Intramuscular fat accumulation and muscle atrophy in the absence of muscle retraction.

OBJECTIVES: Although many clinical and experimental investigations have shed light on muscle atrophy and intramuscular accumulation of fat after rotator cuff disruption, none have reported on their onset in the absence of muscle retraction. METHODS: In 30 rabbits, we detached one supraspinatus (SSP) tendon and repaired it immediately, thus preventing muscle retraction. The animals were killed in groups of 10 at one, two and six weeks. Both shoulders of 15 non-operated rabbits served as controls. We measured the weight and volume of SSP muscles and quantified the cross-sectional area of intramuscular fat (i-fat) histologically. RESULTS: There was significant loss of muscle weight and volume after one week (p = 0.004 and 0.003, respectively), and two weeks (both p < 0.001) in the experimental group; which recovered to control values after six weeks. I-fat accumulated one week after immediate repair, greater than in the control group and statistically significant at the mid-part of the muscle (mean 2.7% vs 1.5%, p = 0.008). I-fat continued to accumulate up to six weeks at all sites of the SSP muscle (all 3, p < 0.001). More fat accumulated closer to the musculotendinous junction than at the mid-part after two and six weeks (p = 0.012 and 0.019, respectively). CONCLUSION: Muscle atrophy and i-fat accumulation occur early after SSP tendon tear and immediate repair. While early repair benefitted muscle recovery, it did not prevent fat accumulation. SSP muscle retraction was not essential to the muscle alterations. The divergent evolution of muscle and fat points to different pathophysiologies. Cite this article: Bone Joint Res 2014;3:117-22.

[Epidemiology and pathophysiology of rotator cuff tears]. / Epidemiologie und Pathophysiologie der Rotatorenmanschettenrupturen.

Tears of the rotator cuff are one of the most common injuries to any tendon in the human body. Various theories and observations have since been made as to the origin of these tears. It is now commonly accepted to distinguish between"extrinsic" and"intrinsic" causes. Extrinsic describes an attrition which might have been caused by a subacromial bony conflict, while intrinsic changes originate within the tendon body itself due to age-related degenerative changes, giving rise to partial and later complete rupture of the tendon body. A better understanding of the underlying pathophysiological mechanism will make it easier for the attending orthopedic surgeon to decide whether a more conservative or operative course will have the best outcome for the situation given.

Different levels of COX-1 and COX-2 enzymes in synoviocytes and chondrocytes during joint contracture formation.

OBJECTIVE: To measure the levels of prostaglandin endoperoxide H synthase (PGHS) isozymes (or cyclooxygenase, COX) in vivo during the development of joint contractures secondary to immobilization in rats. METHODS: Rats had one knee joint immobilized for up to 32 weeks. Three groups were compared: 47 rats had knee joints immobilized, 38 animals had sham surgery, and 13 unoperated animals served as controls. Levels of PGHS-1 and PGHS-2 enzymes were characterized in the chondrocytes and synoviocytes of the knee joint by immunohistochemistry. Immunostaining intensity was quantified by microscopy using conventional analysis. RESULTS: PGHS-1 level was lower in synoviocytes of the anterior capsule compared with shams (1.3 vs 2.0; p < 0.05). PGHS-2 level was also lower in synoviocytes of the posterior capsule (1.8 vs 2.3; p < 0.05), but higher in chondrocytes at the anterior aspect of the tibia compared with shams (1.6 vs 0.8; p < 0.05). PGHS-2 staining was increased in chondrocytes at the posterior, opposed, and anterior aspects of the tibia compared with controls (1.1, 0.6, 0.8 vs 0.2, 0.1, 0.2, respectively; all p < 0.05). CONCLUSION: Immobility induced joint contractures are characterized by a contrasting cellular pattern of PGHS enzyme levels: decreased in the synovium and increased in the chondrocytes. These findings suggest that chondrocytic PGHS isoenzymes are important in cartilage degradation of contractured joints.

Incorporation of perforated and demineralized cortical bone allografts. Part I: radiographic and histologic evaluation.

Massive cortical bone allografts have been found to incorporate slowly into host bone. They are subject to complications such as nonunion, fatigue fracture and infection. In an attempt to improve osteoinduction in cortical bone allografts, laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into the sheep tibia. In this model, mid-shaft tibial bone allografts from out-bred sheep donor animals were prepared by partial demineralization and drilling of 0.33-mm diameter holes with a pulsed, 2.94-microm wavelength Erbium:Yttrium-Aluminum-Garnet laser. Recipient animals of the same out-bred strain were divided into three groups of eight according to the type of cortical allograft used: group 1, fresh-frozen, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Plain films were taken in two standard views at monthly intervals. Incorporation was evaluated at nine months postoperatively. Longitudinal radiographic data was correlated to a histologic and morphometric evaluation of each bone graft. Computer tomography was used for the latter analysis. Results showed that untreated allografts, although surrounded by a periosteal bone cuff, were poorly incorporated. Partial demineralization lead to excessive resorption of allografts, but little new bone formation. Laser perforation and partial demineralization induced complete incorporation of allografts into the host bone. Based on the results of the radiographic, histologic and morphometric evaluation, the development of laser-perforated and partially demineralized bone allografts was proposed for clinical use.

Incorporation of perforated and demineralized cortical bone allografts. Part II: A mechanical and histologic evaluation.

Laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into sheep tibiae. This paper reports results of the mechanical testing of the transplanted bones, which was done at nine months postoperatively. Animals were divided into three groups of eight according to the type of cortical allograft used: group 1, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Thus, changes in flexural rigidity of 24 transplanted whole tibiae were investigated. Starting in the anterior direction at the tibial tuberosity, the flexural rigidity was determined using a nondestructive 4-point bending test. The elliptical distribution of the flexural rigidity was compared to the untreated contralateral control bone of each animal. Mechanical parameters were defined as percentage rates for comparative analysis between groups. Flexural rigidity measurement showed that bones transplanted with untreated allografts were stiffer than contralateral control bones. Partial demineralization of allografts reduced the flexural rigidity of transplanted bones below the level of contralateral control bones. Flexural rigidities of test bones transplanted with laser perforated and partially demineralized allografts were higher than those seen in bones transplanted with partially demineralized allografts. These results were corroborated by the histologic analysis which showed that untreated allografts, although surrounded by a periosteal bone cuff that effectively increased their outer diameter. In contrast, excessive bone resorption was observed in partially demineralized allografts. Laser-perforated and partially demineralized allografts showed histologic evidence of complete incorporation into the host bone. Based on this mechanical evaluation, it was concluded that processing of cortical bone allografts by the combination of perforation and partial demineralization resulted in improved mechanical strength of the transplanted bones as compared to processing by partial demineralization alone.

Growth in width of the metacarpals--an investigation in human fetuses.

The process of growth in width of the human hand during fetal life has never been described. Do metacarpals grow concentrically and separation between the bones occurs through expansion of soft tissues? Or is growth eccentric, a process termed drift by Enlow, a relocation in space of organs? Hands of 10 spontaneously aborted fetuses (age range: between 14.5 and 24 weeks of gestation) were examined paying special attention to the bone bark. A thicker bone bark was taken as an indication of growth in that direction. The thickness of the bone bark was measured at the radial and ulnar sides at the level of the proximal and of the distal physes of the second to fifth metacarpals. A ratio of radial over ulnar bone bark thickness (R/U ratio) was calculated. The third metacarpal grew almost concentrically (R/U ratio 1.12 +/- 0.06). The second metacarpal grew in a radial direction (R/U ratio 3.29 +/- 0.19) and the fourth and more so the fifth metacarpal grew in an ulnar direction (R/U ratio 0.70 +/- 0.04 and 0.42 +/- 0.02, respectively). The differences in R/U ratios between every metacarpal were statistically significant for all comparisons P < or = 0.001. Fetal growth in width of the human metacarpals is eccentric and not concentric. It is concluded that during growth in width the metacarpals move away from the midline of the hand and that growth occurs through eccentric bone apposition rather than through soft tissue expansion.

Bone growth increases the knee flexion contracture angle: A study using rats.

OBJECTIVES: To assess the impact of bone growth on the flexion contracture angle at the knee, to measure the bone growth pattern in rats, and to assess the impact of immobility on bone growth. DESIGN: Experimental, controlled study. SETTING: Bone and joint laboratory. ANIMALS: Sixty Sprague-Dawley rats. INTERVENTIONS: Knee joints of 40 rats were immobilized unilaterally in flexion. Sham-operated animals (n = 20) were controls. MAIN OUTCOME MEASURES: The contracture angle and the femur and tibia lengths on radiographs. RESULTS: The angle of flexion increased over time and was largely explained by bone growth (r =.725, p <.01). Femur and tibia grew in rats until they were 11 months old. Immobility enhanced growth in bone length, especially of the femur, after 16 and 32 weeks of immobility (p <.05). CONCLUSIONS: Knee flexion contracture angle increased as a consequence of normal bone growth, a situation that is also encountered in skeletally immature children. The continued growth in length of bones in children may influence the progression of contractures and add to the therapeutic challenge. Ongoing bone growth should be considered when interpreting reports that use animal models for bone and joint diseases.

Optimization of perfusion decalcification for bones and joints in rats.

Decalcification of osseous tissues by perfusion of decalcifying solution into the vascular system has never been applied to the study of peripheral joints. To optimize perfusion methods, rats were decalcified by direct immersion or by one of two perfusion techniques: 1) systemic perfusion circulating the decalcifying solution from the ascending aorta; and 2) regional perfusion circulating the solution to the lower extremities from the abdominal aorta. The process of decalcification was monitored by serial radiographic examinations. After decalcification, bone and joint samples were stained for histochemistry and immunohistochemistry. With systemic perfusion, the decalcification time, dependent on weight, was markedly reduced compared to immersion. Regional perfusion decalcification was faster than all other methods studied. Microstructural preservation was comparable and immunostaining quality often improved. Applications of this work will improve the study of basic skeletal and articular problems.

Early reactions after reimplantation of the tendon of supraspinatus into bone. A study in rabbits.

In 14 rabbits we determined the origin of the cells effecting healing of the tendon of supraspinatus inserted into a bony trough. After two weeks both the cellularity of the underlying bone and the thickness of the subacromial bursa were significantly increased in the operated compared with the control shoulders. The cellularity of the stump of the tendon, however, was significantly decreased in the operated shoulders. In this model, both the underlying bone and the subacromial bursa but not the stump of the tendon contributed to the process of repair. We conclude that the medial stump should be debrided judiciously but that cutting back to bleeding tissue is not necessary during repair of the rotator cuff. Moreover, great care should be taken to preserve the subacromial bursa since it seems to play an important role in the healing process.

Synovial adhesions are more important than pannus proliferation in the pathogenesis of knee joint contracture after immobilization: an experimental investigation in the rat.

OBJECTIVE: To measure intraarticular pannus proliferation after early and prolonged joint immobility using an animal model. METHODS: Forty rats underwent unilateral immobilization of a knee joint with an internal fixator for periods of 2, 4, 8, 16, and 32 weeks. Twenty rats received sham surgery. The knee joints were harvested and processed for histological examination. The synovial intima length and the subintimal area were measured on standardized sagittal sections with image analysis software. The measurements were recorded with regard to their location (anterior or posterior; superior or inferior). RESULTS: Intra and interrater reliabilities for all measurements were > 87.9%. The synovial intima length was smaller in immobilized knees than in controls at all time points. At 4 and 32 weeks, the difference was statistically significant (p < 0.05). The differences were marked in the posterior synovium, where the intima length of immobilized knees was significantly smaller than in controls after 4, 8, 16, and 32 weeks of immobilization (p < 0.05). The subintimal area was comparable in immobilized and control knees at all time points. CONCLUSION: We standardized the quantification of intraarticular pannus in a joint contracture model after immobility of up to 32 weeks' duration. This study revealed a significant decrease in synovial intima length but no change in the subintimal area of immobilized knees compared with controls. The decrease in synovial intima length with immobility suggests that adhesions of synovium villi rather than pannus proliferation are the major pathophysiological changes leading to contracture after immobility.

Contractures secondary to immobility: is the restriction articular or muscular? An experimental longitudinal study in the rat knee.

OBJECTIVES: To measure articular structures' contribution to the limitation of range of motion after joint immobility. STUDY DESIGN: Experimental, controlled study involving 40 adult rats that had one knee joint immobilized in flexion for durations of 2, 4, 8, 16, and 32 weeks; 20 rats underwent a sham procedure. The angular displacement was measured both in flexion and extension at three different torques. Myotomy of transarticular muscles allowed isolation of the arthrogenic component of the contracture. RESULTS: A contracture developed in all immobilized knees. The articular structures were incrementally responsible for the limitation in range of motion (from 12.6 degrees +/-6.7 degrees at 2 weeks to 51.4 degrees +/-5.4 degrees at 32 weeks). The myogenic restriction proportionately decreased over time (from 20.1 degrees +/-8.4 degrees at 2 weeks to only 0.8 degrees +/-7.2 degrees at 32 weeks). The increase in the arthrogenic component of contracture was predominant in extension. CONCLUSION: This study quantified the increasing role of arthrogenic changes in limiting the range of motion of joints after immobility, especially as the period of immobility extended past 2 weeks. These data provide a better understanding of joint contracture development and can be used to guide therapeutic approaches.

Extent and direction of joint motion limitation after prolonged immobility: an experimental study in the rat.

OBJECTIVES: To test the hypotheses that contractures progress at different rates in relation to the time after immobilization, that immobilization in flexion leads to loss of extension range of motion, and that joints of sham-operated animals are better controls than the contralateral joint of experimental animals. STUDY DESIGN: Experimental, controlled study in which 40 adult rats had one knee joint immobilized at 135 degrees of flexion for up to 32 weeks and 20 animals underwent a sham procedure. At intervals of 2, 4, 8, 16, and 32 weeks, 8 experimental and 4 sham-operated animals were killed and their knee motion measured in flexion and extension. RESULTS: In the experimental group, the range of motion decreased in the first 16 weeks of immobility at an average rate of 3.8 degrees per week (p<.0001) to reach 61.1 degrees of restriction. A plateau was then observed from which the contracture did not progress further. The loss in range of motion occurred in extension, not in flexion. CONCLUSION: This study defined an acute stage of contractures starting at the onset of immobility and lasting 16 weeks, during which the range of motion was progressively restricted, and a chronic stage during which no additional limitation was detected. The loss in motion was attributed to posterior knee structures not under tension during immobilization in flexion. Contrary to the hypothesis, the contralateral joint was validated as a control choice for range-of-motion experiments.

Correlation of radiographic measurements with biomechanical test results.

The refracture of bone after the removal of an internal fixation device is not common, but it can be a serious complication. The development of a simple radiographic tool to determine the condition of a healing bone would be of clinical importance. As a basis to developing this tool, it was important to determine whether a correlation exists between radiographic results and the stiffness index of bones during fracture healing. This experimental investigation in beagles showed that at 24 weeks after a unilateral femoral osteotomy, plain radiographs could be used to determine the stiffness index of healing bone. The results showed the cortex to callus ratio (the thickness of the cortex, including the periosteal callus, normalized by the thickness of the cortex of the bone not surgically treated) correlated positively with the stiffness index of the bones. This measurement might be useful in the clinical assessment of fracture healing. From a clinical point of view, caution is recommended after the removal of fracture plates when the cortex to callus ratio is low because such a low ratio may be associated with a low stiffness index of the healing bone. This information allows clinicians to adapt the postoperative regimen accordingly.

Role of bone bark during growth in width of tubular bones. A study in human fetuses.

The morphologic features of bone bark, a structure surrounding the distal and proximal ends of long bones, were studied in the distal femur, proximal tibia, and proximal fibula of 77 spontaneously aborted human fetuses varying in gestational age from 10 to 20 weeks. Standard histologic techniques used in addition to in situ immunohistochemical staining allowed the examination of the structure of the bone bark and localization of Types 1, 2, and 3 collagens at different gestational ages. The bone bark was shaped like a cylindrical sheath of bone lamellae of varying thickness. The epiphyseal end of the bone bark, known as the groove of Ranvier, was covered outwardly by a fibrous layer and inwardly by the epiphyseal cartilage and contained mesenchymal cells, chondroblastic precursor cells, and densely packed cells differentiating into osteoblasts. Neither the cell density in the groove nor the thickness of the bone bark were identical circumferentially, indicating an unequal growth in width. In addition, the presence of periosteal apposition and endosteal resorption of the bone bark on one side and of endosteal bone deposition accompanied by periosteal resorption of the bone bark on the opposite side support the concept of a spatial drift of bones. These observations furnish histologic proof that groove and bone bark, although assuring an equal growth in length, contribute to an unequal and eccentric growth in width.

Histologic evidence of degeneration at the insertion of 3 rotator cuff tendons: a comparative study with human cadaveric shoulders.

We determined on histologic examination the degree of degeneration at the insertion of 3 rotator cuff tendons in 76 cadaveric shoulders, 17 of which had a partial tear of the supraspinatus. Fiber thinning, the presence of granulation tissue, and incomplete tearing of fibers, all evidence of degeneration, were quantified separately for each tendon. Among the shoulders that were intact on macroscopy, no significant difference in degeneration score could be found. In all 3 tendons degeneration was more prominent on the articular sides compared with the bursal sides (P < .0001). The degeneration score of partially torn supraspinatus was significantly higher than that of the intact tendons (P < .0001). The extent of granulation tissue, 1 criterion of degeneration, seemed to contribute mostly to this difference. Intrinsic degeneration occurred foremost in the articular side of the rotator cuff and might constitute the primary cause of rotator cuff tearing.

Mechanical properties of perforated and partially demineralized bone grafts.

Changes in flexural rigidity and compression strength of 18 sheep tibias were investigated after laser perforation and partial demineralization. Test bones were divided into three groups: Group 1, no treatment; Group 2, laser hole grid; and Group 3, laser hole grid and partial demineralization. Starting in the anterior direction at the tibial tuberosity, the flexural rigidity was determined using a nondestructive four-point bending test. The elliptical distribution of the flexural rigidity before and after a specific treatment was compared. After the bending test, a cylindrical center section of each test bone was loaded axially to failure to determine subsequent changes in compression strength. Results showed that perforation alone produced minimal reduction of rigidity and insignificant changes in compression strength. However, additional partial demineralization resulted in larger reductions. In compression testing, perforated and partially demineralized bone specimen showed marked decrease of the ultimate failure stress. The observed increase in failure strain appeared to be related to compression of the laser holes. The findings of this study suggest that partial demineralization and perforation can be applied to diaphyseal bone grafts and that their decreased mechanical properties are a function of the bone volume reductions produced by both processes.

Histomorphic analysis of the development of the pars interarticularis and its association with isthmic spondylolysis.

STUDY DESIGN: Descriptive study analyzing the histologic development of human fetal lumbar spine pars interarticularis. OBJECTIVES: To delineate the histologic development of the fetal pars interarticularis and correlate the development of the pars with isthmic spondylolysis. SUMMARY OF BACKGROUND DATA: Isthmic spondylolysis is present in 5% to 6% of the population. The incidence at birth is zero but rises sharply to 5% at age 4 to 5 years. The origin of the lesion is unknown; however, the current theory states that isthmic spondylolysis results from a fatigue fracture through a congenitally weak pars interarticularis. No study exists investigating the histologic development of the pars. METHODS: Microscopic examination of lumbar spines of human fetuses aged 8-20 weeks' gestation was performed. Specimens were dissected en bloc, decalcified, embedded in paraffin wax, sectioned and fixed on glass slides, and stained. RESULTS: The pars begins to ossify at 12 to 13 weeks' gestation by endochondral ossification. The ossification center originates in the region of the pars in lower lumbar vertebrae, resulting in uneven distribution of trabeculation and cortication in this region. The ossification center arises at the end of the pedicle in upper lumbar segments giving rise to uniform trabeculation through the pars. CONCLUSIONS: Uneven distribution of isthmic ossification results in formation of a potential stress riser in the region of the pars in lower lumbar vertebrae, which could be susceptible to fatigue fracture.

Pathogenesis of soft-tissue contracture in club foot.

We investigated the pathogenesis of soft-tissue contracture in club foot, using immunohistochemistry to study 41 biopsy specimens and 12 normal deltoid ligaments from cadavers. Five biopsy specimens were studied by electron microscopy (EM) to determine the presence of myofibroblasts. All 41 specimens of club foot stained positively for vimentin as against only one of the 12 control specimens. By contrast, there was no difference in staining for desmin or alpha-smooth muscle actin. EM showed some variability in the appearance of ligamentous cells. Most contained bundles of microfilaments in the cytoplasm and many had abundant pinocytotic vesicles, but no basal lamina or plasmalemmal attachment plaques. Cells of the medial ligamentous tissue in patients with club foot contain vimentin and others have myofibroblastic characteristics. Both features may contribute to recurrence after soft-tissue release.

Structural disorders at the insertion of the supraspinatus tendon. Relation to tensile strength.

We examined macroscopically and microscopically 55 cadaver rotator-cuff tendons attached to their humeral heads to determine the distance between the edge of the articular cartilage and the tendon insertion of the supraspinatus (the width of the sulcus) and the score of regressive changes at the sulcus. In 33 specimens we measured the tensile strength. The width of the sulcus was correlated with the score of regressive changes and with the ultimate tensile strength of the supraspinatus tendon. The width of the sulcus correlated positively with the score of regressive changes (r = 0.66, p < 0.0001), but there was a negative correlation between the latter and the ultimate tensile strength (r = -0.81, p = 0.001) and between the width of the sulcus and the ultimate tensile strength (r = -0.74, p = 0.004). We believe that the width of the sulcus is a simple and useful clinical indicator of the integrity and the tensile strength of the supraspinatus tendon.

Intraarticular tissue proliferation after immobility: methods of assessment and preliminary results in rat knee joints.

OBJECTIVE: To develop methods to quantify intraarticular connective tissue proliferation after immobility and to report results in an animal model of joint contracture. METHODS: Six rats had their right knee joints immobilized with an internal fixator for 3 weeks. The joints were harvested and sectioned. We measured the length of synovial intima and the subintimal area with image analysis software. Proliferating synoviocytes were identified by immunohistochemistry using the "proliferating cell nuclear antigen" antibody and were counted under optical microscopy on whole joint sections. Two outcomes were analyzed: the subintimal area and a synoviocyte proliferation index (number of proliferating synoviocytes/synovial intima length). Both were obtained for posterior and anterior aspects of the knee. RESULTS: Intra and interobserver reliabilities over 87% were found with these measurement techniques. Subintimal area means were not statistically different between immobilized and contralateral knees. In all subjects, the synoviocyte proliferation index (SPI) posteriorly was higher in immobilized than in contralateral knees (p<0.05), due to an increase in proliferating cells (p<0.05) rather than a change in synovial intima length (p>0.05). The SPI anteriorly was comparable in immobilized and contralateral knees (p>0.05). CONCLUSION: We developed methods to measure intraarticular connective tissue proliferation in a contracture model in vivo. Proliferative changes in the posterior aspect of the knee suggest local mediation of connective tissue proliferation in the contracture process. These methods and preliminary results will benefit investigators assessing interventions in similar models.