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Abstract
Objective
To determine the effects of transforming growth factor-β1 (TGF-β1) on the synthesis of DNA, collagen, and proteoglycans (PG) by equine chondrocytes.
Sample Population
Articular cartilage obtained from multiple joints of a 4-month-old foal.
Procedure
Chondrocytes were isolated by collagenase digestion, cultured in monolayer, trypsinized, and implanted at a cellular density of 10 × 106 chondrocytes/ml in a three-dimensional fibrin matrix. Chondrocytes in culture were supplemented with TGF-β1 at concentrations of 0, 1, 5, or 10 ng/ml in serum-free medium or medium containing fetal bovine serum (FBS). Total PG accumulation, [35S]-labeled PG synthesis, PG monomer hydrodynamic size, type II collagen production, total DNA content, and [3H]thymidine incorporation into DNA were determined at 7 and 14 days of culture.
Results
Chondrocytes maintained a rounded phenotype, dedifferentiating slightly to a more fibroblastic appearance only in medium containing FBS and 10 ng of TGF-β1/ml. Type II collagen immunoreaction on day 14 was decreased in the pericellular matrix in cultures containing FBS and 1, 5, and 10 ng of TGF-β1/ml, and in all serum-free culture conditions compared to FBS and 0 ng of TGF-β1/ml. Total proteoglycan accumulation and [35S]-labeled proteoglycan synthesis in cultures on days 7 and 14 were increased by the addition of exogenous TGF-β1 in serum-free conditions and decreased by TGF-β1 in FBS-supplemented conditions. Calculation of the partition coefficients for PG indicated that there was synthesis of low molecular weight PG in serum-free conditions and larger sized proteoglycans in FBS-supplemented conditions. Proteoglycan molecular size was unchanged by the addition of TGF-β1. Total DNA content of chondrocytes increased with the addition of TGF-β1 in FBS-supplemented conditions and decreased in serum-free conditions.
Conclusions
In a solid three-dimensional fibrin matrix, the effects of TGF-β1 on chondrocyte biological activity depend on the culture duration and on the presence of FBS in the medium. Stimulatory effects of TGF-β1 were most pronounced in serum-free culture conditions with high concentration of TGF-β1 (5 and 10 ng/ml) on day 7 and with low concentration of TGF-β1 (1 ng/ml) on day 14.
Clinical Relevance
TGF-β1 may not be a suitable growth factor for enhancement of equine articular grafting in sites exposed to serum. (Am J Vet Res 1997;58:66–70)
Summary
Equine neonatal chondrocytes were cultured in three-dimensional fibrin matrices under conditions of immediate implantation or implantation following monolayer culture for 6 days, and 3 cell concentrations (1 × 105, 1 × 106, and 5 × 106 chondrocytes/ cm3). Equine fibrinogen was collected by cryoprecipitation and polymerized by use of activated bovine thrombin. The fibrin implants were harvested and analyzed histologically and biochemically at 3, 7, and 14 days after the chondrocytes were implanted in fibrin. The differentiation ratio (ratio of rounded, chondrocyte-like cells to stellate, fibroblast-like cells) was statistically higher for implants that received 5 × 106 precultured cells at all time periods than for implants that received 1 × 105 or 1 × 106 precultured cells. The differentiation ratio was statistically higher for implants that received 5 × 106 immediately implanted cells than for other implants at 7 days after implantation. At 14 days, implants that received 5 × 106 precultured chondrocytes had a higher differentiation ratio than did implants that received 5 × 106 chondrocytes that had not been precultured. Among implants that received precultured chondrocytes, total glycosaminoglycan and chondroitin sulfate content was lowest for implants that received only 1 × 105 cells. Among implants that received chondrocytes that had not been precultured, glycosaminoglycan content was not significantly different among the 3 cell concentrations, and chondroitin sulfate content was different only between implants that received 5 × 106 vs 1 × 106 cells. Only after the longest incubation period and at the highest cell concentration studied did preculturing of chondrocytes improve maintenance of phenotype. Preculturing did not appear to influence proteoglycan synthesis.
Abstract
Objective
To isolate mesenchymal stem cells from adult horses and determine specific monolayer culture conditions required to enhance biochemically and phenotypically defined chondrocytic differentiation.
Animals
2 adult horse bone marrow donors without skeletal or hematologic abnormalities.
Procedure
Bone marrow was aspirated from the sternebra, and mesenchymal stem cells were isolated by centrifugation and cultured in monolayers. Subcultures were established in 24-well plates on day 13. Culture medium was harvested every 2 days, and culture of 12 of the 24 wells was terminated on day 6 and of the remaining wells on day 12. Medium proteoglycan content was determined for all samples, and proteoglycan monomeric size was determined for pooled samples from days 2-6 and 8-12. Total nucleated cell numbers were determined at culture termination on days 6 and 12. Histologic, histochemical, and collagen immunohistochemical analyses of multiwell chamber slides harvested on day 6 or 12 were performed.
Results
Mesenchymal cells were an abundant cellular constituent of bone marrow aspirates, and separation of hematopoietic elements was achieved by centrifugation and delayed medium exchange. The remaining mesenchymal stem cells progressed from large, spindyloid, fibroblastic-appearing cells to a rounder shaped cell which formed colony plaques; isolated cells remained more spindyloid. Mesenchymal cell transformation toward a chondrocytic phenotype was verified by a shift in expression from collagen type I to type II, and an increase in quantity and molecular size of proteoglycans synthesized over time.
Conclusions
Mesenchymal stem cells obtained from adult horses have the capacity to undergo chondrogenic differentiation in monolayer cultures and may provide a locally recruitable or transplantable autogenous cell source for articular cartilage repair. (Am J Vet Res 1998;59:1182-1187).
Objective
To determine clinical and radiographic findings in and treatment and outcome of large animals with shoulder joint luxations.
Design
Retrospective study.
Animals
5 horses, 3 goats, 1 calf, 1 sheep, 1 Himalayan tahr, 1 pot-bellied pig, 1 reindeer, and 1 white-tailed deer.
Procedure
Medical records and radiographs were reviewed to determine signalment, history, physical examination findings, type of luxation, treatment, and outcome. Owners and referring veterinarians were contacted for follow-up information.
Results
Goats, sexually intact males, and animals < 1 year old were overrepresented, compared with the general hospital population during the study period. Closed reduction was attempted in 3 animals and was successful in 1. Open reduction and internal stabilization was attempted in 4 animals, including 1 in which closed reduction was unsuccessful. Long-term stabilization of the joint was achieved in 3 animals, but overall results were poor because of osteoarthritis and chronic lameness. Three animals were not treated, and 5 were euthanatized because of a poor prognosis.
Clinical Implications
Large animals with shoulder joint luxation and concurrent fractures had a poorer prognosis than did those with shoulder joint luxation alone. (J Am Vet Med Assoc 1998;213:1608–1611)
Abstract
Objective—To investigate the efficacy and safety of a low-volume, single-catheter, continuous peripheral neural blockade (CPNB) technique to locally deliver bupivacaine to alleviate signs of severe forelimb pain resulting from experimentally induced tendonitis in horses.
Design—Randomized controlled experimental trial.
Sample—14 horses and 5 forelimbs from equine cadavers.
Procedures—Horses underwent collagenase-induced superficial digital flexor tendonitis in the midmetacarpal region of 1 forelimb. To deliver analgesia, a closed-tip catheter was placed from lateral to medial, approximately 12 cm distal to the accessory carpal bone, between the suspensory ligament and accessory ligament of the deep digital flexor tendon. Success of catheter placement and anesthetic delivery was documented ex vivo in 5 forelimbs from equine cadavers. Effective analgesia in affected forelimbs of horses from continuous (n = 7) versus intermittent (7) local anesthetic delivery (intermittent peripheral neural blockade; IPNB) was compared over a 3-day period.
Results—Horses that received CPNB in the affected forelimb were less lame than horses that received IPNB. A lower proportion of CPNB-treated horses had behavioral and physiologic signs of pain, compared with IPNB-treated horses. Neither technique completely blocked the sensation of pain or resulted in swelling in the distal portion of the forelimb, vasodilation, or an increase in lameness. After removal, Staphylococcus aureus was cultured from 1 catheter tip.
Conclusions and Clinical Relevance—For short-term treatment, CPNB was more effective than IPNB for reduction in signs of severe pain in the distal aspect of the forelimb of horses.
Abstract
Objective—To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis.
Animals—8 horses.
Procedures—Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed.
Results—Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons.
Conclusions and Clinical Relevance—ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.
SUMMARY
Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals. This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 × 106 viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 C and 5% CO2. Histologic and histo-chemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22.
Glycosaminoglycan (GAG) assay by dimethyl-methylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P ≤ 0.0001) increase in total GAG content was observed by day 3 (0.329 M-μg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 ± 0.062 μg/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0354 ± 0.037 μg/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 ± 0.027 μg/mg on day 10. Fluctuations in K5 content were not significant until an increase on day 22.
Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.
SUMMARY
Sodium hyaluronate reduces adhesions after tendon repair in rodents and dogs, and has been used in limited clinical trials in people. To evaluate its effect on tendon healing and adhesion formation in horses and to compare these effects with those of a compound of similar viscoelastic properties, a study was performed in horses, using a model of collagenase injection in the flexor tendons within the digital sheath.
Eight clinically normal horses were randomly allotted to 2 groups. Adhesion formation between the deep digital flexor tendon and the tendon sheath at the pastern region was induced in the forelimbs of all horses. Using tenoscopic control, a 20-gauge needle was inserted into the deep digital flexor tendon of horses under general anesthesia and 0.2 ml of collagenase (2.5 mg/ml) was injected. The procedure was repeated proximally at 2 other sites, spaced 1.5 cm apart. A biopsy forceps was introduced, and a 5-mm tendon defect was created at each injection site. Group-A horses had 120 mg of sodium hyaluronate (NaHA) gel injected into the tendon sheath of one limb. Group-B horses had methylcellulose gel injected at the same sites. The contralateral limbs of horses in both groups served as surgical, but noninjected, controls. Horses were euthanatized after 8 weeks of stall rest.
Ultrasonographic evaluation revealed improved tendon healing after NaHa injection, but no difference in peritendinous adhesion formation. Tendon sheath fluid volume and hyaluronic acid (ha) content were greater in NaHA-treated limbs. Gross pathologic examination revealed considerably fewer and smaller adhesions when limbs were treated with NaHA. However, significant difference in pull-out strengths was not evident between NaHA-treated and control limbs. Histologically, the deep digital flexor tendon from the NaHA-treated limbs had reduced inflammatory cell infiltration, improved tendon structure, and less intratendinous hemorrhage. Treatmerit with methylcullulose had no significant effect on tendon healing, adhesion size, quantity, or strength or on the volume and composition of the tendon sheath fluid. Sodium hyaluronate, administered intrathecally, appears to have a pharmaceutically beneficial action in this collagenase-induced tendinitis and adhesion model in horses.
