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- Author or Editor: Alan J. Nixon x
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Summary
Medical records from 119 horses that had undergone arthroscopic surgery for removal of axial osteochondral fragments of the palmar/plantar proximal aspect of the proximal phalanx were reviewed. Standardbred racehorses represented 109 (92%) of those affected. Ninety-three (78%) of the horses were < 3 years old. Gender distribution was consistent with that of the equine hospital population. Fragments most commonly were observed in the hind limbs (155/164; 95%), specifically, on the medial aspect of the left hind limb (72/164; 44%). Bilateral fragmentation occurred in 21 of 119 (18%) horses, and 15 of 119 (13%) horses had fragments in the medial and lateral aspect within the same joint. Fifteen (13%) horses had a concurrent osteochondritis dissecans lesion on the distal intermediate ridge of the tibia, and 30 of 119 (25%) had radiographic signs of osteoarthritis involving the centrodistal (distal intertarsal) and tarsometatarsal articulations.
In 55 of 87 (63%) racehorses and in 100% of the 9 nonracehorses, performance returned to preoperative levels after surgery. Fragment numbers or distribution, concurrent osteochondritis dissecans lesions of the distal intermediate ridge of the tibia, or tarsal osteoarthritis were not significantly associated with outcome. Abnormal surgical findings, consisting of articular cartilage fibrillation or synovial proliferation, were significantly (P < 0.0001) associated with adverse outcome; these findings were documented in 31% of the 32 horses without successful outcome and in only 2% of the 55 horses with successful outcomes.
Abstract
Objective—To assess the effects of supraphysiologic concentrations of insulin-like growth factor-I (IGF-1) on morphologic and phenotypic responses of chondrocytes.
Sample Population—Articular cartilage obtained from 2 young horses.
Procedure—Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content was determined by bisbenzimide-fluorometric assay, and elution of IGF-1 into medium was determined by IGF-1 radioimmunoassay.
Results—Both 12.5 and 25 µg of IGF-1/ml enhanced phenotypic expression of chondrocytes without inducing detrimental cellular or metabolic effects. Highest concentration of IGF-1 (25 µg/ml) significantly increased total DNA content, glycosaminoglycan (GAG) content, GAG synthesis, and size of proteoglycan monomers produced, compared with cultures supplemented with 12.5 µg of IGF-1/ml or untreated cultures. Histologic examination confirmed these biochemical effects. Matrix metachromasia, type-II collagen in situ hybridization and immunoreaction were increased in cultures treated with 25 µg of IGF-1/ml, compared with cultures supplemented with 12.5 µg of IGF-1/ml or untreated cultures.
Conclusions and Clinical Relevance—Chondrocytes exposed to high concentrations of IGF-1 maintained differentiated chondrocyte morphology and had enhanced synthesis of matrix molecules without inducing apparent detrimental effects on chondrocyte metabolism. These results suggest that application of such composites for in vivo use during cartilage grafting procedures should provide an anabolic effect on the grafted cells. (Am J Vet Res 2002;63:301–305)
Abstract
Objective—To clarify the diagnosis of avulsion of the lateral palmar intercarpal ligament (LPICL), correlate avulsion of this ligament with lameness, determine the prevalence of avulsion of the LPICL in a hospital population, and evaluate the response to surgical removal of the avulsion fragment in horses.
Design—Retrospective study.
Animals—37 horses with avulsion of the LPICL.
Procedure—Medical records and radiographs of horses with avulsion of the LPICL were reviewed; follow-up information was obtained from race records and from owners via a telephone survey.
Results–Of 6,418 horses evaluated for forelimb lameness from March 1, 1990, to December 31, 2001, 37 (0.5%) had avulsion of the LPICL; each horse had a discrete fragment associated with avulsion of the ligament origin from the ulnar carpal bone. Twenty-six horses underwent arthroscopic fragment removal; 20 of 22 (91%) horses for which follow-up information was available returned to work. Of 9 horses treated conservatively, 5 returned to work. Odds ratio calculations indicated that horses treated surgically were 8 times as likely to return to work than those not treated surgically. Twelve horses had LPICL avulsion without concurrent osteochondral fragmentation in the same or additional joints; follow-up information was available for 9 of those horses, of which 8 returned to athletic work.
Conclusions and Clinical Relevance—In horses, discrete avulsion of the LPICL can be a cause of lameness and arthroscopic debridement may be the treatment of choice. Prognosis for return to work of horses with avulsion of the LPICL is good. (J Am Vet Med Assoc 2005;226:760–766)
Abstract
Objective—To evaluate the effects of recombinant human platelet–derived growth factor-BB (rhPDGF-BB) on the metabolic function and morphologic features of equine superficial digital flexor tendon (SDFT) in explant culture.
Animals—6 euthanized horses (2 to 5 years old).
Methods—Forelimb SDFT explants were cultured for 6 days as untreated control specimens or treated with rhPDGF-BB (1, 10, 50, or 100 ng/mL of medium). Treatment effects on explant gene expression were evaluated via real-time PCR analysis of collagen type I, collagen type III, PDGF-A, and PDGF-B mRNA. Explants were assayed for total collagen, glycosaminoglycan, and DNA content; histologic changes were assessed via H&E staining and immunohistochemical localization of collagen types I and III.
Results—No morphologic or proliferative changes were detected in tendon explant sections. After high-dose rhPDGF-BB treatment, gene expression of collagen types I and III was increased and decreased, respectively. Expression of PDGF-A and PDGF-B mRNA was significantly increased at 24 hours, but later decreased to have few or negative autoinductive effects. Although PDGF gene expression waned after 48 hours of culture, collagen type I gene expression was significantly increased at 48 hours and reached peak value on day 6. Glycosaminoglycan and DNA content of explants were unchanged with rhPDGF-BB treatment.
Conclusions and Clinical Relevance—Results suggest that rhPDGF-BB use may be of benefit in the repair of equine tendon, particularly through induction of collagen type I mRNA. Positive autoinductive effects of PDGF-BB in equine SDFT explants were detected early following culture medium supplementation, but these diminished with time.
Summary
Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 × 106 cells/g of cartilage (wet weight), compared with a yield of 17.83 × 106 cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham’s F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, α-ketoglutarate, and l-glutamine. The medium was buffered with hepes, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at −196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.
Abstract
Objective
To isolate, clone, and determine primary nucleotide sequence of equine insulin-like growth factor I (IGF-I) and to examine IGF-I gene expression in tissues and cartilage from horses.
Animals
Horses of various ages.
Methods
Total RNA was isolated from tissues and purified. Complementary DNA (cDNA) was derived by reverse transcription and polymerase chain reaction (PCR) amplification and subcloned to plasmid vectors for sequencing and comparison with other species. Total RNA from various tissues was probed with radiolabeled cDNA or complimentary RNA constructs by use of northern blotting, tube hybridization, or PCR procedures to determine IGF-I expression patterns.
Results
Nucleotide sequence of equine IGF-I was 90% homologous to that of cows, 88% homologous to that of humans and sheep, and 77% homologous to that of rats. Amino acid sequence was identical to that of humans, cows, dogs, and pigs. A larger PCR product (IGF-IB) was consistent with alternate splicing with retention of IGF-I exon 4 sequence, similar to rats and mice. Northern blot analysis revealed multiple IGF-I transcripts; predominant sizes were 1.6 and 4.5 kb. The IGF-I message was commonly detected in liver, kidney, and cartilage from young foals and was diminished in cartilage from a 12-month-old horse.
Conclusions
Nucleotide sequences of equine pre-propeptides were different from those of other species, but the sequence coding the mature IGF-I peptide was more closely homologous. The larger IGF-IB form differed substantially in the carboxy-terminal. The biological action of the cleaved terminal was speculated to be autocrine feedback. Expression of IGF-I was apparent in many tissues, including cartilage, and was greater in immature horses. (Am J Vet Res 1999;60:1234–1241)
Abstract
Objective
To compare chondrocyte proliferation and metabolism in three-dimensional fibrin cultures formed from polymerized autogenous fibrinogen with that of commercially manufactured fractionated fibrinogen.
Animals
Fibrinogen and chondrocytes for in vitro experimentation derived from 2 horses, ages 12 and 14 months, donated for reasons unrelated to skeletal or hematologic abnormalities.
Procedure
Fibrinogen was isolated from whole blood, using plasma cryoprecipitation and centrifugation, and fractionated fibrinogen was purchased. Each was mixed with 10 × 106 chondrocytes/0.5 ml of fibrinogen, and was polymerized by addition of 0.5 ml of calcium-activated thrombin. Thirty 1-ml fibrin-chondrocyte disks were formed from each fibrinogen source and cultured for 0 (n = 6), 7 (n = 12), or 14 (n = 12) days. Chondrocyte metabolism and cell proliferation in each fibrin type were objectively assessed by assays for total proteoglycan content, [35S]proteoglycan accumulation, proteoglycan monomer size, and total DNA. Cell morphology and cartilage-specific cell function was evaluated by routine histologic, alcian blue histochemical, type-II collagen immunohistochemical, and type-II collagen in situ hybridization methods.
Results
Histologic examination indicated better retention of chondrocyte morphology in autogenous composites. Autogenous fibrinogen also stimulated greater chondrocyte proliferation (DNA content increased 1.4-fold on day 14) and supported higher proteoglycan accumulation (increased 1.4-fold on day 14), compared with commercial, fractionated fibrinogen. Abundant intracellular type-II procollagen mRNA was detected in autogenous fibrin cultures by in situ hybridization, and translation was confirmed by extensive pericellular type-II collagen accumulation.
Conclusions
Autogenous fibrinogen has an inherent capacity to maintain chondrocyte phenotypic metabolism that is reduced or absent in commercially prepared fibrinogen. Enhanced, differentiated cell function may be useful for in vivo applications, but represents an added variable that may confound in vitro experiments, and should be considered when designing studies of chondrocyte function. (Am J Vet Res 1998;59:514–520)
Summary
Postoperative abdominal fluid changes were compared in 2 groups of horses; those undergoing double small-colon resection and anastomosis (n = 10) and those undergoing exploratory celiotomy alone (n = 5). Peritoneal fluid was collected before surgery and on postoperative days 1, 3, 5, and 7. Total and differential nucleated cell counts, rbc numbers, and total protein and fibrinogen concentrations were evaluated. In both groups, all values were significantly higher than normal on the first postoperative day (after small-colon resection and anastomoses, wbc = 130,350 ± 23,310 cells/µl, rbc = 7,389,000 ± 6,234,000 cells/µl, total protein = 3.63 ± 0.16 g/dl; after exploratory celiotomy alone, wbc = 166,620 ± 34,340 cells/µl, rbc = 295,000 ± 86,070 cells/µl, total protein 4.38 ± 0.54 g/dl). The number of total peritoneal nucleated cells and rbc significantly decreased after the first postoperative day, whereas total protein and fibrinogen concentrations, percent neutrophils, and percent mononuclear cells remained unchanged. None of the values had returned to normal by postoperative day 7 (after small-colon resection and anastomoses, wbc = 45,600 ± 8,765 cells/µl, rbc= 95,390 ± 53,380 cells/µl, total protein = 4.39 ± 0.23 g/dl; after exploratory celiotomy alone, wbc= 43,340 ± 7,746 cells/µl, rbc = 12,860 ± 11,790 cells/µl, total protein = 3.92 ± 2.20 g/dl.) The resection and anastomosis group had a significantly lower total protein concentration on the first postoperative day and a significantly higher mean total rbc count over the entire 7-day postoperative evaluation than did horses that underwent celiotomy alone. Other values in the 2 groups of horses did not differ significantly. As a result, there was insufficient evidence to conclude that resection and anastomosis of the small colon in healthy horses causes a different inflammatory response than does manipulation of the intestine alone.
Abstract
Objective—To characterize the nucleotide sequence of equine platelet-derived growth factor (PDGF)-A and -B and analyze temporal expression of these genes in equine tendon after induced tendinitis injury.
Animals—18 mature horses.
Procedures—Genes for equine PDGF-A and -B were reverse transcribed and sequenced from synovial tissue mRNA obtained from a 3-year-old horse. Collagenase-induced lesions were created in the tensile region of the superficial digital flexor tendon in 14 horses; 3 horses served as uninjured control animals. Tendons were harvested and total RNA was isolated from experimental horses 1, 2, 4, 8, and 24 weeks after collagenase injection. Temporal gene expression for PDGF-A and -B was determined by use of quantitative PCR analysis.
Results—Equine PDGF-A shared 83.8% sequence and 87.5% peptide homology with human PDGF-A, with a discrepancy of 70 bp from the human sequence. Equine PDGF-B was similar in length to the human gene, sharing 90.3% and 91.7% nucleotide and peptide identity, respectively. Expression of PDGF-A mRNA in collagenase-induced tendinitis lesions was unchanged, compared with expression for normal control tendon, and remained steady throughout the 24-week study. Expression of PDGF-B mRNA decreased over time, and the expression at 24 weeks was significantly reduced, compared with expression in normal and acutely injured tendon.
Conclusions and Clinical Relevance—Injured tendon mounts a minimal constitutive PDGF-A or -B mRNA response. Serial exogenous treatment with either PDGF isoform within the first 2 to 4 weeks after tendon injury may bolster the meager PDGF paracrine-autocrine intrinsic response to injury.
Abstract
Case Description—3 horses were referred for treatment of subchondral cystic lesions of 1 or both medial femoral condyles.
Clinical Findings—All horses had clinically apparent lameness confirmed to be due to a radiographically evident subchondral cystic lesion of the medial femoral condyle with a large articular component (> 15 mm) and shallow subchondral depth (< 10 mm). Arthroscopic assessment of affected cartilage revealed undulating cartilage with a relatively smooth surface and extensive residual perimeter attachment.
Treatment and Outcome—Resorbable polydioxanone pins were used arthroscopically to reattach the cartilage overlying the subchondral cystic lesions. A biologic graft (bone marrow aspirate concentrate or allogeneic chondrocytes) was injected into the depths of the cystic cavity following cartilage reattachment. Follow-up examination confirmed radiographic resolution of the lesion and elimination of clinical signs within the treated femorotibial joint.
Clinical Relevance—Lesions with a large area of affected articular cartilage have been associated with a decreased rate of return to athletic function following arthroscopic debridement, likely secondary to the loss of subchondral architecture and the production of imperfect fibrocartilage repair. Salvage of the affected cartilage in a select population of horses with progressively expanding but shallow subchondral cystic lesions of the medial femoral condyle is possible and may improve radiographic and clinical outcome.
