Cartilage matrix changes in the developing epiphysis: early events on the pathway to equine osteochondrosis?

REASONS FOR PERFORMING STUDY: The earliest osteochondrosis (OC) microscopic lesion reported in the literature was present in the femorotibial joint of a 2-day-old foal suggesting that OC lesions and factors initiating them may arise prior to birth. OBJECTIVE: To examine the developing equine epiphysis to detect histological changes that could be precursors to OC lesions. METHODS: Osteochondral samples from 21 equine fetuses and 13 foals were harvested from selected sites in the scapulohumeral, humeroradial, metacarpophalangeal, femoropatellar, femorotibial, tarsocrural and metatarsophalangeal joints. Sections were stained with safranin O and picrosiruis red to assess cartilage changes and structural arrangement of the collagen matrix. RESULTS: Extracellular matrix changes observed included perivascular areas of paleness of the proteoglycan matrix associated with hypocellularity and, sometimes, necrotic chondrocytes. These changes were most abundant in the youngest fetuses and in the femoropatellar/femorotibial (FP/FT) joints. Indentations of the ossification front were also observed in most specimens, but, most frequently, in scapulohumeral and FP/FT joints. A cartilage canal was almost always present in these indentations. The vascular density of the cartilage was higher in the youngest fetuses. In these fetuses, the most vascularised joints were the metacarpo- and metatarsophalangeal joints but their cartilage canals regressed quickly. After birth, the most vascularised cartilage was present in the FP/FT joint. Articular cartilage differentiated into 4 zones early in fetal life and the epiphyseal cartilage also had a distinct zonal cartilage structure. A striking difference was observed in the collagen structure at the junction of the proliferative and hypertrophic zones where OCD lesions occur. CONCLUSION: Matrix and ossification front changes were frequently observed and significantly associated with cartilage canals suggesting that they may be physiological changes associated with matrix remodelling and development. The collagen structure was variable through the growing epiphysis and a differential in biomechanical properties at focal sites may predispose them to injury.

Perichondrial-mediated TGF-beta regulation of cartilage growth in avian long bone development.

We previously observed using cultured tibiotarsal long-bone rudiments from which the perichondrium (PC) and periosteum (PO) was removed that the PC regulates cartilage growth by the secretion of soluble negative regulatory factors. This regulation is "precise" in that it compensates exactly for removal of the endogenous PC and is mediated through at least three independent mechanisms, one of which involves a response to TGF-beta. PC cell cultures treated with 2 ng/ml TGF-beta1 produced a conditioned medium which when added to PC/PO-free organ cultures effected precise regulation of cartilage growth. In the present study, we have investigated the possibility that TGF-beta itself might be the negative regulator which is produced by the PC cells in response to their treatment with TGF-beta1. Using a TGF-beta responsive reporter assay, we determined that PC cell cultures, when treated with 2 ng/ml or greater exogenous TGF-beta1, produce 300 pg/ml of active TGF-beta. Then we observed that this concentration (300 pg/ml) of active TGF-beta1, when added to PC/PO-free tibiotarsal organ cultures, effected precise regulation of cartilage growth, whereas concentrations of TGF-beta1 either greater or less than 300 pg/ml produced abnormally small cartilages. These results suggest that one mechanism by which the PC effects normal cartilage growth is through the production of a precisely regulated amount of TGF-beta which the PC produces in response to treatment with exogenous TGF-beta itself.

Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb.

The vertebrate forelimb and hindlimb are serially homologous structures; however, their distinctive morphologies suggest that different mechanisms are associated with each limb type to give rise to limb-type identity. Three genes have been implicated in this process; T-box transcription factors Tbx5 and Tbx4, which are expressed in the forelimb and hindlimb, respectively, and a paired-type homeodomain transcription factor Pitx1, expressed in the hindlimb. To explore the roles of Pitx1 and Tbx4 in patterning the hindlimb, we have ectopically misexpressed these genes in the mouse forelimb using transgenic methods. We have developed a novel technique for visualising the structure and organisation of tissues in limbs in 3D using optical projection tomography (OPT). This approach provides unparalleled access to understanding the relationships between connective tissues during development of the limb. Misexpression of Pitx1 in the forelimb results in the transformation and translocation of specific muscles, tendons, and bones of the forelimb so that they acquire a hindlimb-like morphology. Pitx1 also upregulates hindlimb-specific factors in the forelimb, including Hoxc10 and Tbx4. In contrast, misexpression of Tbx4 in the forelimb does not result in a transformation of limb-type morphology. These results demonstrate that Pitx1, but not Tbx4, determines the morphological identity of hindlimb tissues.

Multiple mechanisms of perichondrial regulation of cartilage growth.

We previously observed that the perichondrium (PC) and the periosteum (PO) negatively regulate endochondral cartilage growth through secreted factors. Conditioned medium from cultures of PC and PO cells when mixed (PC/PO-conditioned medium) and tested on organ cultures of embryonic chicken tibiotarsi from which the PC and PO have been removed (PC/PO-free cultures) effect negative regulation of growth. Of potential importance, this regulation compensates precisely for removal of the PC and PO, thus mimicking the regulation effected by these tissues in vivo. We have now examined whether two known negative regulators of cartilage growth (retinoic acid [RA] and transforming growth factor-beta1 [TGF-beta1]) act in a manner consistent with this PC/PO-mediated regulation. The results suggest that RA and TGF-beta1, per se, are not the regulators in the PC/PO-conditioned medium. Instead, they show that these two factors each act in regulating cartilage growth through an additional, previously undescribed, negative regulatory mechanism(s) involving the perichondrium. When cultures of perichondrial cells (but not periosteal cells) are treated with either agent, they secrete secondary regulatory factors into their conditioned medium, the action of which is to effect precise negative regulation of cartilage growth when tested on the PC/PO-free organ cultures. This negative regulation through the perichondrium is the only activity detected with TGF-beta1. Whereas, RA shows additional regulation on the cartilage itself. However, this regulation by RA is not "precise" in that it produces abnormally shortened cartilages. Overall, the precise regulation of cartilage growth effected by the action of the perichondrial-derived factor(s) elicited from the perichondrial cells by treatment with either RA or TGF-beta1, when combined with our previous results showing similar--yet clearly different--"precise" regulation by the PC/PO-conditioned medium suggests the existence of multiple mechanisms involving the perichondrium, possibly interrelated or redundant, to ensure the proper growth of endochondral skeletal elements.

Heterochronic differences of Hoxa-11 expression in Xenopus fore- and hind limb development: evidence for lower limb identity of the anuran ankle bones.

The wrist (carpus) and ankle (tarsus) of most tetrapods, as well as the wrist of anurans, contains relatively small nodular skeletal elements. The anuran tarsus, however, comprises a pair of long bones, the proximal tarsals tibiale and fibulare, which resemble the lower leg bones, tibia and fibula (zeugopodium). In this paper we investigate whether the proximal tarsals of Xenopus are of zeugopodial character identity, i.e. whether they develop under the influence of the same genes that pattern the lower limb. We compare Hoxa-11 expression in the forelimb bud with that in the hind limb bud by whole-mount in situ hybridization. Hoxa-11 has been implicated in the development of the lower limb. In Xenopus we note three differences between Hoxa-11 expression in fore- and hind limb buds: (1) Hoxa-11 expression is maintained until the hind limb bud reaches a larger size (2 mm) than that of the forelimb bud (1.5 mm); (2) Hoxa-11 expression is maintained over larger spatial domains than in the forelimb; and (3) Hoxa-11 expression has a pronounced posterior polarity in the hind limb, but not in the forelimb. Hind limb expression of Hoxa-11 can be understood as a heterochronic prolonging of the expression dynamic in the forelimb. Finally we found that the proximal tarsals start to develop within the expression domain of Hoxa-11, while in the forelimb the lower arm elements reach the distal expression limit of Hoxa-11. The gene expression data presented here support the notion of a zeugopodial identity of the proximal tarsal elements in Xenopus.

Regulation of growth region cartilage proliferation and differentiation by perichondrium.

Endochondral bone formation in vertebrates requires precise coordination between proliferation and differentiation of the participating chondrocytes. We examined the role of perichondrium in this process using an organ culture system of chicken embryonic tibiotarsi. A monoclonal antibody against chicken collagen type X, specifically expressed by hypertrophic chondrocytes, was utilized to monitor the terminal differentiation of chondrocytes. Proliferation of chondrocytes was examined by a BrdU-labeling procedure. The absence of perichondrium is correlated with an extended zone of cartilage expressing collagen type X, suggesting that the perichondrium regulates chondrocyte hypertrophy in a negative manner. Removal of perichondrium, in addition, resulted in an extended zone of chondrocytes incorporating BrdU, indicating that the perichondrium also negatively regulates the proliferation of chondrocytes. Partial removal of perichondrium from one side of the tibiotarsus led to expansion of both the collagen type X-positive domain and the BrdU-positive zone at the site of removal but not where the perichondrium remained intact. This suggests that both types of regulation by the perichondrium are local effects. Furthermore, addition of bovine parathyroid hormone (PTH) to perichondrium-free cultures reversed the expansion of the collagen type X-positive domain but not that of the proliferative zone. This suggests that the regulation of differentiation is dependent upon the PTH/PTHrP receptor and that the regulation of proliferation is likely independent of it. Taken together, these results are consistent with a model where perichondrium regulates both the exit of chondrocytes from the cell cycle, and their subsequent differentiation.

The effects of partial thyroidectomy on the development of the equine fetus.

A syndrome of congenital hypothyroidism and dysmaturity has been an important cause of reproductive loss and foal mortality in western Canada. The cause and pathogenesis of this syndrome is under investigation. One issue to be addressed is whether all the anomalies present in affected foals are produced concurrently by the same agent, or if affected foals are primarily hypothyroid in utero which induces the associated lesions. This study was designed to document the effects of fetal thyroidectomy, at about 215 days of gestation, on the growth and development of the equine fetus and to compare the anomalies present to those reported in the spontaneously occurring syndrome of foals in western Canada. Two sham-operated controls and 4 partially thyroidectomised foals were carried to term following surgery. Sham-operated control foals were normally developed. Partially thyroidectomised foals were hypothyroid; had hyperplastic thyroid gland remnants; abnormal behaviour and locomotor skills; and numerous, marked deficiencies in their skeletal development. The anomalies present in partially thyroidectomised foals were comparable to those reported in congenitally hypothyroid neonates of other species, including human infants, and were similar to those described in congenitally hypothyroid and dysmature foals reported in western Canada.

Paralysis and long bone growth in the chick: growth shape trajectories of the pelvic limb.

Growth of chick embryonic femora, tibiotarsi and first phalanges of digit three were measured at one day intervals from day 6 through 16 of incubation. Normal controls were compared to embryos paralyzed at 5 days of incubation. Over the 10 day study period, length of the paralyzed femora, length and width of the paralyzed tibiotarsi and differences in length of the phalanges were observed. Growth in length of phalanx one of digit three was most affected by paralysis over this period. Changes in shape of these bones also occurred during growth. Normal long bones undergo changes in shape as differential growth in length and width occurs. Such changes in shape can be considered as the bone's normal growth "trajectory". Paralyzed bones displayed a different growth trajectory than normal bones. As expected, the long bones of paralyzed embryos were shorter than age-matched controls. Contrary to expectations, however, paralyzed long bones were relatively more stout than age-matched controls.

[Observations of the development of the equine distal interphalangeal joint cavity concerning the pelvic limb of fetuses, fillies and adult horses]. / Zur Entwicklung der Gelenkhöhle des Hufgelenks an der Beckengliedmasse beim Fetus und Fohlen im Vergleich zum adulten Pferd.

Ten horses, younger than 1 year, were used to investigate the articulatio interphalangea distalis of the pelvic limb. The expansion of the dorsal recess was compared to those of adult horses. The recessus dorsalis always formed a shape like the letter 'm' or like a triangle with oblique angles. Consequently the great proximo-distal expansion on the medial side of the dorsal recess very probably has nothing to do with the age of horses.

Cultured embryonic bone shafts show osteogenic responses to mechanical loading.

Pairs of 17-day embryonic chick tibiotarsi were removed and maintained in organ culture. One of each pair was subjected to a single 20-minute period of intermittent loading at 0.4 Hz, producing peak longitudinal compressive strains of 650 microstrain (mu epsilon). In the 18-hour culture period following loading, alkaline phosphatase levels in the osteoblasts of the loaded tibiotarsi were maintained whereas in controls they declined. In situ hybridization using a collagen type I cRNA riboprobe showed a substantial increase in expression of mRNA for collagen type I in the periosteal tissue of bones that were cultured for 18 hours after loading compared with that in similarly cultured controls and bones cultured for 4 hours. These results demonstrate that appropriate loading of embryonic chick bones in organ culture elicits adaptive regulation of matrix synthesis as evidenced by increased expression of the gene for type I collagen and alkaline phosphatase activity. This model may be useful as it must contain all the obligatory steps between strain change in the matrix and modified osteogenic activity.

Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins.

To examine the regulation of collagen types IX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type IX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).

Some observations on the development of the long bones of ratite birds.

Observations were made on the long bones of 16 young ratite birds (10 rheas, 3 emus and 3 ostriches), from 1-day-old to 12 weeks of age. At hatching all long bones contained large cartilaginous cones which were continuous with the growth plates, and an osseous cortex. At one week of age ossification had commenced on the periphery of these embryonic cones and in some bones the cones had become separated from the growth plates. At 3 weeks of age the embryonic cones of cartilage were still present in the proximal and distal tibio-tarsi and narrow cartilaginous bridges connected the cones to the growth plates. Embryonic cones were not present in other long bones of this 3-week-old rhea nor in the long bones of ratites 6, 8 and 12 weeks of age. Other praecocial birds such as turkey poults and chickens have cones of embryonic cartilage in their long bones at hatching and these persist in the tibio-tarsi till one to 2 weeks of age. The presence of large cones of embryonic cartilage in the tibio-tarsal bones of 3-week-old ratite birds is probably a normal phenomenon. Awareness of this feature is necessary for the correct differential diagnosis of the prevalent musculoskeletal disorders of ratite birds.

Epigenesis in developing avian scales. I. Qualitative and quantitative characterization of finite cell populations.

Throughout a period from day 8.5 to day 12.5 of incubation of a chick embryo, a finite cell population of scale epidermis was characterized from various view points such as cellular organization, position, shape, area, number of constituent cells, density, and cell proliferation activity. In this study, the preparation of whole mount specimens was found to be quite valuable. On day 8.5, cells in the prospective scale region could be morphologically distinguished in the tarsometatarsus at a certain distance proximally away from the tarsometatarsal-phalangeal joint. --On day 9.25, about 1,100 cells became highly columnar in shape and densely associated, forming a placode structure. In both distally and proximally adjacent regions of this placode, the cells were semiquadrate in shape and loosely associated, leading to the formation of the interplacode structures. Such contrasting difference in cell organization between placode and interplacode was preserved from day 9.25 to day 11. During this period, both the area and number of constituent cells increased greatly in the placode and only slightly in the interplacode. However, cell proliferation activity was completely suppressed in the placode, and quite active in the interplacode. The activity in cell proliferation proved to be inversely correlated with the density of basal cells. Throughout the present study, it has been demonstrated that the early development of scale epidermis is achieved through a coordinated activity of the two discrete cell populations: the placode and interplacode.