Jerry A. Maynard

The influence of physical activity on ligaments and tendons.

Using either a bone-ligament-bone or a muscle-tendon-bone preparation, numerous investigators have demonstrated that the usual site of separation is in the transitional zone between the ligament (or tendon) and bone; hence, the term junction strength or load at separation is used to describe functional changes in these preparations. Junction strength is decreased with inactivity (immobilization) and increased with chronic activity (training) provided that the exercise program is of an endurance nature. Training also increases junction strength in thyroidectomized and hypophysectomized rats. Besides in junction strength, training results in heavier ligaments and higher ligament weight/length ratios. However, water content, collagen concentrations/dry weight or collagen concentration per weight/length unit are not significantly influenced by repeated bouts of exercise. Although immobilization is associated with lower elastic stiffness values (kg/mm), training appears to have little influence on this measure in normal animals. Rats and dogs with surgically repaired ligaments are weaker and the strength results are markedly lower if the leg is immobilized. Exercise training improves the repair strength of ligaments but does not result in normal values twelve weeks after the surgery. Exogenous administration of ICSH or testosterone results in higher repair strength whereas TSH, thyroxine, ACTH and growth hormone decreases this measure. It was concluded that the mechanical stress produced by chronic exercise is an important determination of the strength of repaired ligaments and of the junctions between ligaments (or tendons) and bones.

Elastic fibers in human intervertebral discs

Although previous studies failed to demonstrate elastic fibers in intervertebral discs, electron microscopy of twenty human intervertebral discs obtained at autopsy and operation revealed characteristic elastic fibers in both the annulus fibrosus and the nucleus pulposus. Their contribution to the mechanical properties of the intervertebral disc remains to be determined.

Pseudoachondroplastic Dwarfism: A Rough-surfaced Endoplasmic Reticulum Storage Disorder

An estimated 70,000 to 100,000 anterior cruciate ligament reconstructions are performed each year in the United States. With the increasing number of anterior cruciate ligament surgeries being performed, a concomitant increase in intraoperative complications can be expected. Complications include those associated with tunnel placement, notchplasty, graft fixation and advancement, suture laceration, graft laceration, guidewire insertion and removal, intra-articular hardware, posterior cruciate ligament laceration, compartment syndrome, and vascular injury.

Legg-Calvé-Perthes disease. Histochemical and ultrastructural observations of the epiphyseal cartilage and physis.

UNLABELLED Biopsy specimens of the lateral aspect of the femoral head and neck were obtained from five children with Legg-Calvé-Perthes disease and were studied using histochemistry and electron microscopy. Beneath the normal articular cartilage there was a thick zone of hyaline (epiphyseal) cartilage containing sharply demarcated areas of hypercellular and fibrillated cartilage with prominent blood vessels. The fibrillated cartilage was strongly positive to alcian blue, weakly positive to periodic acid-Schiff, and positive to aniline blue. The interterritorial matrix in the hypercellular areas was weakly positive to both alcian blue and periodic acid-Schiff. Ultrastructural examination of these areas revealed many irregularly oriented large collagen fibrils and variable amounts of proteoglycan granules. These results suggest that in the fibrillar areas there are: (1) a high proteoglycan content, (2) a decrease in structural glycoproteins, and (3) a different size of collagen fibrils from that of normal epiphyseal cartilage. The hypercellular areas had a decrease in proteoglycans, glycoproteins, and collagen. The lateral physeal margin was often irregular, with a marked reduction of collagen and proteoglycan granules, and contained numerous large lipid inclusions. CLINICAL RELEVANCE The abnormal areas in the epiphyseal cartilage of patients with Legg-Calvé-Perthes disease have different histochemical and structural properties from normal cartilage and from fibrocartilage. This suggests that the disease could be a localized expression of a generalized, transient disorder of epiphyseal cartilage that is responsible for delayed skeletal maturation. The cartilage lesions are similar to those seen in the vertebral plates in patients with juvenile kyphosis. Whether the epiphyseal cartilage abnormalities are primary or are secondary to ischemia remains uncertain; however, it appears that the collapse and necrosis of the femoral head could result from the breakdown and disorganization of the matrix of the epiphyseal cartilage, followed by abnormal ossification.

Oxidant conditioning protects cartilage from mechanically induced damage.

Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the antioxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that antioxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant preconditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 h with tert‐butyl hydrogen peroxide (tBHP) at nonlethal concentrations (25, 100, 250, and 500 µM) for a varying number of times (one, two, or four) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post‐compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied four times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia‐inducible factor‐1alpha (HIF‐1α), and glyceraldehyde 6‐phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated four times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase‐3 (MMP‐3) expression. These findings demonstrate that repeated exposure of cartilage to sublethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide‐induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.

CD44 expression in the developing and growing rat intervertebral disc

CD44 has been identified at the time of extracellular matrix formation and expansion in several sites of the developing embryo (Wheatley et al. [ 1993 ] Development 119:295–306). The nucleus pulposus, consisting of a hydrated extracellular matrix tissue at birth, not previously closely analyzed, was examined for expression of CD44 in the developing and aging rat intervertebral disc. CD44 was identified solely on notochordal cells from the first onset of intervertebral disc formation (day 15 embryo) through the loss of notochordal cells from the nucleus pulposus (12–24 months of age). No CD44 expression was found in the notochordal cells prior to disc formation or in any cells other than the notochordal cells in the annulus fibrosus or nucleus pulposus of the intervertebral disc. Using reverse transcriptase–polymerase chain reaction methodology, the single 365 amino acid CD44 standard, CD44s, open reading frame was amplified from notochordal cells isolated from the nucleus pulposus. Western blot analysis of a cultured nucleus pulposus notochordal cells total protein extract identified a single CD44 species devoid of chondroitin sulfate with a mass of ∼85 kDa, characteristic of CD44s. Cell surface detection for CD44 was co‐localized with hyaluronan and proteoglycans at first appearance of disc formation in the nucleus pulposus.

The ultrastructure of the growth plate in slipped capital femoral epiphysis.

Core biopsy specimens of the proximal femoral growth plate from three patients with slipped capital femoral epiphysis were studied by light and electron microscopy. In the resting zone, the cartilage matrix was composed of large, densely packed collagen fibrils. The cartilage matrix in the zones of chondrocyte hypertrophy where the slippage occurred contained only scattered fine collagen fibrils in a homogeneous ground substance. The change in composition of cartilage matrix in the distal region of the epiphyseal plate may predispose that region to slippage.

The effects of exercise training and denervation on the morphology of intrafusal muscle fibers.

SummaryThe morphological effects of daily bouts of exercise and denervation on teres minor intrafusal muscle fibers were investigated in male Sprague-Dawley rats. After denervation, nuclear bag and nuclear chain muscle fiber cross-sectional area atrophied only 25 and 33% of the amount experienced by extrafusal fibers. Of the two fiber types, the nuclear chain fibers appeared to be more responsive to the effects of exercise than the nuclear bag fibers; however, this trend for enlargement had no statistical significance. Length measurements did not reveal any marked changes of any fiber type to the experimental conditions of this study. It was concluded that the possible differences in function and innervation of the nuclear bag and nuclear chain fibers could partly account for these findings.

Proteoglycans of human scoliotic intervertebral disc

To determine the effects of scoliosis, irrespective of its etiology, on the intervertebral disc, we isolated the proteoglycans from the nucleus pulposus, annulus fibrosus, and cartilaginous end-plates of patients with idiopathic scoliosis, paralytic scoliosis, and cerebral palsy. The chemical composition of the proteoglycans, the proportions of aggregates and monomers, the sizes of the monomers, and the limiting viscosity numbers of the purified preparations were determined. The results showed that: (1) The scoliotic nucleus pulposus contained normal amounts of proteoglycan aggregates and monomers. The molecules that were isolated from the nucleus pulposus of a sixyear-old patient had a normal composition, but those from two adolescents with scoliosis contained more protein and less carbohydrate than the proteoglycans of normal tissue. (2) The disc level or location of the tissue in the scoliotic curve (concave or convex) did not affect either the amount or the molecular-weight distribution of proteoglycan aggregates from the fibrocartilaginous portion of the annulus fibrosus. (3) The convex portion of the end-plates appeared histologically normal, but in all specimens studied the tissue contained only traces or greatly reduced amounts of proteoglycan aggregates. The monomers were smaller than normal. Premature closure of the growth plate on the concave side of the curve was observed histologically in the only specimen that was available for study (from an eleven-year-old child with a 70-degree lumbar curve) . The proteoglycans extracted from the remaining hyaline cartilage, however, behaved as do their counterparts from normal tissue. CLINICAL RELEVANCE: Proteoglycans have important roles in determining the shock-absorbing ability of the nucleus pulposus, the elastic behavior of the annulus fibrosus, and the resilience to compression of the cartilaginous end-plates. In addition, proteoglycans play a role in the process of endochondral ossification occurring in the growth-plate portion of the end-plate. S Supported by a grant from the Hearst Foundation. t Read in part at the Annual Meeting of the Orthopaedic Research Society, New Orleans, Louisiana, January 19, 1982. Department of Orthopaedic Surgery, Biochemistry Research Laboratory, 180 Medical Laboratories, University of Iowa, Iowa City, Iowa 52242. The results of this study indicate that the scoliotic curve, irrespective of its etiology, produces secondary changes in the proteoglycans of the intervertebral disc that could alter the properties of the tissues and might be important in the progression of the disease. Scoliosis can occur in patients with generalized connective-tissue disorders (Marfan’s disease , osteogenesis imperfecta , Ehler-Danlos syndrome , and others) or in association with paralysis of the trunk, spina bifida, cerebral palsy, or congenital hemivertebra. Most often, however, scoliosis occurs in otherwise normal individuals and it is called “idiopathic”29”2. The intervertebral disc, a major load-carrying component of the spine, is formed by three distinct structures: the nucleus pulposus, the annulus fibrosus, and the cartilaginous end-plates. In a normal spine the nucleus pulposus is located centrally or slightly posteriorly (lumbar region), and in the young it is a gelatinous structure formed by a loose network of collagen fibers embedded in a gel of proteoglycans. Its water content, ranging from 70 to 90 per cent, is highest at birth and decreases with age’3. When a compressive load is applied to the spine, a fluid pressure develops within the nucleus and pushes the annulus fibrosus radially outward and the centers of the end-plates away from each other’3. The mechanical behavior of the intervertebral disc is largely dictated by its two major macromolecular components: proteoglycans and collagen. While the negatively charged groups of the glycosaminoglycan chains are largely responsible for the fluid pressure in the nucleus pulposus”, the collagenous structure of the annulus fibrosus is ideally suited to accommodate the complex stresses caused by compression of the nucleus From a mechanical point of view, the cartilaginous end-plates may be considered the weakest structure of the intervertebral disc. They have been shown to fracture under compressive loads that leave both the nucleus and the annulus intact5’27. Together with the periphery of the annulus they represent, however, the main route for the nutrition of the discs20”4, the largest avascular structures of the body. The end-plates also provide for growth and ossification of the vertebral bodies and are the sites of insertion of the fibers of the annulus fibrosus. During the 816 ANGIOLA PEDRINI-MILLE ET AL. THE JOURNAL OF BONE AND JOINT SURGERY growth years the end-plates are formed by a layer of hyaline cartilage overlying a growth plate. Endochondral growth and ossification occur uniformly across the entire vertebral body until ossification centers appear in the outer regions of the end-plates at around the age of twelve yearsl7. The resulting epiphyseal ring is totally ossified and joined to the body of the vertebra by the age of seventeen to twenty-five years. This coincides with the cessation ofgrowth, although hyaline cartilage persists in the central portions of the end-plates8”0. Normal endochrondral growth i nvolves proliferation, maturation, hypertrophy, and degeneration of chondrocytes leading to calcification of the intercellular matrix. The synthesis of proteoglycans, collagen, and possibly non-collagenous matrix proteins changes in a characteristic fashion at different stages of the process23. In order for these events to proceed normally, an optimum pressure is also required. In 1862 Heuter and Volkmann noted an inverse relationship between compressive forces parallel to the axis of epiphyseal growth and the rate of growth of the cartilage, and suggested that a decrease in compression would lead to an acceleration of growth i:t, This was proved by Hert1’, who also demonstrated that a decrease of the compressive forces beyond a point at which the epiphysis could effectively be considered to be under traction leads to dedifferentiation of the chondrocytes or dosure of the growth plate. Irrespective of its etiology, the scoliotic curve severely affects the mechanical properties of the spine. Structures on the concave side of the curve are subject to much greater loads than are their counterparts on the convex side. In 1973 we reported that the nucleus pulposus of children with idiopathic scoliosis contained more collagen and less glycosaminoglycans than normal tissue, while the composition of the corresponding annulus fibrosus was normal2’. This was confirmed by Zaleske et al. 16, who found the same abnormalities in tissues of patients with scoliosis secondary to myelomeningocele. Normal nucleus pulposus contains only type-Il collagen whereas annulus fibrosus contains both type-I and type-Il collagen, their proportions changing in a gradient-like fashion from the outer zone, where type I predominates, to the inner region, where 70 per cent of the collagen is type II I0#{149} The relative amounts of the two types of collagen have recently been found to be severely altered in scoliotic annuli taken from the convex side of the curve, although a somewhat abnormal pattern of distribution was also seen in the outer and middle regions of the annulus fIbrosus from the concave side2t. The present study of the proteoglycans of the nucleus pulposus, annulus fibrosus, and end-plates of the scoliotic spine was motivated by the involvement of such molecules in determining the mechanical properties of a tissue and by their role in endochondral ossification. Our main purpose was to see whether the uneven distribution of forces caused by the scoliotic curve, irrespective of its etiology, would affect the proteoglycans of the intervertebral disc. Materials and Methods Intervertebral discs were obtained at autopsy, within twelve hours of death, from skeletally normal individuals and at surgery from eight patients. Five patients had idiopathic scoliosis: one girl, six years and six months old, with a 90-degree curve and three adolescent girls and one boy, fifteen to eighteen years old, with curves measuring between 45 and 50 degrees. One patient had paraplegia: a fifteen-year-old boy who had been completely paralyzed below the sixth thoracic vertebra since the age of five years. The thoracolumbar curve. first noted when he was seven years old, remained rather mild and non-progressive up to the age of thirteen years and then increased approximately 50 degrees in the sixteen months preceding surgery, when it measured 90 degrees. Two girls had cerebral palsy: one fifteen-year-old girl with a right lumbar curve of 70 degrees and one eleven-yearold girl with a rapidly progressing curve of 74 degrees.

Pseudoachondroplasia: biochemical and histochemical studies of cartilage.

Cartilage from patients with pseudoachondroplasia is characterized by unique inclusions in the cisternae of the endoplasmic reticulum and proteoglycan abnormalities have been suggested in this form of dwarfism. To elucidate the nature of the proteoglycan defect, we determined the amount of the individual glycosaminoglycans present in iliac-crest cartilage of three patients and extracted the proteoglycan monomers from one of the samples. Sections of iliac-crest cartilage and proximal fibular growth plates were examined by electron microscopy and also stained with hematoxylin and eosin, safranin O-fast green, and alcian blue in the presence of increasing concentrations of magnesium chloride (zero to one molar). The chondrocytes of the iliac crest and fibular physes were arranged in clusters more than in columns and contained characteristic endoplasmic reticulum inclusions, which were particularly large in the hypertrophic cells. The cartilage stained very poorly with hematoxylin and eosin and with safranin O-fast green. The alcian-blue stain was abolished from perilacunar areas and from longitudinal septa by magnesium chloride concentrations that were lower than those required by normal tissue. The proteoglycans of iliac-crest cartilage were found to be significantly enriched in keratan sulphate and had a below-normal ratio of chondroitin-4-sulphate to chondroitin-6-sulphate, although the amount of the two isomeric chondroitin sulphates combined was within normal limits. The urinary excretion of glycosaminoglycan by the three patients was normal. Pseudoachondroplasia appears to be a generalized cartilage disorder involving abnormalities of proteoglycans, probably related to the core protein or to enzymes that are responsible for the formation of the glycosaminoglycan chains.(ABSTRACT TRUNCATED AT 250 WORDS)