Cell growth characteristics and differentiation frequency of adherent equine bone marrow-derived mesenchymal stromal cells: adipogenic and osteogenic capacity

OBJECTIVES: To characterize equine bone marrow (BM)-derived mesenchymal stem cell (MSC) growth characteristics and frequency as well as their adipogenic and osteogenic differentiation potential. STUDY DESIGN: In vitro experimental study. ANIMALS: Foals (n=3, age range, 17-51 days) and young horses (n=5, age range, 9 months to 5 years). METHODS: Equine MSCs were harvested and isolated from sternal BM aspirates and grown up to passage 10 to determine cell-doubling (CD) characteristics. Limit dilution assays were performed on primary and passaged MSCs to determine the frequency of colony-forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). RESULTS: Initial MSC isolates had a lag phase with a significantly longer CD time (DT=4.9+/-1.6 days) compared with the average DT (1.4+/-0.22 days) of subsequent MSC passages. Approximately 1 in 4224+/-3265 of the total nucleated BM cells displayed fibroblast colony-forming activity. Primary MSCs differentiated in response to adipogenic and osteogenic inductive conditions and maintained their differentiation potential during subsequent passages. CONCLUSIONS: The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of foals and young adult horses are similar to those documented for BM MSCs of other mammalian species. CLINICAL RELEVANCE: The results have direct relevance to the use of BM as a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.     

Subconjunctival bone marrow‐derived mesenchymal stem cell therapy as a novel treatment alternative for equine immune‐mediated keratitis: A case series

Equine immune‐mediated keratitis (IMMK) leads to increased corneal opacity and inflammation secondary to an alteration of the local immune system. Bone marrow‐derived mesenchymal stem cells (BM‐MSC) have been shown to modulate the immune system by downregulating inflammation. Four horses with unilateral IMMK poorly responsive to traditional medical treatments underwent novel, autologous subconjunctival BM‐MSC therapy. Bone marrow was harvested and processed as previously described for equine orthopedic disease. Horses received autologous subconjunctival BM‐MSC injections approximately every 3‐4 weeks for 1‐5 treatments total. Horses were maintained on their current medical treatment regimen throughout the BM‐MSC treatment period. Three horses had a positive response to therapy as demonstrated by an increase in corneal clarity, a decrease in neovascularization and a reduction in surface irregularity. One horse was nonresponsive to therapy. These experimental results demonstrate the safety and potential efficacy of an innovative solution for IMMK.     

Isolation of equine bone marrow‐derived mesenchymal stem cells: a comparison between three protocols

Reason for performing the study: There is a need to assess and standardise equine bone marrow (BM) mesenchymal stem cell (MSC) isolation protocols in order to permit valid comparisons between therapeutic trials at different sites. Objective: To compare 3 protocols of equine BM MSC isolation: adherence to a plastic culture dish (Classic) and 2 gradient density separation protocols (Percoll and Ficoll). Materials and methods: BM aspirates were harvested from the sternum of 6 mares and MSCs isolated by all 3 protocols. The cell viability after isolation, MSC yield, number of MSCs attained after 14 days of culture and the functional characteristics (self‐renewal (CFU) and multilineage differentiation capacity) were determined for all 3 protocols. Results: The mean ± s.d. MSC yield from the Percoll protocol was significantly higher (6.8 ± 3.8%) than the Classic protocol (1.3 ± 0.7%). The numbers of MSCs recovered after 14 days culture per 10 ml BM sample were 24.0 ± 12.1, 14.6 ± 9.5 and 4.1 ± 2.5 × 10 ⁶ for the Percoll, Ficoll and Classic protocols, respectively, significantly higher for the Percoll compared with the Classic protocol. Importantly, no significant difference in cell viability or in osteogenic or chondrogenic differentiation was identified between the protocols. At Passage 0, cells retrieved with the Ficoll protocol had lower self‐renewal capacity when compared with the Classic protocol but there was no significant difference between protocols at Passage 1. There were no significant differences between the 3 protocols for the global frequencies of CFUs at Passage 0 or 1. Conclusions and clinical relevance: These data suggest that the Percoll gradient density separation protocol was the best in terms of MSC yield and self‐renewal potential of the MSCs retrieved and that MSCs retrieved with the Ficoll protocol had the lowest self‐renewal but only at passage 0. Then, the 3 protocols were equivalent. However, the Percoll protocol should be considered for equine MSC isolation to minimise culture time.     

Congenital Malformations of the First Sternal Rib

During the dissection and skeletal examination of 151 horses, a congenital malformation (CM) of the first sternal rib that influenced the aperture of the Thoracic inlet was noted in six horses. The presentation of this CM was variable between horses in gross anatomic appearance; notably, an absent first sternal rib, bifid tuberculum costae, bifid sternochondral articulation onto the sternum, flared shaft, normal first sternal rib inserting onto the cranial branch of a bifid sternochondral articulating second sternal rib, straight costal shaft, and an articulating rudimentary tuberculum costae with a ligamentous extension replacing the bony shaft and attaching to a rudimentary sternochondral articulation onto the sternum. Of the 151 horses examined, the CM of the first sternal rib was restricted to 6/60 Thoroughbred horses, and only in those that were affected by either the unilateral or bilateral transposition of the caudal ventral tubercle from C6 onto the ventral surface of C7. The normal anatomic presentation of the thoracic inlet was altered, along with associative musculature including neurological pathways. These CMs are likely to produce clinical and functional ramifications of the thoracic inlet, thoracic limb, and thoracic viscera, with the probability of altering postural and locomotive function as noted in four horses demonstrating the CM.     

Assessment of mild hindlimb lameness during over ground locomotion using linear discriminant analysis of inertial sensor data

REASONS FOR PERFORMING STUDY: Hindlimb lameness is common and can be difficult to diagnose or quantify in evaluating response to nerve blocks. An objective measure of lameness can also be used to evaluate the effectiveness of the treatment's contribution to evidence-based medicine. The inertial sensor system can be used to capture 6 degree of freedom movement during over ground locomotion and here was used to quantify tuber coxae movement in nonlame and lame horses. HYPOTHESIS: Tuber coxae movement is useful for discriminating between nonlame and lame horses. OBJECTIVES: To measure left and right tuber coxae movement in lame and nonlame horses during over ground locomotion and to implement a linear discriminant analysis to discriminate between lame and nonlame horses. METHODS: Two inertial sensors were attached to the skin over left and right tuber coxae of 21 horses (9 mildly and 12 not lame). Horses were trotted on a hard surface. A total of 1021 strides were collected. For each stride 34 features were extracted from the dorsoventral and craniocaudal movement and used in 2 different classification scenarios (lame vs. nonlame or left lame, right lame and nonlame) using linear discriminant analysis. RESULTS: Six degree of freedom inertial sensors were successfully used to collect kinematic data continuously from left and right tuber coxae in horses during over ground locomotion. These data were used for an automated classification of lameness. In the first scenario, a sensitivity of 89% was achieved with a specificity of 75%. In the second scenario, all horses could be correctly assigned to the correct class in a simple 3 class reclassification test. POTENTIAL RELEVANCE: A mobile system that reliably detects and quantifies hindlimb lameness in horses during unconstrained locomotion could be a valuable tool to perform an evidence-based assessment of lameness in horses in a clinical setting, e.g. before and after nerve blocks or before and after surgery.     

Collection of bone grafts from the tuber coxae of the horse.

Autogenous bone grafts were obtained from the tuber coxae of 9 horses. The method used involved an oblique incision to expose the lateral aspect of the tuber coxae. The periosteum was incised and reflected in order to make a 5- by 2.5-cm opening in the lateral cortex for graft retrieval. The method provided good visualization, ample grafting material, and freedom from postsurgical complications.     

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.     

Bone marrow response to large volume blood collection in the horse

Evaluation of erythropoietic regeneration in horses is difficult unless serial bone marrow aspirates are performed. To investigate the acute and chronic erythropoietic regenerative response of equine bone marrow following acute removal or loss of blood, sequential bone marrow aspirates over 4 weeks were taken from the sternum of five horses from which 20 ml kg(-1)of blood had been removed. We found that the total number of erythroid cells counted (expressed as a percentage of the total number of erythroid and myeloid cells counted) expanded initially by 13.7 per cent within 3 days after blood removal, the erythroid response peaking by 9 days with a further 13.5 per cent increase. This peak coincided with the lowest M:E ratio. Concomitantly, a shift from proliferative phase cells to maturing phase cells occurred, which appeared to persist beyond 31 days post collection. Thus, we found that the equine bone marrow mounted a regenerative erythropoietic response more slowly than previously determined and, also, regeneration of the erythroid compartment was incomplete 31 days after blood removal of this magnitude.     

Basic three-dimensional kinematics of the vertebral column of horses walking on a treadmill

OBJECTIVE: To determine kinematic movements of the vertebral column of horses during normal locomotion. ANIMALS: 5 Dutch Warmblood horses without apparent lameness or problems associated with the vertebral column. PROCEDURE: Kinematics of 8 vertebrae (T6, T10, T13, T17, L1, L3, L5, and S3) and both tuber coxae were determined, using bone-fixated markers. Horses were recorded while walking on a treadmill at a constant speed of 1.6 m/s. RESULTS: Flexion-extension was characterized by 2 periods of extension and flexion during 1 stride cycle, whereas lateral bending and axial rotation were characterized by 1 peak and 1 trough. The range of motion for flexion-extension was fairly constant for vertebrae caudal to T10 (approximately 7 degrees). For lateral bending, the cranial thoracic vertebrae and segments in the pelvic region had the maximal amount of motion, with values of up to 5.6 degrees. For vertebrae between T17 and L5, the amount of lateral bending decreased to <4 degrees The amount of axial rotation increased gradually from 4 degrees for T6 to 13 degrees for the tuber coxae. CONCLUSIONS: This direct measurement method provides 3-dimensional kinematic data for flexion-extension, lateral bending, and axial rotation of the thoracolumbar portion of the vertebral column of horses walking on a treadmill. Regional differences were observed in the magnitude and pattern of the rotations. Understanding of the normal kinematics of the vertebral column in healthy horses is a prerequisite for a better understanding of abnormal function.     

In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors

OBJECTIVE: To compare the osteogenic potential of cancellous bone of conventional graft sites with that of one nonconventional site (fourth coccygeal vertebra) and to investigate the tibial periosteum as a donor site with respect to osteogenic potential. STUDY DESIGN: In vitro osteogenic cell culture system. SAMPLE POPULATION: Eight adult horses. METHODS: Cancellous bone or tibial periosteum was aseptically collected and cut into bone chips or periosteal strips of 1 to 2 mm(3) for primary explant cultures. After 2 weeks, primary tissue cultures that yielded a population of osteogenic cells were counted and subcultured at 1 x 10(5) cells/35-mm dish in osteogenic media. After 7 to 10 days, subcultures were stained with Von Kossa (VK) to assess mineralized bone nodule formation. VK-positive bone nodules were counted as osteoprogenitors and compared among 3 donor sites, which provided consistent primary osteogenic cells (tuber coxae, fourth coccygeal vertebra, periosteum) using ANOVA (P <.05). RESULTS: Sternal and tibial bone yielded viable osteogenic cells from 25% and 50% of horses, respectively, whereas yields from tuber coxae, coccygeal vertebra, and periosteum were 75%, 100%, and 100%, respectively. Tuber coxae and periosteum had significantly greater numbers of osteoprogenitors compared with fourth coccygeal vertebra. CONCLUSIONS: Among the conventional donor sites, tuber coxae most consistently yielded viable osteogenic cells with an acceptable percentage of osteoprogenitors. Sternal and tibial sites were unreliable in providing osteogenic cells. Two new donor sites, the fourth coccygeal vertebra and tibial periosteum, were tissues with good osteogenic potential. CLINICAL RELEVANCE: When a source of transplantable viable osteoprogenitor cells is desired, use of the tuber coxae as a conventional donor site is warranted. Use of tibial periosteum or fourth coccygeal vertebra as reliable sources of transplantable osteoprogenitors should be considered.     

Use of high-speed cinematography and computer generated gait diagrams for the study of equine hindlimb kinematics

High-speed cinematography with computer aided analysis was used to study equine hindlimb kinematics. Eight horses were filmed at the trot or the pace. Filming was done from the side (lateral) and the back (caudal). Parameters measured from the lateral filming included the heights of the tuber coxae and tailhead, protraction and retraction of the hoof and angular changes of the tarsus and stifle. Abduction and adduction of the limb and tarsal height changes were measured from the caudal filming. The maximum and minimum values plus the standard deviations and coefficients of variations are presented in tabular form. Three gait diagrams were constructed to represent stifle angle versus tarsal angle, metatarsophalangeal height versus protraction-retraction (fetlock height diagram) and tuber coxae and tailhead height versus stride (pelvic height diagram). Application of the technique to the group of horses revealed good repeatability of the gait diagrams within a limb and the diagrams appeared to be sensitive indicators of left/right asymmetries.     

Effect of transforming growth factor beta1 on chondrogenic differentiation of cultured equine mesenchymal stem cells

OBJECTIVE: To determine the morphologic and phenotypic effects of transforming growth factor beta1 (TGFbeta1) on cultured equine mesenchymal stem cells (MSC) and articular chondrocytes. SAMPLE POPULATION: Bone marrow aspirates and articular cartilage samples from a 2-year-old and two 8-month-old horses. PROCEDURE: After initial isolation and culture, MSC and chondrocytes were cultured in Ham's F-12 medium supplemented with TGF-beta1 at a concentration of 0, 1, 5, or 10 ng/ml. Medium was exchanged on day 2, and cells were harvested on day 4. Medium was assayed for proteoglycan (PG) content. Total RNA was isolated from cell cultures, and expression of aggrecan, decrin, collagen type-I, and collagen type-II mRNA was assessed by means of Northern blot analyses. Cell cultures were stained with H&E or toluidine blue and examined histologically. Additional cultures were examined after immunohistochemical staining for type-I and -II collagen. RESULTS: MSC cultures exposed to TGF-beta1 had an increased cellular density with cell layering and nodule formation that was most pronounced in cultures treated with 5 ng of TGF-beta1/ml. Expression of collagen type-II mRNA in MSC cultures exposed to 5 ng of TGF-beta1/ml was 1.7 times expression in control cultures, and expression of collagen type-I mRNA was 2.8 times expression in control cultures. Treatment of MSC with TGF-beta1 led to dose-related increases in area and intensity of type-II collagen immunoreaction. CONCLUSION: Results suggest that TGF-beta1 enhances chondrogenic differentiation of bone marrow-derived MSC in a dose-dependent manner.     

Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.     

Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells

Reasons for performing study: Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse. Objectives: To describe an optimised safe technique for obtaining bone marrow‐derived MSCs from the sternum of the Thoroughbred horse. Methods: The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony‐forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation. Results: The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony‐forming units of mesenchymal stem cells with trilineage capabilities. Conclusions: Accurate placement of a Jamshidi needle into the medullary cavity of the 4th–6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse. Potential clinical relevance: Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell‐based therapies.     

Use of a centrifugation-based, point-of-care device for production of canine autologous bone marrow and platelet concentrates

OBJECTIVE: To analyze a centrifugation-based, point-of-care device that concentrates canine platelets and bone marrow-derived cells. ANIMALS: 19 adult sexually intact dogs. PROCEDURES: Anticoagulated peripheral blood (60 mL) and 60 mL of anticoagulated bone marrow aspirate (BMA) were concentrated by centrifugation with the centrifugation-based, point-of-care device to form a platelet and a bone marrow concentrate (BMC) from 11 dogs. Blood samples were analyzed on the basis of hemograms, platelet count, and PCV. The BMA and BMC were analyzed to determine PCV, total nucleated cell count, RBC count, and differential cell counts. The BMC stromal cells were cultured in an osteoinductive medium. Eight additional dogs were used to compare the BMC yield with that in which heparin was infused into the bone marrow before aspiration. RESULTS: The centrifugation-based, point-of-care device concentrated platelets by 6-fold over baseline (median recovery, 63.1%) with a median of 1,336 x 10(3) platelets/microL in the 7-mL concentrate. The nucleated cells in BMCs increased 7-fold (median recovery, 42.9%) with a median of 720 x 10(3) cells/microL in the 4-mL concentrate. The myeloid nucleated cells and mononuclear cells increased significantly in BMCs with a significant decrease in PCV, compared with that of BMAs. Stromal cell cultures expressed an osteoblastic phenotype in culture. Infusion of heparin into the bone marrow eliminated clot formation and created less variation in the yield (median recovery, 61.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Bone marrow-derived cell and platelet-rich concentrates may form bone if delivered in an engineered graft, thus decreasing the need for cancellous bone grafts.     

Comparison of Chondrogenic Potential in Equine Mesenchymal Stromal Cells Derived from Adipose Tissue and Bone Marrow

Objective— To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs). Study Design— In vitro experimental study. Animals— Adult Thoroughbred horses (n=11). Methods— BM (5 horses; mean [±SD] age, 4±1.4 years) or adipose tissue (6 horses; mean age, 3.5±1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis±transforming growth factor‐3 (TGFβ₃) and bone morphogenic factor‐6 (BMP‐6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross‐sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays. Results— Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline‐like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion— Equine MSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species. Clinical Relevance— Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.     

Scintigraphic abnormalities of the pelvic region in horses examined because of lameness or poor performance: 128 cases (1993-2000)

OBJECTIVE: To identify scintigraphic abnormalities in the pelvic region of horses examined because of hind limb lameness or poor performance and determine the clinical relevance of areas of abnormal radiopharmaceutical uptake (ARU) in these horses. DESIGN: Retrospective study. ANIMALS: 128 horses. PROCEDURE: Medical records were reviewed, and information on signalment, history, admitting complaints, physical examination findings, and results of lameness examinations was recorded. Clinical relevance of areas of ARU was determined by comparison with results of other diagnostic tests. For horses with clinically relevant areas of ARU, follow-up information was obtained through telephone interviews with owners and trainers and analysis of race records. RESULTS: Areas of ARU were identified in the tuber coxae (25 horses), ischiatic tuber (9), hip joint (10), third trochanter (10), ilium (5), sacral tuber region (22), greater trochanter (1), cranial femoral cortex (1), skeletal muscle surrounding the pelvis (34), or multiple areas (11). In 44 horses, areas of ARU were associated with the primary cause of lameness; in 51, areas of ARU were not associated with the primary cause of lameness; and in 33, the primary cause of lameness was not determined. Thirty-six of the 44 horses with clinically relevant areas of ARU were available for follow-up; 15 (42%) had a good outcome. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that pelvic scintigraphy may be useful in identifying abnormalities in horses with hind limb lameness or poor performance.     

Assessment of a bone biopsy technique for measuring tiludronate in horses: a preliminary study

This study assessed the feasibility of measuring tiludronate in horses using a minimally invasive bone biopsy technique. Eight horses were treated with intravenous (IV) tiludronate [1 mg/kg bodyweight (BW)], either once (n = 4) or twice, 28 d apart (n = 4). The horses that were treated once were euthanized on days 1, 43, 57, or 92 and those that were treated twice, were euthanized on days 112, 154, 194, or 364. Bone samples were taken bilaterally from each horse at 4 sites: the third metacarpal bone (MCIII), the 13th rib (R13), the tuber coxae (TC), and the cuboid bone (CB). Test samples were taken with a 5-mm diameter dental drill, while larger reference samples were taken with an osteotome. The concentrations of tiludronate were measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The TC was the easiest site to sample, and no technical difficulties were encountered for extraction and measurement. Drill sampling at the MCIII was difficult. Moreover, both the extraction and measurement caused technical problems and results were unreliable in most cases (93%). Drill samples obtained from the R13 were very small and access to the CB required considerable dissection, which would not be feasible in vivo. Forty-six percent and 36% of the tiludronate measurements performed on the R13 and CB samples, respectively, were unreliable. The ratio of tiludronate concentrations ranged from 73% to 185% (median: 118%) in the TC, 65% to 208% (median: 81%) in the R13, and 26% to 110% (median: 57%) in the CB. In all but 1 horse, the highest concentrations of tiludronate were found in the TC. It was concluded that bone biopsies performed at the TC were adequate for measuring tiludronate in horses and should be considered in future for repeated measurements over time in living animals.