Evaluation of serum concentrations of biochemical markers of bone metabolism and insulin-like growth factor I associated with treadmill exercise in young horses

OBJECTIVE: To evaluate changes in serum concentrations of biochemical markers of bone metabolism and insulin-like growth factor I (IGF-I) associated with treadmill exercise in young horses. ANIMALS: 12 two-year-old Thoroughbred mares. PROCEDURE: During a 20-week study period, 6 horses were exercised on a treadmill 3 times a week (exercise group) and 6 horses received walking exercise 6 days a week (controls). Serum concentrations or activity of biochemical markers and IGF-I were assessed biweekly. Bone mineral density and content of the first phalanx were measured by dual-energy X-ray absorbiometry (DEXA) on completion of the study. RESULTS: Compared with values in controls, bone mineral density and content were higher and serum concentrations of osteocalcin (a marker of bone formation) and the carboxy-terminal telopeptide of type I collagen (a marker of bone resorption; ICTP) were lower in exercised horses. Serum concentration and activity of the bone formation markers carboxy-terminal propeptide of type I collagen and bone-specific alkaline phosphatase (BAP) were not different between the 2 groups. Serum IGF-I concentration was lower in the exercise group, compared with control values; there was a significant correlation between change in IGF-I values and changes in osteocalcin, ICTP, and BAP values at the end of the study. CONCLUSIONS AND CLINICAL RELEVANCE: Treadmill exercise over 20 weeks induced adaptive changes in bones of 2-year-old Thoroughbreds; training appears to increase bone mineral density, thereby enhancing mechanical strength of bone, but decreases bone turnover. Results indicated an association between changes in serum IGF-I concentration and bone cell activity in horses.     

Evaluation of serum osteocalcin and CTX-I in Ardenner horses with special reference to juvenile interphalangeal joint disease

The first aim of this study was to establish a profile of age-related normal serum concentrations of osteocalcin (OC) in Ardenner horses. For this first part, blood samples from 49 healthy Ardenner horses were collected. The second aim was to study two biochemical markers of bone metabolism, OC and a carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I), in 30 young Ardenner horses during 1 year. Amongst them, 17 showed lesions of juvenile degenerative joint disease in the distal forelimbs. A specific radioimmunoassay for equine OC was used to measure the serum concentration of the marker. The serum concentration of CTX-I was measured using a commercially available human assay validated for use in the horse. The effect of age, sex, season and health status (with or without lesions) was assessed. Levels of OC fall between birth and the adult stage: this decrease being most marked between birth and 1 year of age. This age-related decrease of OC was confirmed in the 30 young Ardenner horses, but CTX-I levels remained constant in this group. The Levels of the two markers changed significantly with the season with higher concentrations during the winter. No significant difference was shown either between the two sexes or between the two health statuses.     

Biochemical markers of bone metabolism in growing thoroughbreds: a longitudinal study

This study describes longitudinal changes in serum levels of biochemical markers of bone cell activity in a group of 24 thoroughbred foals from birth to 18 months of age. The markers of bone formation included the type I collagen carboxy-terminal propeptide (PICP), the bone-specific isoenzyme of alkaline phosphatase (BAP), and osteocalcin (OC). Levels of the cross-linked telopeptide of type I collagen (ICTP), a marker of bone resorption, and the N-terminal propeptide of type III collagen (PNIIIP), a marker of soft tissue turnover, were also measured. Levels of all markers fell significantly between birth and 18 months of age (70-80 per cent); this decrease being most marked between 0 and 6 months. However, a transient increase in levels of the markers then occurred between 6 and 14 months of age. The timing of this increase was specific for each parameter. ICTP and OC concentrations increased between October and December. PICP concentrations increased between December and April whereas the increase in PIIINP was coincident with the peak in weight gain between April and June. Changes in BAP concentration were less distinct at this time. Season was shown to have significant effects on the biochemical markers independent from the effect of age. Concentrations of all markers decreased with increasing body weight and at any given age heavier horses had lower marker levels. These results show that biochemical markers of bone cell activity and soft tissue turnover follow characteristic patterns of change in growing thoroughbreds influenced by age, season and bodyweight. The demonstration that the reference ranges for the biochemical markers change from month to month means that single samples from individuals are of little value for monitoring bone cell activity in growing thoroughbreds.     

Effect of adipose-derived nucleated cell fractions on tendon repair in horses with collagenase-induced tendinitis

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.     

Biochemical markers of bone metabolism in draught andwarmblood horses

Concentrations of the cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and osteocalcin (OC) have been determined in the serum of one hundred clinically healthy adult Draught or Warmblood horses. The correlation between these two markers has been evaluated and the influence of gender, age and type of horse described. No significant variations were observed between animals of different sex, but a significant inverse correlation (P<0.0001) with age was observed for both measured parameters. After correction for age, serum levels of OC were found to be lower in Draught [adjusted least square mean (LSM)=6.612μg. L⁻¹] than in Warmblood horses (adjusted LSM=8.596μg.L⁻¹), whereas levels of ICTP were higher in Draughts (adjusted LSM=8.035pg.L⁻¹) than in Warmbloods (adjusted LSM=6.643μg.L⁻¹). A significant correlation (P<0.0001) was observed between OC and ICTP. This correlation was stronger if the type of horse was taken into account in the statistical model. The ratio OC:ICTP was independent of gender and age. A higher OC:ICTP ratio in Warmbloods compared to the Draught horses might reflect a higher bone remodelling level of horses submitted to regular daily work. It was concluded that ICTP and OC are influenced by the type of horse, and probably reflect a physiological difference in bone remodelling between these animals.     

Circadian variation in biochemical markers of bone cell activity and insulin-like growth factor-I in two-year-old horses

Studies in humans have found circadian changes to be one of the most important sources of controllable preanalytical variability when evaluating bone cell activity using biochemical markers. It remains unclear whether similar circadian changes influence bone marker concentrations in the horse. The aim of this study was to characterize changes in serum concentrations of three biochemical markers of bone cell activity over a 24-h period in six 2-yr-old Thoroughbred mares, and to determine circadian variability in IGF-I, which regulates bone turnover. Three bone markers were measured in serum: osteocalcin, a marker of bone formation, the carboxy-terminal propeptide of type-I collagen (a marker of bone formation), and the carboxy-terminal telopeptide of type-I collagen (a marker of bone resorption). Data were analyzed using the cosinor technique, which fits a 24-h cycle to each dataset. A significant circadian rhythm was observed for osteocalcin (P = 0.028), with an estimated amplitude of 7.6% of the mean (95% confidence interval 1.3% to 16.3%), and an estimated peak time of 0900. However, the observed rhythm for the carboxy-terminal telopeptide of type-I collagen (amplitude = 7.4%) was not significant (P = 0.067), and there were no significant changes in concentrations of the carboxy-terminal propeptide of type-I collagen over the 24-h study period (P = 0.44). There was a small but significant circadian rhythm for IGF-I (P = 0.04), with an estimated amplitude of 3.4% (95% confidence interval 0.2 to 7.1%) and peak at 1730. Further studies are now required to determine the potential association between circadian changes in IGF-I and osteocalcin in the horse. Although no significant circadian variation was found in concentrations of the car-boxy-terminal propeptide of type-I collagen and the carboxy-terminal telopeptide of type-I collagen, this may in part be a result of the age of the animals that were still skeletally immature. Future studies should aim to determine whether these markers develop a circadian rhythm at a later age when growth is complete. In the meantime, consistency in time of sampling should continue to be considered best practice when measuring biochemical markers of bone turnover in the horse.     

Relationship between stages of the estrous cycle and bone cell activity in Thoroughbreds

OBJECTIVE: To investigate the relationship between stage of estrous cycle and bone cell activity in Thoroughbreds. SAMPLE POPULATION: Blood samples collected from forty-seven 2-year-old Thoroughbred mares in training for racing. PROCEDURES: Blood samples were collected monthly (in April through September) from the mares. Stage of estrus was determined by assessing serum progesterone concentration. Bone cell activity was determined by measuring concentrations of 2 markers of bone formation (osteocalcin and the carboxy-terminal propeptide of type I collagen [PICP]) and a marker of bone resorption (the cross-linked carboxy-terminal telopeptide of type I collagen [ICTP]) in sera. RESULTS: When the relationship between stage of the estrous cycle and markers of bone cell activity was examined, serum concentrations of both osteocalcin and ICTP were significantly higher in mares that were in the luteal phase, compared with mares that were at other stages of the estrous cycle. Stage of estrus did not affect serum PICP concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that bone cell activity in Thoroughbred mares fluctuates during the estrous cycle; serum concentrations of markers of bone formation and bone resorption are increased during the luteal phase. Further studies are required to determine whether these changes are of clinical importance and increase the risk of injury for mares in training during the breeding season. As in humans, stage of estrus must be considered as a source of uncontrollable variability in serum bone marker concentrations in horses.     

Serum osteocalcin and CTX-MMP concentration in young exercising thoroughbred racehorses

Bone responds to exercise with changes in bone (re-)modelling, which might be monitored non-invasively with biochemical bone markers. The aim of this study was to evaluate the influence of exercise on serum osteocalcin and serum carboxy-terminal cross-linked telopeptide of type I collagen generated by matrix metalloproteinases (CTX-MMP) concentration in young racehorses. Seventy-one 2 to 4-year-old Thoroughbreds were included in this prospective infield study. Blood sampling was performed six times (i.e. six sampling cycles) during a 9-month period. Serum samples were analysed with commercial osteocalcin and CTX-MMP radioimmunoassays. Two-year-old racehorses had higher serum osteocalcin and CTX-MMP values than 3-year-old horses. Gender and training amplitude did not significantly influence serum osteocalcin and CTX-MMP values. Two-year-old horses showed an increase in osteocalcin values between cycles 2 and 3 and an increase in serum CTX-MMP values between cycles 1 and 2. Serum osteocalcin and CTX-MMP concentrations decreased between cycles 4 and 5, and 5 and 6. Three-year-old horses showed an increase in serum osteocalcin levels between cycles 3 and 4 and an increase in serum CTX-MMP concentrations between cycles 1 and 2, and 3 and 4. Serum osteocalcin levels decreased between cycles 5 and 6, whereas serum CTX-MMP levels decreased between cycles 4 and 5, and 5 and 6. Two- and three-year-old horses showed a decreased osteocalcin/CTX-MMP ratio between cycles 1 and 2. Moreover, 2-year-old horses showed an increase in the osteocalcin/CTX-MMP ratio between cycles 2 and 3. Sore shin formation did not significantly influence serum osteocalcin and CTX-MMP values. Serum osteocalcin and CTX-MMP are promising bone markers for monitoring exercise induced changes in equine bone metabolism.     

Use of biochemical markers of bone metabolism in veterinary medicine

Effective, non-invasive bone assessment methods for screening, diagnosis and follow-up of the skeleton are more and more requested in veterinary medicine. In contrast to clinical parameters, invasive methods and imaging techniques, indices of bone turnover is a tool for bone metabolism evaluation of the whole skeleton. Biochemical bone markers therefore provide a more real-time assessment of the bone status with simple blood- or urine-analysis. This article surveys currently available biochemical marker of bone metabolism used in veterinary medicine. Additionally, information is provided about physiological and pathological, as well as therapeutic variations of biochemical bone marker concentrations in various species.     

A Marine Mineral Supplement Alters Markers of Bone Metabolism in Yearling Arabians

This study tested whether the supplement (Aquacid), high in calcium and other minerals, can alter markers of bone metabolism and mineralization of the equine third metacarpus bone. Radiographs were taken of the left third metacarpus of 14 yearlings. Radiographic bone aluminum equivalence (RBAE) of each cortex was calculated to estimate mineral content. Blood samples were also taken at this time. Horses were ranked according to RBAE and gender, were pair-matched, and randomly assigned to two treatment groups. Each group was provided one of two mineral supplements in addition to their normal diet. The treated group (Aq) received 75 g Aquacid/horse/d, which provided an additional 15 g of calcium. The control group (Co) received 39.5 g of limestone to provide similar amounts of calcium. The study lasted for 112 days, with blood being taken every 28 days. At day 56 and 112, additional radiographs was taken to track changes in RBAE. Blood was analyzed for osteocalcin (a bone formation marker) and serum C-telopeptide crosslaps of type I collagen (a bone resorption marker) to detect alterations in bone metabolism. Using day 0 values as a covariate for bone markers, there was a trend (P = .07) for osteocalcin concentrations to be greater in Aq horses than in Co. Likewise, C-telopeptide crosslaps of type I collagen concentrations were greater (P < .0001) in Aq horses than in Co. There were minimal differences in RBAE values. These findings suggest Aquacid, while not altering bone mass, increases bone turnover and may aid in repairing damaged bone and preventing injuries.     

The Use of Pneumatic Impact Lithotripsy in a Laparoscopic Retrieval Pouch for Removal of Large Cystoliths in Two Female Horses

Two mares, aged 15 and 21 years, were examined because of urinary incontinence, intermittent hematuria, and urine scalding. On admission of both horses, physical parameters were within normal limits and urine scalding of the skin at the ventral perineum was noted. Transrectal palpation and cystoscopy revealed a large type I cystolith (>10 cm) with associated hyperemia and focal ulceration of the bladder mucosa. In horse 1, hemogram, serum biochemical analysis, and renal ultrasound were not performed because of owner finances. In horse 2, results from hematological and serum biochemical analysis were unremarkable and renal ultrasonography did not reveal any abnormalities. Pneumatic impact lithotripsy in a laparoscopic retrieval pouch was performed under cystoscopic guidance after caudal epidural anesthesia, with the horses standing and under sedation. A laparoscopic retrieval device was passed alongside a flexible endoscope into the urinary bladder and the cystolith was manipulated into the pouch. A customized single stainless-steel rod scaler attached to an air compressor was used for fragmentation of the cystolith contained within the retrieval pouch. Lithotripsy time was 42 minutes for horse 1 and 31 minutes for horse 2. Both horses were released from hospital the day of surgery. Both horses were continent and voided normal streams of urine for the duration of the follow-up periods of 27 and 19 months for horse 1 and horse 2, respectively. Pneumatic impact lithotripsy in a laparoscopic retrieval pouch provided a time-efficient and minimally invasive surgical treatment option for removal of large cystoliths in mares.     

Response of serum biochemical markers of bone metabolism totraining in the juvenile racehorse

Sixteen Quarter Horse-type geldings were used to examine the response of biochemical markers of bone metabolism to forced exercise prior to and during race training. The study began when the average age of the horses was 15 months. Horses were exercised on a high-speed treadmill for 14 weeks, and were subsequently placed into race training. Serum was collected and assayed for concentrations of osteocalcin (BGP), the carboxyterminal telopeptide of type I collagen (ICTP) and the carboxyterminal propeptide of type I procollagen (PICP). When data were normalized from the onset of race training, ICTP and PICP concentrations were higher in the pre-exercised horses (P<.05 and P<.1, respectively) indicating higher rates of bone turnover. Overall, bone turnover appeared to be decreased during race training, as concentrations of PICP and ICTP were lower when compared to values seen during the pre-training Phase.     

Biochemical Markers of Bone Modeling and Remodeling in Juvenile Racehorses at Varying Mineral Intakes

In this study, blood-borne biochemical markers were used to track comparative rates of bone turnover in horses fed differing amounts of Ca, P and Mg. Bone turnover was tracked by serum osteocalcin; bone resorption by the carboxyterminal telopeptide of type I collagen (ICTP); and bone formation by the carboxyterminal propeptide of type I procollagen (PICP). Twenty-one longyearling Quarter Horses were blocked by gender and age, randomly assigned to one of four diets and subjected to 128 d of race training. The study was conducted in 32-d periods, each consisting of 28 d of race training followed by a 4-d fecal and urine collection, or a 4-d rest period. Blood samples were taken weekly during the training period. Serum and plasma samples were analyzed for biochemical markers of bone activity using RIA procedures. Onset of training resulted in elevated blood concentrations of ICTP, PICP and osteocalcin. Concentrations of ICTP and PICP were greater during the first 64 d of training, indicating increased bone activity during the first half of the training period. Horses with the greatest intake of minerals exhibited greater concentrations of PICP (bone formation marker) and lesser concentrations of ICTP (bone breakdown marker). Further, ICTP, PICP and osteocalcin concentrations decreased dramatically following 4-d of confinement and relative inactivity. Therefore it appears that feeding minerals at levels greater than current NRC recommendations provided a protective effect on the developing skeleton of the young racehorse. Additionally, the biochemical markers used in this study were sensitive enough to track daily changes in bone activity resulting from daily changes in stress to the skeleton.     

Biochemical markers of bone metabolism and risk of dorsal metacarpal disease in 2-year-old Thoroughbreds

REASONS FOR PERFORMING STUDY: Dorsal metacarpal disease (DMD) is a common problem in 2-year-old racehorses and results in loss of a significant number of days from training. Biochemical markers of bone cell activity measured early in the training season could have value for identifying 2-year-old Thoroughbred racehorses that develop DMD. OBJECTIVES: To determine the association between serum concentrations of osteocalcin, the carboxyterminal propeptide of type I collagen (PICP) and the carboxyterminal cross-linked telopeptide of type I collagen (ICTP) measured early in the training season and the risk of DMD. METHODS: Blood samples were collected from 165 two-year-old Thoroughbreds during late November/early December. Osteocalcin and PICP were measured as markers of bone formation, and ICTP as a marker of bone resorption. Training and veterinary records for each horse were monitored over the following training/racing season (10 months). Cases were defined as an episode where signs of DMD were sufficiently severe for a horse to miss at least 5 consecutive days of training. Classification tree and logistic regression analysis were used to identify the most important factors suitable for prediction of DMD risk. RESULTS: There were 24 cases of DMD during the season (14.6% cumulative incidence), with an average time to recognition of approximately 6 months (May). The earliest recognised case was in February and the latest in September. Osteocalcin and ICTP concentrations in the early stages of the training season were significantly higher in horses that subsequently developed DMD (P = 0.017 and 0.019, respectively). DMD cases were also significantly older compared to noncases (21.04 vs. 20.44 months, P = 0.023). Using a multivariable logistic regression model, it was possible to postulate a set of diagnostic rules to predict the likelihood of DMD injury during the season. This suggested that horses with ICTP concentrations above 12365 ug/l and older than 20.5 months are 2.6 times more likely to develop DMD. CONCLUSIONS: The measurement of the bone resorption marker ICTP could be useful for identification of 2-year-olds at increased risk of developing DMD. POTENTIAL RELEVANCE: These findings, together with other strategies such as modification of training regimens, e.g. early introduction of short distances of high-speed exercise into the training programme, could help reduce the days lost to training as a result of DMD.     

Long-term effects of intermittent equine parathyroid hormone fragment (ePTH-1-37) administration on bone metabolism in healthy horses

Intermittent administration of parathyroid hormone (PTH) is an anabolic therapy for osteoporotic conditions in humans. This study evaluated the effects of equine PTH fragment (ePTH-1-37) administration on bone metabolism in 12 healthy horses. Six horses each were treated once daily for 120days with subcutaneous injections of 0.5μg/kg ePTH-1-37 or placebo. Blood was collected to determine ionized calcium (Ca(++)), total Ca (Ca(T)), inorganic phosphorus, serum equine osteocalcin (eOC), carboxy-terminal telopeptide of type I collagen (ICTP), bone-specific alkaline phosphatase, and carboxy-terminal cross-linked telopeptide of type I collagen. Bone mineral density (BMD) was determined with dual X-ray absorptiometry of the metacarpus and calcaneus. Significantly higher blood Ca(++) and plasma Ca(T) concentrations were measured 5h after ePTH-1-37 administration compared to placebo. Higher serum eOC concentrations were found for ePTH-1-37 treatment at days 90 (P<0.05) and 120 (P=0.05). Significantly higher serum ICTP levels were observed with ePTH-1-37 treatment at days 60 and 90. For both study groups, BMD increased significantly in the calcaneus. Long-term use of ePTH-1-37 seemed to have no negative effects on bone metabolism in healthy horses. The absence of undesirable side effects is the premise to ensure safety for further clinical investigations in horses with increased bone resorption processes.     

Effects of continuous oral administration of phenylbutazone on biomarkers of cartilage and bone metabolism in horses

OBJECTIVE: To evaluate the effects of continuous oral administration of phenylbutazone on serum and synovial fluid biomarkers of skeletal matrix metabolism in horses. ANIMALS: 11 adult female horses without clinical or radiographic evidence of joint disease. PROCEDURES: Horses were randomly assigned to control or treatment groups. Phenylbutazone was administered orally twice daily at a dose of 4.4 mg/kg for 3 days to the treatment group and subsequently at a dose of 2.2 mg/kg for 7 days. Serum and radiocarpal synovial fluid samples were obtained at baseline and thereafter at regular intervals for 4 weeks. Biomarkers of cartilage aggrecan synthesis (chondroitin sulfate 846) and type II collagen synthesis (procollagen type II C-propeptide) and degradation (collagen type II cleavage) were assayed. Biomarkers of bone synthesis (osteocalcin) and resorption (C-terminal telopeptide of type I collagen) were also measured. RESULTS: No significant differences were found between control and treatment groups or temporally for the biomarkers chondroitin sulfate 846, procollagen type II C-propeptide, collagen type II cleavage, and C-terminal telopeptide of type I collagen in serum or synovial fluid. A significant increase in osteocalcin concentration occurred in synovial fluid during treatment in the treated group. No treatment effect was detected for serum osteocalcin concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that continuous phenylbutazone administration at recommended doses altered some biomarkers in healthy equine joints after short periods of administration. Increased osteocalcin concentration may indicate an undetermined anabolic effect of phenylbutazone administration on periarticular bone or transient induction of osteogenesis in articular chondrocytes or a mesenchymal subpopulation of synoviocytes.     

Use of bone markers in veterinary medicine

Bone metabolism can be monitored in humans and several animal species in vivo by measuring enzymes and other protein products released by osteoblasts and osteoclasts, respectively. The biochemical markers of bone formation currently in use include bone isoenzyme of alkaline phosphatase, osteocalcin and propeptides derived from the N or C terminal ends of the typ I procollagen molecule. The most useful markers of bone resorption are breakdown products of type I collagen. The oldest established method is the measurement of hydroxyproline in urine, which is not specific for bone, because it can be found in all collagen types and is also derived from diets. The measurement of collagen crosslinks, deoxypyridinoline and pyridinoline, is comparatively more specific to monitor bone resorption. Deoxypyridinoline and pyridinoline are used in human medicine for diagnosis and evaluation of bone diseases and in predicting the occurrence of fractures and rates of bone loss. The carboxyterminal telopeptide of type I collagen, which has been used in several animal species, is also a promising bone marker. This article reviews the use of different bone markers in veterinary medicine and the possibilities for diagnosing and preventing bone diseases.     

Arthroscopic Subchondral Bone Plate Microfracture Technique Augments Healing of Large Chondral Defects in the Radial Carpal Bone and Medial Femoral Condyle of Horses

OBJECTIVE: To evaluate the effect of arthroscopic subchondral bone microfracture on healing of large chondral defects in horses. STUDY DESIGN: Short- (4 months) and long-term (12 months) in vivo experimental chondral defect model. ANIMALS: 10 horses, aged 2 to 5 years. METHODS: Each horse had a 1 cm2 full-thickness chondral defect created in both radial carpal bones and both medial femoral condyles. One carpus and one femoral condyle of each horse had the subchondral bone plate under the defect perforated using an orthopedic awl. All horses were exercised, five horses were evaluated after 4 months and five horses after 12 months. Gross, histologic, and histomorphometric examination of defect sites and repair tissues was performed, as was collagen typing of the repair tissue. RESULTS: On gross observation a greater volume of repair tissue filled treated defects (74%) compared with control defects (45%). Histomorphometry confirmed more repair tissue filling treated defects, but no difference in the relative amounts of different tissue types was observed. There was an increased percentage of type II collagen in treated defects compared with control defects and evidence of earlier bone remodeling as documented by changes in porosity. CONCLUSIONS: In full-thickness chondral defects in exercised horses, treatment with subchondral bone microfracture increased the tissue volume in the defects and the percentage of type II collagen in the tissue filling the defects when compared to nontreated defects. CLINICAL RELEVANCE: No negative effects of the microfracture technique were observed and some of the beneficial effects are the basis for recommending its use in patients cases with exposed subchondral bone.     

The influence of sex on biochemical markers of bone turnover in dogs

Biochemical markers of bone turnover have been shown to be useful as inexpensive and noninvasive tools for monitoring skeletal health. The reference range for bone markers in dogs has been set by different age groups. However, other sources of biological variations were not fully investigated in dogs. To explore whether sex influences the interpretation of bone marker data we examined serum bone markers in 33 male and 25 female dogs. The bone markers selected for this study were: bone alkaline phosphatase (BALP) and osteocalcin (OC) as indicators of bone formation, and C-terminal telopeptide (CTx) of type I collagen as marker of bone resorption. All concentrations of bone markers were lower, but still within the reference range reported for dogs. We found statistically significant differences of the median OC and CTx serum concentrations between males and females. The results of this study suggest that there are sex differences in biochemical markers of bone turnover in dogs which should be considered in interpretation of bone marker data.     

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.