Objective—To characterize the mechanical properties of subchondral bone (SCB) of the distopalmar aspect of the condyles of the third metacarpal bone (MC3) and their correlations with structural aspects of MC3s in Thoroughbred racehorses.
Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.
Procedures—MC3s were collected from horses with mild (n = 6) and with severe (6) SCB changes, as determined by micro–computed tomography (CT). Specimens of SCB plate and trabecular bone were cut from the distopalmar aspect of condyles and sagittal ridge and examined with 3-dimensional micro-CT. Specimens were tested in compression, and elastic modulus, yield stress, yield strain, and toughness were calculated. Apparent and true bone mineral density, bone volume fraction, trabecular thickness, trabecular separation, and connectivity were also calculated. Differences in mechanical properties among various classifications of bone were evaluated. Correlations between structural and mechanical variables were also assessed.
Results—No differences were detected between left and right forelimbs. Specimens from condyles had higher values for elastic modulus, yield stress, and toughness than did specimens of sagittal ridge. In SCB with severe changes attributable to SCB disease, SCB plate was weaker and trabecular bone was stronger than in SCB with mild changes. Microstructural and mechanical properties were significantly correlated.
Conclusions and Clinical Relevance—A marked gradient in mechanical properties of SCB from horses, which could be involved in the pathogenesis of condylar fractures, was detected. Mechanical properties of SCB from the distal aspect of MC3s can be predicted to some extent via micro-CT.
Objective—To characterize the microstructure of subchondral bone (SCB) plate and trabecular bone (TBB) of the distopalmar aspect of the condyles of third metacarpal bones (MC3s) from Thoroughbred racehorses at 2 different stages of SCB disease via micro–computed tomography (CT).
Sample Population—12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons.
Procedures—MC3s were collected from horses with mild (n = 6) or severe (6) SCB disease, as determined via micro-CT. Cubic (6 × 6 × 6-cm) specimens of SCB plate and TBB were cut from the palmar aspect of condyles and sagittal ridges and examined with 3-dimensional micro-CT. For each specimen, apparent bone mineral density (aBMD), true BMD (tBMD), bone volume fraction (BVF), trabecular thickness (TBT), trabecular separation (TBS), and connectivity (CN) were calculated.
Results—Condyles had higher aBMD, tBMD, BVF, and TBT and lower TBS than did the sagittal ridge. In bone specimens with mild SCB changes, SCB plate had higher aBMD, TBT, and CN and lower TBS than did TBB. In bone specimens with severe SCB disease, TBB had higher aBMD and TMD and lower TBS than it did in bone specimens with mild disease, and values were similar to those for SCB plate in bone specimens with severe disease.
Conclusions and Clinical Relevance—The microstructure of SCB of the distopalmar aspect of metacarpal condyles of horses varied according to the severity of changes identified via micro-CT. With mild SCB disease, sclerosis existed in the SCB plate of the condyles; with severe disease, sclerosis also invaded condylar TBB.
Objective—To evaluate the effects of glucosamine on equine articular chondrocytes and synoviocytes at concentrations clinically relevant to serum and synovial fluid concentrations.
Sample Population—Articular cartilage and synovium with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte and synoviocyte cell cultures from 8 horses were treated with glucosamine (0.1 to 20 μg/mL) with or without interleukin-1 (IL-1; 10 ng/mL) for 48 hours. Negative control cultures received no glucosamine or IL-1, and positive control cultures received only IL-1. Cultures were assayed for production of proteoglycan (via media containing sulfur 35 (35S)-labeled sodium sulfate and Alcian blue precipitation), prostaglandin E2 (PGE2; via a colorimetric assay), cyclooxygenase-2 (via real-time reverse-transcriptase PCR assay), microsomal PGE2 synthase (mPGEs; via real-time reverse-transcriptase PCR assay), and matrix metalloproteinase (MMP)-13 (via a colorimetric assay).
Results—Glucosamine had no impact on proteoglycan production or MMP-13 production under noninflammatory (no IL-1) or inflammatory (with IL-1) conditions. Glucosamine at 0.1 and 0.5 μg/mL significantly decreased IL-1–stimulated production of mPGEs by chondrocytes, compared with that of positive control chondrocytes. Glucosamine at 0.1 and 5 μg/mL significantly decreased IL-1–stimulated production of mPGEs and PGE2, respectively, compared with that of positive control synoviocytes.
Conclusions and Clinical Relevance—Glucosamine had limited effects on chondrocyte and synoviocyte metabolism at clinically relevant concentrations, although it did have some anti-inflammatory activity on IL-1–stimulated articular cells. Glucosamine may have use at clinically relevant concentrations in the treatment of inflammatory joint disease.
Objective—To evaluate the effects of methylprednisolone acetate (MPA) on proteoglycan production by equine chondrocytes and to investigate whether glucosamine hydrochloride modulates these effects at clinically relevant concentrations.
Sample Population—Articular cartilage with normal gross appearance from metacarpophalangeal and metatarsophalangeal joints of 8 horses (1 to 10 years of age).
Procedures—In vitro chondrocyte pellets were pretreated with glucosamine (0, 1, 10, and 100 μg/mL) for 48 hours and exposed to MPA (0, 0.05, and 0.5 mg/mL) for 24 hours. Pellets and media were assayed for proteoglycan production (Alcian blue precipitation) and proteoglycan content (dimethylmethylene blue assay), and pellets were assayed for DNA content.
Results—Methylprednisolone decreased production of proteoglycan by equine chondrocytes at both concentrations studied. Glucosamine protected proteoglycan production at all 3 concentrations studied.
Conclusions and Clinical Relevance—Methylprednisolone, under noninflammatory conditions present in this study, decreased production of proteoglycan by equine chondrocytes. Glucosamine had a protective effect against inhibition of proteoglycan production at all 3 concentrations studied. This suggested that glucosamine may be useful as an adjunct treatment when an intra-articular injection of a corticosteroid is indicated and that it may be efficacious at concentrations relevant to clinical use.
Objective—To determine whether expansion of equine mesenchymal stem cells (MSCs) by use of fibroblast growth factor-2 (FGF-2) prior to supplementation with dexamethasone during the chondrogenic pellet culture phase would increase chondrocytic matrix markers without stimulating a hypertrophic chondrocytic phenotype.
Sample Population—MSCs obtained from 5 young horses.
Procedures—First-passage equine monolayer MSCs were supplemented with medium containing FGF-2 (0 or 100 ng/mL). Confluent MSCs were transferred to pellet cultures and maintained in chondrogenic medium containing 0 or 10−7M dexamethasone. Pellets were collected after 1, 7, and 14 days and analyzed for collagen type II protein content; total glycosaminoglycan content; total DNA content; alkaline phosphatase (ALP) activity; and mRNA of aggrecan, collagen type II, ALP, and elongation factor-1α.
Results—Treatment with FGF-2, dexamethasone, or both increased pellet collagen type II content, total glycosaminoglycan content, and mRNA expression of aggrecan. The DNA content of the MSC control pellets decreased over time. Treatment with FGF-2, dexamethasone, or both prevented the loss in pellet DNA content over time. Pellet ALP activity and mRNA were increased in MSCs treated with dexamethasone and FGF-2–dexamethasone. After pellet protein data were standardized on the basis of DNA content, only ALP activity of MSCs treated with FGF-2–dexamethasone remained significantly increased.
Conclusions and Clinical Relevance—Dexamethasone and FGF-2 enhanced chondrogenic differentiation of MSCs, primarily through an increase in MSC numbers. Treatment with dexamethasone stimulated ALP activity and ALP mRNA, consistent with the progression of cartilage toward bone. This may be important for MSC-based repair of articular cartilage.
Objective—To identify changes over time in relative expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in synovial fluid from healthy calves and calves with experimentally induced septic arthritis.
Animals—12 Holstein calves.
Procedures—In 7 calves, Escherichia coli was injected in the right tarsal joint on day 1. Joint lavage was performed on day 2, and calves were treated with ceftiofur from days 2 through 21. Synovial fluid samples were collected on days 1 (before inoculation), 2 (before joint lavage), 3, 4, 8, 12, 16, 20, and 24. In the remaining 5 calves, joint lavage was performed on day 2 and synovial fluid samples were collected from the left tarsal joint. Relative expression of MMP-2 and MMP-9 was determined by means of gel zymography.
Results—On day 1, MMP-2 was detected in all synovial fluid samples but MMP-9 was not detected. In calves with septic arthritis, values for relative expression of MMP-9 monomer and dimer were significantly increased on days 2 through 20 and days 2 through 24, respectively, and relative expression of MMP-2 was significantly increased on days 3 through 20. There were significant linear associations between relative expression of the monomer and dimer forms of MMP-9 and between neutrophil count and relative expression of the MMP-9 monomer and dimer forms.
Conclusions and Clinical Relevance—Results indicated that relative expression of MMP-9 and MMP-2 increased in synovial fluid from calves with experimentally induced septic arthritis, with relative expression remaining high for several days after infection.
Objective—To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants.
Sample Population—96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses.
Procedures—Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones.
Results—LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments.
Conclusions and Clinical Relevance—Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.
Objective—To determine whether plasma concentrations of bone turnover markers in growing Hanoverian foals are influenced by age, housing conditions, or osteochondrosis.
Animals—165 healthy foals and 119 foals with osteochondrosis.
Procedures—Foals were allocated according to birth date and housing management into groups of early-born (born before March 31, 2001; n = 154 foals, 88 of which were healthy and 66 of which had osteochondrosis) and late-born (born after March 31, 2001; 130 foals, 77 of which were healthy and 53 of which had osteochondrosis) foals. Plasma osteocalcin and carboxyterminal propeptide of type I collagen concentrations were analyzed as markers of bone formation, and carboxyterminal telopeptide of type I collagen concentration was analyzed as a marker of bone resorption. Foals underwent radiographic evaluation to screen for osteochondrosis.
Results—Plasma concentrations of osteocalcin, carboxyterminal propeptide of type I collagen, and carboxyterminal telopeptide of type I collagen decreased with age, but these changes were more distinct in late-born foals than in early-born foals. Neither sex nor predisposition to develop osteochondrosis affected the pattern of bone marker changes in either group.
Conclusions and Clinical Relevance—An age-related decrease in concentrations of bone markers was seen during the first 200 days of life. Changes in bone marker concentrations were similar for foals with osteochondrosis and healthy foals. The correlation between the decrease in bone marker concentration and date of birth indicates that there are differences in skeletal development between early- and late-born foals.
Objective—To determine the effect of 2 hydroxyapatite pin coatings on heat generated at the bone-pin interface and torque required for insertion of transfixation pins into cadaveric equine third metacarpal bone.
Sample Population—Third metacarpal bone pairs from 27 cadavers of adult horses.
Procedures—Peak temperature of the bone at the cis-cortex and the hardware and pin at the trans-cortex was measured during insertion of a plasma-sprayed hydroxyapatite (PSHA)—coated, biomimetic hydroxyapatite (BMHA)—coated, or uncoated large animal transfixation pin. End-insertional torque was measured for each pin. The bone-pin interface was examined grossly and histologically for damage to the bone and coating.
Results—The BMHA-coated transfixation pins had similar insertion characteristics to uncoated pins. The PSHA-coated pins had greater mean peak bone temperature at the cis-cortex and greater peak temperature at the trans-cortex (70.9 ± 6.4°C) than the uncoated pins (38.7 ± 8.4°C). The PSHA-coated pins required more insertional torque (10,380 ± 5,387.8 Nmm) than the BMHA-coated pins (5,123.3 ± 2,296.9 Nmm). Four of the PSHA-coated pins became immovable after full insertion, and 1 gross fracture occurred during insertion of this type of pin.
Conclusions and Clinical Relevance—The PSHA coating was not feasible for use without modification of presently available pin hardware. The BMHA-coated pins performed similarly to uncoated pins. Further testing is required in an in vivo model to determine the extent of osteointegration associated with the BMHA-coated pins in equine bone.
Objective—To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system.
Animals—6 healthy horses, 6 months to 5 years of age.
Procedures—Bone marrow–derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm2 and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye–binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs.
Results—Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification.
Conclusions and Clinical Relevance—FGF-2 treatment of bone marrow–derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair.