Soluble factors released by ATDC5 cells affect the formation of calcium phosphate crystals.

During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures ( approximately 40 microm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.

(Over)training effects on quantitative electromyography and muscle enzyme activities in standardbred horses.

Too intensive training may lead to overreaching or overtraining. To study whether quantitative needle electromyography (QEMG) is more sensitive to detect training (mal)adaptation than muscle enzyme activities, 12 standardbred geldings trained for 32 wk in age-, breed-, and sex-matched fixed pairs. After a habituation and normal training (NT) phase (phases 1 and 2, 4 and 18 wk, respectively), with increasing intensity and duration and frequency of training sessions, an intensified training (IT) group (phase 3, 6 wk) and a control group (which continued training as in the last week of phase 2) were formed. Thereafter, all horses entered a reduced training phase (phase 4, 4 wk). One hour before a standardized exercise test (SET; treadmill), QEMG analysis and biochemical enzyme activity were performed in muscle or in biopsies from vastus lateralis and pectoralis descendens muscle in order to identify causes of changes in exercise performance and eventual (mal)adaptation in skeletal muscle. NT resulted in a significant adaptation of QEMG parameters, whereas in muscle biopsies hexokinase activity was significantly decreased. Compared with NT controls, IT induced a stronger adaptation (e.g., higher amplitude, shorter duration, and fewer turns) in QEMG variables resembling potentially synchronization of individual motor unit fiber action potentials. Despite a 19% decrease in performance of the SET after IT, enzyme activities of 3-hydroxyacyl dehydrogenase and citrate synthase displayed similar increases in control and IT animals. We conclude that 1) QEMG analysis is a more sensitive tool to monitor training adaptation than muscle enzyme activities but does not discriminate between overreaching and normal training adaptations at this training level and 2) the decreased performance as noted in this study after IT originates most likely from a central (brain) rather than peripheral level.

Iron ions derived from the nitric oxide donor sodium nitroprusside inhibit mineralization.

Sodium nitroprusside (SNP) is a nitric oxide (NO) donor drug, which is therapeutically used as a vasodilating drug in heart transplantations. In our previous study it was found that SNP at a concentration of 100 microM inhibited mineralization in a cell culture system, indicating that the beneficial effects of this drug may also include inhibition of vascular calcification. The aim of this study was to investigate which bioactive compounds generated from SNP inhibit mineralization. ATDC5 cells were grown for 14 days and mineralization was induced by addition of 5 mM phosphate for 24 h. Mineralization was determined by staining precipitated calcium with an alizarin red stain. It was found that the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine were not able to inhibit mineralization and NO scavengers could not antagonize the inhibiting effect of SNP on mineralization. The iron chelator deferoxamine (200 microM) antagonized the inhibiting effect on mineralization mediated by SNP and ammonium iron sulfate inhibited mineralization in a dose-dependent manner (10-100 microM). Furthermore, iron ions (30 microM) were detected to be released from SNP in the cell culture. These data show that the iron moiety of sodium nitroprusside, rather than nitric oxide inhibits mineralization.

Effect of early training on the jumping technique of horses.

OBJECTIVE: To investigate the effects of early training for jumping by comparing the jumping technique of horses that had received early training with that of horses raised conventionally. ANIMALS: 40 Dutch Warmblood horses. PROCEDURE: The horses were analyzed kinematically during free jumping at 6 months of age. Subsequently, they were allocated into a control group that was raised conventionally and an experimental group that received 30 months of early training starting at 6 months of age. At 4 years of age, after a period of rest in pasture and a short period of training with a rider, both groups were analyzed kinematically during free jumping. Subsequently, both groups started a 1-year intensive training for jumping, and at 5 years of age, they were again analyzed kinematically during free jumping. In addition, the horses competed in a puissance competition to test maximal performance. RESULTS: Whereas there were no differences in jumping technique between experimental and control horses at 6 months of age, at 4 years, the experimental horses jumped in a more effective manner than the control horses; they raised their center of gravity less yet cleared more fences successfully than the control horses. However, at 5 years of age, these differences were not detected. Furthermore, the experimental horses did not perform better than the control horses in the puissance competition. CONCLUSIONS AND CLINICAL RELEVANCE: Specific training for jumping of horses at an early age is unnecessary because the effects on jumping technique and jumping capacity are not permanent.

Influence of age, site, and degenerative state on the speed of sound in equine articular cartilage.

OBJECTIVE: To determine the speed of sound (SOS) in equine articular cartilage and investigate the influence of age, site in the joint, and cartilage degeneration on the SOS. SAMPLE POPULATION: Cartilage samples from 38 metacarpophalangeal joints of 38 horses (age range, 5 months to 22 years). PROCEDURE: Osteochondral plugs were collected from 2 articular sites of the proximal phalanx after the degenerative state was characterized by use of the cartilage degeneration index (CDI) technique. The SOS was calculated (ratio of needle-probe cartilage thickness to time of flight of the ultrasound pulse), and relationships between SOS value and age, site, and cartilage degeneration were evaluated. An analytical model of cartilage indentation was used to evaluate the effect of variation in true SOS on the determination of cartilage thickness and dynamic modulus with the ultrasound indentation technique. RESULTS: The mean SOS for all samples was 1,696 +/- 126 m/s. Age, site, and cartilage degeneration had no significant influence on the SOS in cartilage. The analytical model revealed that use of the mean SOS of 1,696 m/s was associated with maximum errors of 17.5% on cartilage thickness and 70% on dynamic modulus in an SOS range that covered 95% of the individual measurements. CONCLUSIONS AND CLINICAL RELEVANCE: In equine articular cartilage, use of mean SOS of 1,696 m/s in ultrasound indentation measurements introduces some inaccuracy on cartilage thickness determinations, but the dynamic modulus of cartilage can be estimated with acceptable accuracy in horses regardless of age, site in the joint, or stage of cartilage degeneration.

Variation in free jumping technique within and among horses with little experience in show jumping.

OBJECTIVE: To quantify variation in the jumping technique within and among young horses with little jumping experience, establish relationships between kinetic and kinematic variables, and identify a limited set of variables characteristic for detecting differences in jumping performance among horses. ANIMALS: Fifteen 4-year-old Dutch Warmblood horses. PROCEDURE: The horses were raised under standardized conditions and trained in accordance with a fixed protocol for a short period. Subsequently, horses were analyzed kinematically during free jumping over a fence with a height of 1.05 m. RESULTS: Within-horse variation in all variables that quantified jumping technique was smaller than variation among horses. However, some horses had less variation than others. Height of the center of gravity (CG) at the apex of the jump ranged from 1.80 to 2.01 m among horses; this variation could be explained by the variation in vertical velocity of the CG at takeoff (r, 0.78). Horses that had higher vertical velocity at takeoff left the ground and landed again farther from the fence, had shorter push-off phases for the forelimbs and hind limbs, and generated greater vertical acceleration of the CG primarily during the hind limb push-off. However, all horses cleared the fence successfully, independent of jumping technique. CONCLUSIONS AND CLINICAL RELEVANCE: Each horse had its own jumping technique. Differences among techniques were characterized by variations in the vertical velocity of the CG at takeoff. It must be determined whether jumping performance later in life can be predicted from observing free jumps of young horses.

Evaluation of consistency of jumping technique in horses between the ages of 6 months and 4 years.

OBJECTIVE: To determine whether differences in jumping technique among horses are consistent at various ages. ANIMALS: 12 Dutch Warmblood horses. PROCEDURE: Kinematics were recorded during free jumps of horses when they were 6 months old (ie, no jumping experience) and 4 years old (ie, the horses had started their training period to become show jumpers). Mean +/- SD height of the horses was 1.40 +/- 0.04 m at 6 months of age and 1.70 +/- 0.05 m at 4 years of age. RESULTS: Strong correlations were found between values from 6-month-old foals and 4-year-old horses for variables such as peak vertical acceleration generated by the hind limbs (r, 0.91), peak rate of change of effective energy generated by the hind limbs (r, 0.71), vertical velocity at takeoff (r, 0.65), vertical displacement of the center of gravity during the airborne phase (r, 0.81), and duration of the airborne phase (r, 0.70). CONCLUSIONS AND CLINICAL RELEVANCE: Although there are substantial anatomic and behavioral changes during the growing period, certain characteristics of jumping technique observed in naïve 4-year-olds are already detectable when those horses are foals.

Computerised analysis of standardised ultrasonographic images to monitor the repair of surgically created core lesions in equine superficial digital flexor tendons following treatment with intratendinous platelet rich plasma or placebo.

The effectiveness of new therapies to treat tendon injuries is difficult to determine and is often based on semi-quantitative methods, such as grey level analysis of ultrasonographic images or subjective pain scores. The alternatives are costly and long-lasting end-stage studies using experimental animals. In this study, a method of ultrasonographic tissue characterisation (UTC), using mathematical analysis of contiguous transverse ultrasonographic images, was used for intra-vital monitoring of the healing trajectory of standardised tendon lesions treated with platelet rich plasma (PRP) or placebo. Using UTC it was possible to detect significant differences between the groups in the various phases of repair. At end stage, over 80% of pixels showed correct alignment in the PRP group, compared with just over 60% in the placebo group (P<0.05). UTC also showed significant differences in the course of the healing process between PRP treated and placebo treated animals throughout the experiment. It was concluded that computerised analysis of ultrasonographic images is an excellent tool for objective longitudinal monitoring of the effects of treatments for superficial digital flexor tendon lesions in horses.

Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo-controlled experimental study.

Tendon injuries are notorious for their slow and functionally inferior healing. Intratendinous application of platelet-rich plasma (PRP) has been reported to stimulate the repair process of tendon injuries, but there is little conclusive evidence for its effectiveness. A placebo-controlled experimental trial was performed to test the hypothesis that a single intratendinous PRP treatment enhances the quality of tendon repair, as evidenced by improved biochemical, biomechanical, and histological tissue properties. In six horses, tendon lesions were created surgically in the Superficial Digital Flexor Tendons (SDFT) of both front limbs, one of which was treated with PRP and the other with saline. After 24 weeks, the tendons were harvested for biochemical, biomechanical, and histological evaluations. Collagen, glycosaminoglycan, and DNA content (cellularity) was higher in PRP-treated tendons (p = 0.039, 0.038, and 0.034, respectively). The repair tissue in the PRP group showed a higher strength at failure (p = 0.021) and Elastic Modulus (p = 0.019). Histologically, PRP-treated tendons featured better organization of the collagen network (p = 0.031) and signs of increased metabolic activity (p = 0.031). It was concluded that PRP increases metabolic activity and seems to advance maturation of repair tissue over nontreated experimentally induced tendon lesions, which suggests that PRP might be beneficial in the treatment of clinical tendon injuries.