) production. Similar reductions in PGE(2) by LPS-stimulated alveolar macrophages after in vitro tilmicosin treatment were noted. This in vitro tilmicosin treatment was not associated with reduction of the expression of LPS-induced COX-2. Alveolar macrophage phospholipase A(2) activity induced by LPS was significantly reduced by prior tilmicosin treatment in vitro. Tilmicosin (in vivo and in vitro) appears to reduce the PGE(2) eicosanoid response of LPS-stimulated alveolar macrophages by reducing the in vitro substrate availability without altering in vitro COX-2 mRNA expression.

Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study.

Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. Discussion/Conclusion. FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering.

Effects of low-temperature hydrogen peroxide gas plasma sterilization on in vitro cytotoxicity of poly(ϵ-caprolactone) (PCL).

Our objective was to determine whether low-temperature hydrogen peroxide (H2O2) gas plasma sterilization of porous three-dimensional poly(ϵ-caprolactone) (PCL) constructs significantly inhibits cellular metabolism of canine chondrocytes. Porous cylindrical constructs were fabricated using fused deposition modeling and divided into four sterilization groups. Two groups were sterilized with low-temperature H2O2 gas plasma (LTGP) and constructs from one of those groups were subsequently rinsed with Dulbecco's Modified Essential Media (LTGPDM). Constructs in the other two groups were disinfected with either 70% isopropyl alcohol or exposure to UV light. Canine chondrocytes were seeded in 6-well tissue-culture plates and allowed to adhere prior to addition of PCL. Cellular metabolism was assessed by adding resazurin to the tissue-culture wells and assessing conversion of this substrate by viable cells to the fluorescent die resorufin. This process was performed at three times prior to addition of PCL and at four times after addition of PCL to the tissue-culture wells. Metabolism was not significantly different among the different tissue-culture wells at any of the 3 times prior to addition of PCL. Metabolism was significantly different among the treatment groups at 3 of 4 times after addition of PCL to the tissue culture wells. Metabolism was significantly lower with constructs sterilized by LTGP than all other treatment groups at all 3 of these times. We conclude that LTGP sterilization of PCL constructs resulted in significant cytotoxicity to canine chondrocytes when compared to PCL constructs disinfected with either UV light exposure or 70% isopropyl alcohol.

In vivo outcomes of tissue-engineered osteochondral grafts.

Tissue-engineered osteochondral grafts have been synthesized from a variety of materials, with some success at repairing chondral defects in animal models. We hypothesized that in tissue-engineered osteochondral grafts synthesized by bonding mesenchymal stem cell-loaded hydrogels to a porous material, the choice of the porous scaffold would affect graft healing to host bone, and the quality of cell restoration at the hyaline cartilage surface. Bone marrow-derived allogeneic mesenchymal stem cells were suspended in hydrogels that were attached to cylinders of porous tantalum metal, allograft bone, or a bioactive glass. The tissue-engineered osteochondral grafts, thus created were implanted into experimental defects in rabbit knees. Subchondral bone restoration, defect fill, bone ingrowth-implant integration, and articular tissue quality were compared between the three subchondral materials at 6 and 12 weeks. Bioactive glass and porous tantalum were superior to bone allograft in integrating to adjacent host bone, regenerating hyaline-like tissue at the graft surface, and expressing type II collagen in the articular cartilage.

Effects of growth factors on equine synovial fibroblasts seeded on synthetic scaffolds for avascular meniscal tissue engineering.

Across species, the avascular portion of the knee meniscus cannot heal spontaneously if severely injured. The most common treatment is meniscectomy which results in osteoarthritis. The objective of this study was to assess the fibrochondrogenic potential of equine fibroblast-like synoviocytes (FLS) seeded on scaffolds under the influence of growth factors in vitro to determine the potential of developing a novel cell-based repair strategy. Cultured FLS were seeded onto synthetic scaffolds in a rotating bioreactor under the influence of three growth factor regimens: none, basic fibroblast growth factor (bFGF) alone, and bFGF plus transforming growth factor (TGF-beta(1)) and insulin-like growth factor (IGF-1). Constructs were analyzed for mRNA expression and production of fibrochondroid extracellular matrix constituents. Type II collagen and aggrecan mRNA were significantly higher in growth factor-treated groups (p<0.05). Despite sub-optimal extracellular matrix production, FLS can exhibit fibrochondral characteristics and may have potential for cell-based tissue engineering for avascular meniscal regeneration.