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Biomedical subjects

Alicia L Bertone

Publications and source records attributed to Alicia L Bertone.

16 recordsLinked to original sources

Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells.

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.

Animals↗

Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.

Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.

Adenoviridae↗

Computed tomographic documentation of a comminuted fourth carpal bone fracture associated with carpal instability treated by partial carpal arthrodesis in an Arabian filly.

OBJECTIVE: To report treatment of a unilateral comminuted fourth carpal bone (C4) fracture associated with carpal instability by partial carpal arthrodesis (PCA) of the middle carpal joint (MCJ) and carpometacarpal joint (CMCJ). STUDY DESIGN: Case Report. ANIMALS: An 8-month-old Arabian filly. METHODS: A C4 slab fracture was diagnosed radiographically; however, fracture comminution was conclusively diagnosed after computed tomographic (CT) imaging. PCA of the MCJ and CMCJ was performed with 2 narrow dynamic compression plates. RESULTS: PCA provided appropriate carpal stability and correct limb alignment immediately after surgery. Complete bony fusion with substantial carpal flexion and no lameness at walk or light trot was observed 8 months after surgery. CONCLUSIONS: Carpal CT was successfully used to define fracture configuration after standard radiographic examination failed to delineate comminution. PCA was selected because of joint instability and lateral carpal collapse of MCJ and CMCJ and can be successfully used to treat comminuted C4 slab fractures associated with carpal instability. Moderate MCJ osteoarthritis without radiocarpal joint involvement allows pain-free, substantial carpal flexion and thus, return to low-level pleasure riding may be possible. CLINICAL RELEVANCE: CT imaging may more adequately characterize traumatic carpal bone injury, particularly, when carpal bone fracture configuration cannot be determined on standard radiographs. Early PCA of the MCJ and CMCJ is an useful alternative to treat comminuted C4 slab fractures that cannot be reconstructed.

Animals↗

Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery.

OBJECTIVE: To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). SAMPLE POPULATION: Articular cartilage, synovium, and bone marrow from 15 adult horses. PROCEDURES: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection. RESULTS: -Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors. CONCLUSION AND CLINICAL RELEVANCE: Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.

Adenoviridae↗

Gross, histologic, and gene expression characteristics of osteoarthritic articular cartilage of the metacarpal condyle of horses.

OBJECTIVE: To identify patterns and correlations of gross, histologic, and gene expression characteristics of articular cartilage from horses with osteoarthritis. ANIMALS: 10 clinically normal horses and 11 horses with osteoarthritis of the metacarpal condyles. PROCEDURES: Metacarpophalangeal joints were opened and digitally photographed, and gross lesions were scored and quantified. Representative cartilage specimens were stained for histologic scoring. Total RNA from dorsal and palmar articular surfaces was processed on an equine gene expression microarray. RESULTS: Histologic scores were greater in both regions of osteoarthritic joints, compared with corresponding regions in control joints. Cartilage from the palmar aspect of diseased joints had the highest histologic scores of osteoarthritic sites or of either region in control joints. A different set of genes for dorsal and palmar osteoarthritis was identified for high and low gene expression. Articular cartilage from the dorsal region had surface fraying and greater expression of genes coding for collagen matrix components and proteins with anti-apoptotic function, compared with control specimens. Articular cartilage from the palmar region had greater fraying, deep fissures, and less expression of genes coding for glycosaminoglycan matrix formation and proteins with anti-apoptotic function, compared with cartilage from disease-free joints and the dorsal aspect of affected joints. CONCLUSIONS AND CLINICAL RELEVANCE: Metacarpal condyles of horses with naturally occurring osteoarthritis had an identifiable and regional gene expression signature with typical morphologic features.

Animals↗

Effects of the omega-3 fatty acid, alpha-linolenic acid, on lipopolysaccharide-challenged synovial explants from horses.

OBJECTIVE: To determine the effects of pretreatment with alpha-linolenic acid, an omega-3 polyunsaturated fatty acid, on equine synovial explants challenged with lipopolysaccharide (LPS). ANIMALS: 8 mature mixed-breed horses (4 mares and 4 geldings). PROCEDURE: Synovial explants were assigned to receive 1 of 7 concentrations of alpha-linolenic acid, ranging from 0 to 300 microg/mL. At each concentration, half of the explants were controls and half were challenged with 0.003 microg of LPS as a model of synovial inflammation. Cell inflammatory response was evaluated by measurement of prostaglandin E2 production via an ELISA. Synovial cell viability, function, histomorphologic characteristics, and cell membrane composition were evaluated by use of trypan blue dye exclusion, hexuronic acid assay for hyaluronic acid, objective microscopic scoring, and high-performance liquid chromatography, respectively. RESULTS: Challenge with LPS significantly increased production of prostaglandin E2 and decreased production of hyaluronic acid. Treatment with alpha-linolenic acid at the highest dose inhibited prostaglandin E2 production. Cell viability and histomorphologic characteristics were not altered by treatment with alpha-linolenic acid or LPS challenge. Treatment with alpha-linolenic acid increased the percentage of this fatty acid in the explant cell membranes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that investigation of alpha-linolenic acid as an anti-inflammatory medication for equine synovitis is warranted.

Analysis of Variance↗

Association between subjective lameness grade and kinetic gait parameters in horses with experimentally induced forelimb lameness.

OBJECTIVE: To evaluate the association between subjective lameness grades and kinetic gait parameters and assess the variability in kinetic parameters in horses with experimentally induced forelimb lameness. ANIMALS: 32 horses. PROCEDURES: Forelimb lameness was induced in each horse via injection of lipopolysaccharide into 1 metacarpophalangeal joint (40 experimental trials). Subjective lameness grading and 13 kinetic gait parameters (force plate analysis) were assessed before (baseline) and at 12, 18, and 24 hours after lipopolysaccharide injection. While horses were trotting, kinetic gait analysis was performed for 8 valid repetitions at each time point. Repeated-measures analyses were performed with 8 repetitions for each kinetic parameter as the outcome, and lameness grades, time points after lipopolysaccharide injection, and repetition order as explanatory variables. Sensitivity and specificity of kinetic parameters for classification of horses as sound or lame (in relation to subjective lameness scores) were calculated. Between- and within-horse variabilities of the 13 kinetic parameters were assessed by calculation of coefficients of variation. RESULTS: Subjective lameness grades were significantly associated with most of the kinetic parameters. Vertical force peak and impulse had the lowest between- and within-horse coefficients of variation and the highest correlations with subjective lameness grade. Vertical force peak had the highest sensitivity and specificity for lameness classification. Vertical force peak and impulse were significantly decreased even in horses with mild or unobservable lameness. CONCLUSIONS AND CLINICAL RELEVANCE: Among the kinetic gait parameters, vertical force peak and impulse had the best potential to reflect lameness severity and identify subclinical forelimb gait abnormalities.

Animals↗

Comparison of proteoglycan and collagen in articular cartilage of horses with naturally developing osteochondrosis and healing osteochondral fragments of experimentally induced fractures.

OBJECTIVE: To compare articular cartilage from horses with naturally developing osteochondrosis (OC) with normal articular cartilage and healing cartilage obtained from horses with experimentally induced osteochondral fractures. SAMPLE POPULATION: 109 specimens of articular cartilage from 78 horses. PROCEDURE: Morphologic characteristics, proteoglycan (PG), and type II collagen were analyzed in articular cartilage of OC specimens (group 1), matched healing cartilage obtained 40 days after experimentally induced osteochondral fractures (group 2), and matched normal cartilage from the same sites (group 3). RESULTS: 79 specimens of OC cartilage were obtained from horses. Ex vivo PG synthesis was significantly greater in the femoral cartilage, compared with synthesis in the tibial cartilage, and significantly greater for groups 1 and 2, compared with group 3. For groups 1 and 2, femoral fragments had significantly greater PG content, compared with PG content in tibial fragments. Keratan sulfate content was significantly less in group 3, compared with groups 1 and 2. Cartilage from the OC specimens had loss of structural architecture. The OC tissue bed stained positive for chondroitin sulfate and type II collagen, but the fracture bed did not. CONCLUSIONS AND CLINICAL RELEVANCE: Our analyses could not distinguish articular cartilage from horses with OC and a healing fracture. Both resembled an anabolic, reparative process. Immunohistochemical analysis suggested a chondromyxoid tissue in the OC bed that was morphologically similar to fibrous tissue but phenotypically resembled hyaline cartilage. Thus, tissue in the OC bed may be degenerative cartilage, whereas tissue in the fracture bed may be reparative fibrous callus.

Animals↗

Response of induced bone defects in horses to collagen matrix containing the human parathyroid hormone gene.

OBJECTIVE: To determine whether human parathyroid hormone (hPTH) gene in collagen matrix could safely promote bone formation in diaphyseal or subchondral bones of horses. ANIMALS: 8 clinically normal adult horses. PROCEDURE: Amount, rate, and quality of bone healing for 13 weeks were determined by use of radiography, quantitative computed tomography, and histomorphometric analysis. Diaphyseal cortex and subchondral bone defects of metacarpi were filled with hPTH(1-34) gene-activated matrix (GAM) or remained untreated. Joints were assessed on the basis of circumference, synovial fluid analysis, pain on flexion, lameness, and gross and histologic examination. RESULTS: Bone volume index was greater for cortical defects treated with hPTH(1-34) GAM, compared with untreated defects. Bone production in cortical defects treated with hPTH(1-34) GAM positively correlated with native bone formation in untreated defects. In contrast, less bone was detected in hPTH(1-34) GAM-treated subchondral bone defects, compared with untreated defects, and histology confirmed poorer healing and residual collagen sponge. CONCLUSIONS AND CLINICAL RELEVANCE: Use of hPTH(1-34) GAM induced greater total bone, specifically periosteal bone, after 13 weeks of healing in cortical defects of horses. The hPTH(1-34) GAM impeded healing of subchondral bone but was biocompatible with joint tissues. Promotion of periosteal bone formation may be beneficial for healing of cortical fractures in horses, but the delay in onset of bone formation may negate benefits. The hPTH(1-34) GAM used in this study should not be placed in articular subchondral bone defects, but contact with articular surfaces is unlikely to cause short-term adverse effects.

Analysis of Variance↗

Generation and performance of an equine-specific large-scale gene expression microarray.

OBJECTIVE: To create high-quality sequence data for the generation of an equine gene expression microarray and evaluate array performance by use of lipopolysaccharide (LPS) exposure of synoviocytes. SAMPLE POPULATION: Public nucleotide sequence database from Equus caballus and synoviocytes from clinically normal adult horses. PROCEDURE: Computer procurement of equine gene sequences, probe design, and manufacture of an oligomicroarray were performed. Array performance was evaluated by use of patterns for equine synoviocytes in response to LPS. RESULTS: Starting with 18,924 equine gene sequences, 3,098 equine 3' sequences were annotated and met the inclusion criteria for an expression microarray. An equine oligonucleotide expression microarray was created by use of 68,266 of the 25-oligomer probes to uniquely identify each gene. Most genes in the array (68%) were expressed in equine synoviocytes. Repeatability of the array was high (r, > 0.99), and LPS upregulated (> 5-fold change) 84 genes, many of which were inflammatory mediators, and downregulated (> 5-fold change) 14 genes. An initial pattern of gene expression for effects of LPS on synoviocytes consisted of 102 genes. CONCLUSIONS AND CLINICAL RELEVANCE: Use of a computer algorithm to curate an equine sequence database generated high-quality annotated species-specific gene sequences and probe sets for a gene expression oligomicroarray, which was used to document changes in gene expression associated with LPS exposure of equine synoviocytes. The equine public database was expanded from 290 annotated genes to > 3,000 provisionally annotated genes. Similar curation and annotation of public databases could be used to create other species-specific microarrays.

Animals↗

Comparison of hematologic values and transforming growth factor-beta and insulin-like growth factor concentrations in platelet concentrates obtained by use of buffy coat and apheresis methods from equine blood.

OBJECTIVE: To evaluate the buffy coat and apheresis methods for preparation of platelet concentrates from equine blood by comparing platelet and growth factor concentrations. ANIMALS: 15 mature mixed-breed geldings. PROCEDURE: Whole blood samples were collected and processed by use of a buffy coat or apheresis method to obtain platelet poor and platelet concentrated fractions. The PCV, WBC count, and platelet count were compared among whole blood samples, platelet poor fractions, concentrates obtained by use of the apheresis method (ie, apheresis platelet concentrates), and concentrates obtained by use of the buffy coat method (ie, buffy coat platelet concentrates). Concentrations of transforming growth factor-beta (ie,TGF-beta1 andTGF-beta2) and insulin-like growth factor were compared between buffy coat and apheresis platelet concentrates. RESULTS: Platelet concentrations were 8.9-fold and 5.2-fold greater in buffy coat and apheresis platelet concentrates, respectively, compared with whole blood. Platelet concentrations were 13.1-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta1 concentrations were 2.8-fold and 3.1-fold greater in buffy coat and apheresis platelet concentrates, respectively, and TGF-beta1 concentrations were 10.5-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta2 concentrations were 3.6-fold greater in apheresis platelet concentrates, compared with whole blood. Platelet concentrations correlated with growth factor concentrations across all blood and platelet fractions. White blood cell counts had a significant positive correlation with TGF-beta1 concentration in buffy coat platelet concentrates. CONCLUSIONS AND CLINICAL RELEVANCE: Platelets and TGF-beta1 can be concentrated reliably from equine blood by use of buffy coat or apheresis methods, without modification of the protocols used for humans.

Analysis of Variance↗

Effect of laser shock peening on fatigue life and surface characteristics of stainless steel cortical bone screws.

OBJECTIVE: To investigate the effect of laser shock peening on the fatigue life and surface characteristics of 3.5-mm-diameter cortical bone screws. SAMPLE POPULATION: 32 stainless steel, 3.5-mm-diameter cortical bone screws. PROCEDURE: Screws were randomly assigned to an untreated control group or 2 power-density treatment groups, 6 gigawatts (GW)/cm2 and 8.5 GW/cm2, for laser shock peening. Number of cycles to failure and findings on scanning electron microscopy-assisted morphometric evaluation, including the mode of failure, surface debris, surface damage, and thread deformation, were compared between control and treated screws. RESULTS: The 6 GW/cm2 treated screws had a significant (11%) improvement in fatigue life, compared with untreated control screws. The 8.5 GW/cm2 treated screws had a significant (20%) decrease in fatigue life, compared with control screws. A mild but significant increase in thread deformation was evident in all treated screws, compared with control screws. The 8.5 GW/cm2 treated screws had significantly more surface irregularities (elevations and pits), compared with control or 6 GW/cm2 treated screws. CONCLUSION AND CLINICAL RELEVANCE: A modest positive increase in fatigue strength was produced by this design of laser shock peening on the midshaft of cortical bone screws. High laser shock peening power densities were detrimental, decreasing screw fatigue strength probably resulting from structural damage. Greater fatigue life of cortical bone screws can be generated with laser shock peening and could reduce screw breakage as a cause of implant failure; however, future studies will be necessary to address biocompatibility, alternative cleaning techniques, alterations in screw strength and pullout characteristics, and effects on susceptibility to corrosion.

Analysis of Variance↗

Nonsteroidal anti-inflammatory drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are substances other than steroids that inhibit a component of the inflammatory cascade. This article is dedicated to those substances which specifically inhibit cyclooxygenase. NSAIDs are used extensively in the veterinary field. This article discusses their pharmacologic mechanism of action, indications, and toxicity. The two isoforms of cyclooxygenase (COX-1 and COX-2) are reviewed along with the newer NSAID which are more effective and less toxic due to more specific COX-2 inhibition. Specific effects on soft tissue, bone, cartilage, and synovium are summarized. Pain modulation is extensively reviewed as well as the antiendotoxic and antithrombotic uses.

Animals↗

Bone gentamicin concentration after intra-articular injection or regional intravenous perfusion in the horse.

OBJECTIVE: To compare intra-articular (IA) and bone gentamicin concentrations achieved after intra-articular administration or regional intravenous perfusion (RIP). STUDY DESIGN: Experimental study. ANIMALS: Twelve healthy adult horses. METHODS: Horses were assigned to 2 treatment groups (n = 6/group): Group 1, 1 g gentamicin administered simultaneously in both left and right metacarpophalangeal joints and group 2, 1 g gentamicin administered simultaneously in both left and right lateral palmar veins. Serum, synovial fluid, and bone biopsy specimens were collected. Gentamicin concentrations were determined by fluorescence polarization immunoassay. Bone, synovial fluid, and serum gentamicin concentrations were compared over time and between groups using 2-way ANOVA. Significance of all tests were evaluated at P <.05. RESULTS: IA metacarpophalangeal joint administration resulted in higher concentration of gentamicin in synovial fluid than RIP administration. Synovial fluid concentration remained above minimum inhibitory concentration (MIC) for common pathogens for over 24 hours with IA and RIP administration. Bone gentamicin concentration remained above MIC for 8 hours with both methods; there was no significant difference in gentamicin concentration in bone with either method. Neither IA nor RIP administration had a significant effect on serum concentration of gentamicin. CONCLUSIONS: In normal horses, there is no difference in bone gentamicin concentration obtained with IA or RIP administration. CLINICAL RELEVANCE: Based on MIC for common equine pathogens, administration of gentamicin intra-articularly or by regional intravenous perfusion should be useful for treatment of osteomyelitis.

Analysis of Variance↗

The effects of radiofrequency energy treatment on chondrocytes and matrix of fibrillated articular cartilage.

BACKGROUND: There is no consensus regarding the safety of radiofrequency energy treatment for chondroplasty. HYPOTHESIS: Use of a radiofrequency thermal probe will produce a dose-dependent detrimental effect on chondrocytes in a setting mimicking the arthroscopic procedure. STUDY DESIGN: Controlled laboratory study. METHODS: Paired patellae from 11 horses were fibrillated; one served as the control while the contralateral patella was treated with 20, 40, and 60 watts of radiofrequency energy for 4 minutes. Proteoglycan synthesis, proteoglycan degradation, and cell viability were measured. Explant histologic sections were scored for cellular characteristics, metachromatic matrix staining intensity, and tissue architecture and were quantitatively analyzed for cell death. RESULTS: Mean peak surface articular cartilage temperatures were 35.47 degrees C (20 watts), 40.76 degrees C (40 watts), and 44.81 degrees C (60 watts). Treatment at all three settings significantly decreased proteoglycan synthesis of the chondrocytes. Proteoglycan degradation increased with increased power setting. Treatment at 40 and 60 watts significantly decreased cell viability to 81% and 73%, respectively. Significant histologic changes occurred in the superficial cartilage zone with 60-watt application. CONCLUSION: Use of radiofrequency energy on mechanically fibrillated articular cartilage induced a dose-dependent detrimental effect on chondrocytes and matrix metabolism. CLINICAL RELEVANCE: Cautious use of radiofrequency energy for treatment of articular cartilage is recommended until long-term effects are evaluated.

Animals↗