These studies examined the effects of heat-inactivated horse serum, insulin, triiodothyronine (T3), and thyroxine (T4), individually and in combination, on collagen and proteoglycan synthesis by primary cell cultures of articular chondrocytes from immature male rabbits. Insulin concentrations of 25 to 100 ng/ml (4.4 to 17.4 x 10(-9) M) increasingly stimulated collagen and proteoglycan synthesis in the absence of serum. The effects of 25 ng/ml (4.4 x 10(-9) M) insulin or 15% heat-inactivated horse serum on collagen synthesis were similar. Triiodothyronine (10(-10) to 10(-6) M) and T4 (10(-8) to 10(-4) M) also stimulated collagen synthesis in the absence of serum, with peak effects at 10(-8) and 10(-6) M, respectively. Biphasic stimulation of proteoglycan synthesis was obtained with 10(-11) to 10(-7) MT3 (maximum at 10(-8) M) and 10(-8) to 10(-5) M T4 (maximum at 10(-7) M). In these experiments, triiodothyronine was 10 to 100 times more potent than T4 in stimulating cartilage matrix production. The cells retained their chondrocytic phenotype under hormonal stimulation, secreting almost exclusively Type II collagen and large, chondroitin sulfate-rich proteoglycans. The addition of insulin to maximally-stimulating concentrations of either T3 or T4 in serum-free medium further stimulated matrix synthesis, suggesting that these hormones modulate chondrocyte metabolism via multiple biosynthetic/receptor pathways.

Animals↗

PubMed1993

Effects of gestation, lactation, and maternal calcium intake on mechanical strength of equine bone.

Skeletal homeostasis during late gestation, lactation, and the post-lactational recovery period is poorly understood. In an experiment using an animal model (the horse), metacarpal breaking strengths (MBS) estimated via transmission ultrasonics were examined during the last 12 weeks of gestation and for 40 weeks after parturition. MBS increased during the last 6-10 weeks of gestation in mares fed amounts of calcium (Ca) recommended by the National Research Council; maximum MBS coincided with the week of parturition. In contrast, MBS in mares fed 20% less Ca remained relatively constant during the last 12 weeks of gestation. In contrast to increases during late gestation, MBS decreased steadily in all mares during the first 12 weeks of lactation. MBS increased after approximately 12 weeks of lactation, but more slowly than they had declined. MBS of the bones of mares fed recommended amounts of Ca were fully restored at 24 weeks post-parturition, but those of Ca-deficient mares had not fully recovered even 20 weeks after milk production had ceased (40 weeks after parturition). Mid-cannon mediolateral diameters of foals born to mares fed Ca-deficient diets were thinner and mechanically weaker at birth (both p < 0.01). These differences in limb bone size and strength persisted during the first 40 weeks of life.

Analysis of Variance↗

PubMed1991

Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture.

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.

Animals↗

PubMed1990

Polysulfated glycosaminoglycan accelerates net synthesis of collagen and glycosaminoglycans by arthritic equine cartilage tissues and chondrocytes.

Low molecular weight polysulfated glycosaminoglycan (PSGAG) stimulated net collagen and glycosaminoglycan synthesis by normal and arthritic equine fetlock cartilage tissues in organ culture. Arthritic tissues were more sensitive to PSGAG stimulation. The rates of cartilage-specific type-II collagen and chondroitin sulfate-rich glycosaminoglycan synthesis by confluent chondrocyte cell cultures obtained from normal and arthritic equine cartilage tissues were increased by 25 and 50 mg of PSGAG/ml. Cells from arthritic cartilage were also more sensitive to the presence of PSGAG. In addition, concentrations of PSGAG (25 and 50 mg/ml) approximate to those in synovial fluid after intra-articular injection of 250 mg of PSGAG inhibited the rate of collagen and glycosaminoglycan degradation in cell culture. These findings suggest that PSGAG may have a role in the healing of mild cartilage degeneration by encouraging the production of replacement hyaline matrix materials, while delaying their subsequent degradation. In contrast, growth of cell cultures was inhibited by PSGAG, suggesting that these compounds may fail to stimulate chondrocyte replication, a prerequisite for tissue regeneration. Nonetheless, these observations provide direct evidence of a truly chondroprotective role for low molecular weight PSGAG in the treatment of equine degenerative joint disease.

Animals↗

PubMed1987

The use of transmission ultrasonics to assess bone status in the human newborn.

The applicability of transmission ultrasonics as a method for assessment of bone status in human newborns was investigated in two studies. Sound transmission velocity (SCV) through the intact distal radius and ulna was compared to midshaft bone mineral content (BMC) and to midshaft mechanical breaking strength (MBS) in 13 postmortem newborns [gestational age (GA) = 20 to 41 wk]. Midshaft MBS, ranged from 1 to 16 kg; BMC, which ranged from 84 to 88 mg/cm in the term infant, was consistent with previous reported photon absorptiometric data. SCV in the distal radius and ulna was correlated with midshaft MBS (r = 0.69 to 0.82) and BMC (r = 0.85 to 0.93) and increased exponentially with midshaft MBS and BMC. GA was correlated with SCV (r = 0.90 to 0.95). Log GA was correlated with midshaft MBS (r = 0.87 to 0.96) and BMC (0.97 to 0.99) in each of the four measured bones. In the second study, SCV through the distal radius and ulna was measured in 85 live newborns ranging in GA from 28 to 43 wk. SCV increased linearly with GA (r = 0.71 to 0.77). These data demonstrate that SCV through the distal radius and ulna increases linearly with GA and that SCV through the distal bones of the forearm is reliably related to midshaft BMC and MBS during the third trimester of gestation. Transmission ultrasonic measurement of SCV provides a rapid, reproducible, nonionizing, and noninvasive method for assessing bone strength and mineralization in human neonates.

Bone Development↗

PubMed1987

Serum triiodothyronine and thyroxine concentrations in weanling horses fed carbohydrate by direct gastric infusion.

Plasma glucose and serum insulin, thyroxine, and triiodothyronine concentrations were monitored in 6 weanling Thoroughbreds after direct gastric infusion of solutions containing sucrose or casein. Neither plasma glucose nor serum hormone concentrations were affected by infusions of water or by infusions of 326 or 424 g of casein/250 kg of body weight. However, glucose and hormone concentrations increased significantly (P less than 0.001) after infusions of 649 or 844 g of sucrose/250 kg. Initial rates of increase were more rapid and increases were subsequently reversed more rapidly when 844 g of sucrose/250 kg was infused than when 649 g of sucrose/250 kg was infused. Soluble carbohydrate in the digestive tract triggered specific responses in the serum thyroid hormone concentrations of weanling horses. Magnitudes and durations of these responses appeared to depend on the amount of carbohydrate present.

Animals↗

PubMed1986

Effects of age and diet on the development of mechanical strength by the third metacarpal and metatarsal bones of young horses.

The application of transmission ultrasonics to the equine cannon holds promise as a method of monitoring metacarpal and metatarsal development, quality and integrity under a variety of experimental and field conditions. The validity of relating the velocities of sound pulses transmitted through the cannon to the mechanical breaking strengths of these bones was tested in two studies. Breaking strengths calculated from the sound velocities through sections of the metacarpal bones from 14 yearling ponies and 12 yearling horses were highly correlated with the mechanical breaking strengths of those sections (r = .907 and .927, respectively; P less than .01). Sound velocities through the cannons of the horses before sacrifice ranged from 2,453 to 3,130 m/s and were correlated with their mechanically determined breaking strengths (193 to 262 X 10(6) N/M2; r = .673; P less than .01). The correlation coefficient increased to .912 when the sound velocities were adjusted for the sound-delaying effects of the overlying soft tissues. In a third study, 13 horses were weaned at 2 to 4 mo of age and were fed diets providing either 100 or 130% of National Research Council (NRC) energy and protein recommendations. Metacarpal and metatarsal development was monitored monthly for 15 mo via transmission ultrasonics. Sound velocities, breaking strengths calculated from velocities adjusted for estimated soft tissue cover, measured bone mediolateral diameters and cannon diameters minus estimated soft tissue increased as quadratic functions of chronologic age (r greater than .840; P less than .0001). None of these variables was significantly affected by diet, leg or sex. These studies have demonstrated that the use of transmission ultrasonics to estimate and monitor metacarpal and metatarsal breaking strengths in the live horse is reliable, reproducible, simple, accurate and valid. They also suggest that NRC energy and protein recommendations meet the requirements for maximum bone growth and development in well-managed young equines.

Aging↗

PubMed1986

Estimation of urinary flow rate in weanling and yearling horses.

In an attempt to validate the use of urinary creatinine concentration as an index of urinary flow rate, a series of timed quantitative urine collections were done on several groups of weanling and yearling ponies and Thoroughbreds. A total of 411 urine samples were generated by 19 ponies and 12 Thoroughbreds. Urinary flow rates and urinary creatinine concentrations were measured. When all the data were examined, urinary flow rates were independent of creatinine concentrations. However, for any given animal, daily urinary creatinine excretion was constant over several days, and urinary creatinine concentrations were related to urinary flow rates in a negative curvilinear fashion. Urinary flow rates in young horses can be estimated from urinary creatinine concentrations, but separate curves relating the 2 variables must first be derived for each animal.

Analysis of Variance↗

PubMed1985

Effects of dietary energy supply on serum thyroxine, tri-iodothyronine and insulin concentrations in young horses.

The effects of meal ingestion on the circulating concentrations of the growth-regulating hormones thyroxine (T4), tri-iodothyronine (T3) and insulin were examined in weanling Thoroughbreds fed 70% (diet A), 100% (diet B) or 130% (diet C) of their energy and protein requirements. Peak insulin concentrations occurred 1, 2 and 3 h after the ingestion of diets C, B and A respectively. Increases in plasma glucose concentrations preceded the increases in serum insulin concentrations. Serum T4 concentrations increased after the ingestion of diets A and B and decreased after diet C. In contrast, serum T3 concentrations were unaffected by ingestion of diet A but increased after the ingestion of diets B and C. The increase was much greater and more rapid in the horses fed diet C. However, the decrease in T4 concentration was five times greater than the increase in T3 concentration. Accelerated insulin secretion after the ingestion of a meal high in energy (carbohydrate) content was therefore associated with decreased T4 secretion and accelerated T4 conversion to T3. However, 6 months later serum T4 and T3 concentrations were unaffected by meal ingestion.

Age Factors↗